The Journal of International Medical Research 2010; 1484 1490 [first published online as 38(4) 9] Short-term Corneal Endothelial Changes after Laser-assisted Subepithelial Keratectomy J ZHOU, S LU, J DAI, Z YU, H ZHOU AND X ZHOU Myopia Key Laboratory of the Health Ministry, Eye and ENT Hospital of Fudan University, Shanghai, China To investigate the short-term effects of laser-assisted subepithelial keratectomy (LASEK) on the corneal endothelium, 10 patients undergoing LASEK for myopia without complications (20 eyes) were observed. Each eye was evaluated by slitlamp biomicroscopy and non-contact specular microscopy pre-operatively, and at 15 min, 1 day and 1 week postoperatively. The decrease in endothelial cell density was statistically significant at 15 min post-operatively, and the difference between pre-operative and 15- min post-operative coefficient of variation of cell size was also statistically significant. The percentage of hexagonal cells was significantly different from baseline levels at 15 min and at 1 day postoperatively. All parameters at 1 week postoperatively were not statistically different from those observed pre-operatively. Immediate changes in endothelial cell number and morphology occurred following LASEK, but endothelial morphology returned to the pre-operative condition after 1 week. Long-term followup is needed to identify further consequences of this procedure. KEY WORDS: CORNEA; ENDOTHELIAL CELL; MYOPIA; LASER-ASSISTED SUBEPITHELIAL KERATECTOMY (LASEK) Introduction Laser-assisted subepithelial keratectomy (LASEK), which was first described by Camellin, 1 is a surgical procedure for correcting refractive errors of the cornea. An increasing number of LASEK procedures are performed annually, because this surgery potentially combines the advantages of photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). Consequently, there is increasing interest in the study of corneal wounds. For over 30 years, corneal photoablation using an excimer laser has been shown to be a safe and effective procedure. 2 4 Despite preliminary experimental results that revealed endothelial cell changes when high corrections were attempted, 5 7 in vivo clinical trials have found no adverse effects to the corneal endothelium after LASIK or PRK. 8 15 In LASEK, the epithelial flap is separated from the Bowman membrane using diluted alcohol and, although this procedure has been studied by several researchers, 16 20 its short-term effects on the corneal endothelium are still relatively unknown. Thus, the present prospective study was performed to evaluate short-term corneal 1484
endothelial changes after LASEK. Patients and methods STUDY POPULATION In this prospective clinical trial, patients who underwent bilateral LASEK surgery at the Eye and ENT Hospital of Fudan University, Shanghai, China, between February and March 2009 were enrolled sequentially. The study was designed in accordance with the Declaration of Helsinki and approved by ethical committees of the Eye and ENT Hospital of Fudan University. Each subject received a full explanation of the study, including all procedures involved, and provided written informed consent before enrolment. Patients were eligible for inclusion if: they were within the age range 18 30 years; they had a manifest spherical equivalent refractive error of 1.00 to 9.00 dioptres (dpt) with no more than 2 dpt of corneal astigmatism; and they had a normal pre-operative examination with stable refraction. Ocular surface disease, keratoconus and other corneal ectasias, stromal dystrophies, endothelial diseases, uveitis, glaucoma, cataracts, retinal diseases, pregnancy and history of collagen vascular disease were exclusion criteria. PRE- AND POST-OPERATIVE EXAMINATIONS Pre-operative examinations included: uncorrected and best-corrected visual acuity; manifest and cycloplegic refraction; applanation tonometry; slit-lamp examination; dilated fundus examination; pupil evaluation; keratometry and computerized corneal topography; pachymetry; and non-contact specular microscopy (Tomey EM-3000; Tomey Corp., Nagoya, Japan). Specular microscopy was also conducted using the Tomy EM-3000 within 15 min of the end of the procedure and at 1 day and 1 week post-operatively, in addition to all subsequent