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Doi:10.1016/j.jcrs.2008.06.016

Effect of intraoperative mitomycin-C on healthy 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 laser-assisted 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 contactlenses were treated with LASEK with intraoperative use of topical MMC 0.02% (15 seconds). Non-contact corneal specular microscopy was performed in all eyes preoperatively and 1, 3, and 6months after surgery. Preoperative pachymetry and ablation depth were measured in all eyes. Re-peated-measures analysis of variance was used to compare the changes in the endothelial centralcell 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 changein 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 was31.45 G 8.26, and the mean HEX was 66.03% G 25.83%. After LASEK, there were no statisticallysignificant changes in CCD, CV, or HEX (P>.05). Multiple linear regression did not identify ablationdepth 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 notaffect the corneal endothelium.
J Cataract Refract Surg 2008; 34:1715–1719 Q 2008 ASCRS and ESCRS Although laser in situ keratomileusis (LASIK) is a ma- photorefractive keratectomy (PRK), the corneal epithe- jor refractive surgery technique because of its advan- lium is kept intact in LASEK; however, postoperative tages (eg, minimal pain, rapid visual rehabilitation) haze remains a major problem after LASEK. During and its low incidence of complications, laser-assisted the initial application of PRK, mitomycin-C (MMC) subepithelial keratectomy (LASEK) is also a valuable was introduced clinically as a topical adjunctive ther- surgical technique for the treatment of eyes with apy to prevent the development of corneal hazeIn high myopia, eyes with a thinner cornea, and eyes animal models, MMC has been shown to suppress with preexisting retinal pathology.Unlike in the activation of keratocytes and fibroblasts and pro-long apoptosis of keratocytes after PRK and afterLASEK.Clinical studies show that PRKandLASEKwith intraoperative use of MMC are safe Accepted for publication June 6, 2008.
and produce excellent visual outcomes with few com-plications, although the toxicity of MMC to endothelial From the Department of Ophthalmology, Changzheng Hospital Affiliated to Second Military Medical University, Shanghai, China.
Whether MMC is toxic to endothelial cells depends No author has a financial or proprietary interest in any material or on its prolonged application and/or its use in higher concentrations.In refractive surgery, the conven- Corresponding author: Rui-Li Wei, MD, Department of Ophthalmol- tional MMC concentration is 0.2 mg/mL (0.02%); ogy, Changzheng Hospital, 415 Fengyang Road, Shanghai 200003, however, the application time varies and is as long as 2 minutes. In this study, we measured corneal endothelial cells before and after LASEK with intra- to prevent overcorrection. A 7.0 mm round cellulose operative administration of a conventional dose of sponge soaked in MMC 0.02% (0.2 mg/mL) solution was MMC 0.02% but for a shorter time (15 seconds) to as- then applied over the ablated surface for 15 seconds.
Next, the cornea was irrigated with BSS to remove the re- sess the effect of MMC on corneal endothelial cells af- maining MMC. Care was taken to avoid exposure of the ter LASEK. To assess endothelial cell function, we epithelial flap, limbus, or conjunctiva to the MMC. The ep- used a corneal specular microscope to evaluate the en- ithelial flap was replaced and the undersurface gently dothelial cell central density (CCD) and 2 morphologic washed with BSS. Finally, a sterile bandage soft contact lens (PureVision, Bausch & Lomb) was placed on the dthe coefficient of variation in cell size (CV) cornea and dexamethasone 0.1% and topical gatifloxacin and percentage of hexagram cells (HEX). We excluded patients who wore contact lenses to ensure that any Ablation was performed using a 193 nm 217z scanning- postoperative change in corneal endothelial health spot excimer laser system with a combined 2.0 mm and 1.0 mm spot (Zyoptix, Bausch & Lomb). Before each treat-ment, the laser was calibrated by a fluence test and theeye-tracking system was tested. The radiant exposure was 0.2 J/cm2 in the treatment plane, and the repetitionfrequency of the laser was 120 Hz.
This prospective interventional nonrandomized observer-masked study comprised 89 consecutive patients (174 eyes)who were scheduled to have LASEK to correct myopia or myopic astigmatism. All patients provided informed con-sent after they received a thorough explanation of the proce- Postoperative medications included tobramycin 3 mg/ dure and its risks according to the Declaration of Helsinki.
mL and dexamethasone 1 mg/mL drops 4 times daily until An institutional review board approved the study. Patients complete reepithelialization. The bandage contact lens was aged 18 to 45 years who had unstable refraction, previous removed 1 week after surgery after the epithelium ocular surgery (refractive or other procedures), suspected completely healed. The steroid drops were tapered over the keratoconus, ocular disease, and systemic disease that might subsequent 2 months as follows: fluorometholone 0.1% alter the wound-healing process (eg, such as diabetes and drops 4 times daily for the first week; prednisolone acetate connective tissue disorders) were excluded from the study.
1% ophthalmic suspension drops 18 times daily (6 drops at Patients who wore contact lenses were also excluded.
a time with a 5-minute interval between drops 3 timesa day) for approximately the next 10 days; fluorometholone0.1% drops 3 times daily for the following 2 weeks, twice daily for another 2 weeks, and once daily for the last 2 weeks.
A series of ophthalmologic examinations was performed Carboxymethylcellulose 0.5% was given as needed during preoperatively. The evaluations included measurement of uncorrected and best spectacle-corrected visual acuity Examinations were scheduled for 1 day, 1 week, and 1, 3, (Snellen chart, Nidek ACP 8 auto chart projector), slitlamp and 6 months after surgery. Observation of the corneal endo- biomicroscopy, tonometry (CT-80, Topcon), corneal pachy- thelium was performed at each visit by the same masked ob- metry (SP-3000 pachymeter, Tomey Laboratories, Inc.), kera- server who used the images obtained with the corneal tometry and corneal topography (Orbscan, Bausch & Lomb), specular microscope preoperatively and postoperatively.
The 3 primary outcome measures were endothelial CCD, Immediately before surgery, 3 photographs of the central cornea were taken with a corneal specular microscope (Top-con SP-2000P, Topcon Corp.). The microscope provides animage of the endothelial layer, an automated endothelial cell count, and noncontact pachymetry measurements.
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 (overtime) was performed to determine whether there were signif- All the LASEK procedures were performed by the same icant changes in the endothelial CCD, CV, or HEX after experienced surgeon (R-L.W.) using the same excimer laser surgery. A paired t test was used to compare the 1-, 3-, and (Technolas 217z, Bausch & Lomb Surgical). Topical propara- 6-month postoperative values of the 3 corneal endothelium caine 0.5% was applied to each eye to anesthetize the cornea.
indices with the preoperative values. Multiple linear regres- A 20% alcohol solution mixed with distilled water in sion analysis was performed to determine whether age, pre- a glass syringe was applied to the cornea within an 8.5 operative refractive error, ablation depth, or residual stromal mm corneal well. The duration of exposure to the alcohol bed (RSB) thickness predicted the postoperative changes in was 15 seconds. A cellulose sponge was used to remove the 3 indices over time. A P value of 0.05 or less was consid- the alcohol, and balanced salt solution (BSS) was instilled to rinse the ocular surface. The flap edges were driedwith a sponge, and the epithelial flap was peeled backwith a crescent blade (Alcon Surgical), leaving a hinge at the 12 o’clock position. The stromal bed was dried witha sponge. Laser ablation was performed using the excimer shows the demographics of the patients and laser with Planoscan software (Bausch & Lomb). The target the preoperative and intraoperative measurements.
refraction to be corrected was the manifest refractive error The minimum postoperative follow-up was 6 months.
J CATARACT REFRACT SURG - VOL 34, OCTOBER 2008 postoperative values of any index at any time point 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 postopera- tive measurements) and the intraoperative ablation depth or RSB thickness (PO.05). There were also no sta- tistically significant correlations between changes in CV and HEX (preoperative versus postoperative) and intra- operative ablation depth or RSB thickness (PO.05).
No intraoperative or postoperative complications RSB Z residual stroma bed; SE Z spherical equivalent (eg, epithelial healing defects, infection) occurred.
Reepithelialization was complete in all eyes by the Mitomycin-C is a bifunctional alkylating agent that in- hibits DNA synthesis and cell mitosis. Topical MMC is changes in the CCD, CV, and HEX. As 75% of patients now commonly used in several fields of ophthalmic were between 23 years and 33 years of age, an analysis surgery including glaucoma, pterygium excision, of the correlation between CCD and patient age was and ocular surface neoplasia treatment. Several clini- performed. Analysis of 1 eye of each patient (right cal studies have found that MMC is effective in reduc- eye if both eyes examined) showed no statistically sig- ing the incidence and severity of postoperative corneal nificant correlation between patient age and preopera- haze after PRK or LASEK because it inhibits fibroblas- tive CCD (r Z À0.187, P Z .079). There was no tic proliferation in the ablated stroma.Although statistically significant correlation between the preop- well tolerated in most cases, MMC has been associated erative CCD and the preoperative SE (r Z À0.086, with several complications such as corneal defects and P Z .261) or the preoperative pachymetry (r Z 0.037, delayed healing.These complications result from the toxicity of MMC to the corpuscular physiological func- Multivariate analysis of repeated measures (over tion. As the human corneal endothelium is essentially time) of the change in CCD, CV, and HEX showed nonregenerative in vivo, the effect of MMC on the en- that MMC treatment did not have a statistically signif- dothelium can be an important index in assessing the icant effect from preoperatively to 1 month, 3 months, or 6 months postoperatively (P Z .474, P Z .153, and In the earliest clinical study of corneal endothelial P Z .060, respectively). When the postoperative values cells, Morales et found that topical application of were individually compared with the preoperative MMC 0.02% for 30 seconds induced loss of corneal en- values without taking into account the repeatability dothelial cells 3 months after PRK in 9 eyes. The limita- of the instrument, there were no statistically significant tion of their study was that the number of eyes was too differences between the preoperative values and small to allow one to reach a convincing conclusion.
Table 2. Changes in corneal endothelium indices after LASEK with intraoperative MMC.
Means G SDCCD 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 the specular microscope changes accordingly. How- Table 3. Surgical factors related to changes in corneal endothe- ever, some studies report that this change is insignifi- lial indices after LASEK with intraoperative MMC.
cant and thus can be missed in corneal endothelial cell observation by corneal specular micro We used the same concentration of MMC (0.2 mg/mL) as in other studies but for a shorter period (15 sec- onds). We found no significant change in the corneal endothelial CCD within 6 months after surgery. These results are consistent with those of Diakonis et Be- cause the HEX value reflects the change in endothelial cell morphology, it is regarded as a sensitive index for assessing endothelial damagTo gain a comprehen- sive understanding of endothelial function, we used the CV and HEX indices in our study. There was no significant change in CV and HEX between preopera- tively and any postoperative time point. This agrees with a recent study by Goldsberry et which foundthat the 3 endothelial indices did not change within 1 CCD Z central cell density; CV Z coefficient of variation in cell size;HEX year after PRK; however, the MMC application time Z percentage of hexagram cells; MMC Z mitomycin-C; RSB Z in that study was shorter (12 seconds). In our 6 months of follow-up of patients with healthy endothelium, useof MMC 0.02% on the ablated stroma for 15 secondsdid not induce significant changes in corneal endothe- Another comparative studyfound no change in lial function (eg, CCD, CV, HEX) over baseline values.
the number of corneal endothelial cells over 12 postop- Early studies of LASIKfound an acute effect on the erative months in 15 eyes that had PRK with intraoper- corneal endothelium. The influence of laser enhance- ative use of topical MMC 0.02% for 15 seconds; the ment may represent transient endothelial cell edema 15 fellow eyes had epithelial LASIK without MMC in within 15 minutes and 1 day after LASIK. The most significant differences were qualitative and quantita- In a subsequent comparative study by de Benito- tive changes in endothelial cell morphology; that is, Llopis et al.,48 eyes were treated with intraoperative fewer corneal endothelial hexagram cells. Moreover, MMC 0.02% for 30 seconds over the ablated zone dur- there was a correlation between the change in the cor- ing LASEK and 32 eyes had LASEK without MMC.
neal endothelium and the RSB thickness. An experi- There was no significant difference between the mental studyfound that 193 nm excimer laser 2 groups in corneal endothelial CCD after surgery.
ablation induced loss of the corneal endothelium.
Both groups had a statistically significant increase in Thus, adequate RSB thickness appears to be important to maintaining corneal endothelial function. With cur- Another studyof a larger cohort (1101 eyes) used rent surgical techniques, it is safe to perform excimer intraoperative MMC 0.02% for longer times (range laser ablation when the RSB is thicker than 250 mm.
30 seconds to 2 minutes depending on ablation depth).
Based on these findings, we hypothesized that The authors found that the number of endothelial cells MMC diffuses into the RSB to reach the corneal endo- thelium. The thinner the RSB, the more easily MMC Several streport that the corneal endothe- diffuses deep into the corneal stroma and induces en- lial cell count decreased as a result of cell hypoxia dothelial toxicity. Our study included RSB thickness induced by contact lens use. The studies discussed and ablation depth (laser enhancement) in the analy- above did not exclude patients with a long preopera- sis of correlation. In our patients, the thickest RSB was tive history of contact lens use; thus, their finding of 480 mm and the thinnest, 264 mm. The largest ablation an increase in the number of corneal endothelial cells depth was 224 mm and the smallest, 52 mm. There was is likely the result of the patients discontinuing contact no significant correlation between the change in endo- lens use after surgery. With this in mind, we excluded thelial indices and the RSB thickness or ablation patients who wore contact lenses preoperatively so we could observe the effects of intraoperative MMC alone Although a 0.02% concentration of MMC was ap- on the health of the corneal endothelium after LASEK.
plied for a short time in our study, a flushing dose of The corneal thickness and keratometry change after steroids (2 weeks postoperatively for approximately LASEK or LASIK, and the image magnification of 10 days) seemed to inhibit the development of haze.
J CATARACT REFRACT SURG - VOL 34, OCTOBER 2008 A study of the long-term effect of steroids is underway endothelium after photorefractive keratectomy. Am J Ophthalmol in our laboratory. In terms of the endothelium, there 10. Diakonis VF, Pallikaris A, Kymionis GD, Markomanolakis MM.
were no significant changes between preoperatively Alterations in endothelial cell density after photorefractive kera- and postoperatively in CCD, CV, and HEX, 3 important tectomy with adjuvant mitomycin. Am J Ophthalmol 2007; indices for assessing endothelial cell function. In addi- tion, there was no significant correlation between the 11. de Benito-Llopis L, Teus MA, Ortega M. Effect of mitomycin-C on 3 indices and the 2 LASEK-related factors of ablation the corneal endothelium during excimer laser surface ablation.
J Cataract Refract Surg 2007; 33:1009–1013 12. Lee DH, Chung HS, Jeon YC, Boo SD, Yoon YD, Kim JG. Photo- In summary, prophylactic intraoperative applica- refractive keratectomy with intraoperative mitomycin-C applica- tion of MMC (0.02% for 15 seconds) did not seem to af- tion. J Cataract Refract Surg 2005; 31:2293–2298 fect the corneal endothelium. Therefore, its use to 13. Wiffen SJ, Hodge DO, Bourne WM. The effect of contact lens inhibit the development of haze in LASEK and PRK wear on the central and peripheral corneal endothelium. Cornea2000; 19:47–51 14. 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 1. Dastjerdi MH, Soong HK. LASEK (laser subepithelial keratomi- postoperatively. Am J Ophthalmol 2001; 131:1–6 leusis). Curr Opin Ophthalmol 2002; 13:261–263 15. Isager P, Guo S, Hjortdal JØ, Ehlers N. Endothelial cell loss after 2. Sekundo W, Tietjen A. Laserassistierte subepitheliale Keratek- photorefractive keratectomy for myopia. Acta Ophthalmol ¨ bersicht u¨ber den gegenwa¨rtigen Kenntnis- stand. [Laser-assisted subepithelial keratectomy (LasEK).
16. Chiou AG-Y, Kaufman SC, Kaufman HE, Beuerman RW. Clini- Review of the current state of knowledge.] Ophthalmologe cal corneal confocal microscopy. Surv Ophthalmol 2006; 3. Talamo JH, Gollamudi S, Green WR, de la Cruz Z, Filatov V, 17. Goldsberry DH, Epstein RJ, Majmudar PA, Epstein RH, Stark WJ. Modulation of corneal wound healing after excimer la- Dennis RF, Holley G, Edelhauser HF. Effect of mitomycin C on ser keratomileusis using topical mitomycin C and steroids. Arch the corneal endothelium when used for corneal subepithelial 4. Kim T-I, Lee SY, Pak JH, Tchah H, Kook MS. Mitomycin C.
ceramide, and 5-fluorouracil inhibit corneal haze and apoptosis 18. Kim T, Sorenson AL, Krishnasamy S, Carlson AN, Edelhauser HF.
Acute corneal endothelial changes after laser in situ keratomileu- 5. Kim T-I, Pak JH, Lee SY, Tchah H. Mitomycin C induced reduc- tion of keratocytes and fibroblasts after photorefractive keratec- 19. Edelhauser HF. The resiliency of the corneal endothelium to re- tomy. Invest Ophthalmol Vis Sci 2004; 45:2978–2984. Available fractive and intraocular surgery; the Castroviejo Lecture. Cornea 20. Simaroj P, Kosalprapai K, Chuckpaiwong V. Effect of laser in situ 6. Bedei A, Marabotti A, Giannecchini I, Ferretti C, Montagnani M, keratomileusis on the corneal endothelium. J Refract Surg 2003; 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 8. Rajan MS, O’Brart DPS, Patmore A, Marshall J. Cellular effects of mitomycin-C on human corneas after photorefractive keratec- tomy. 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 J CATARACT REFRACT SURG - VOL 34, OCTOBER 2008

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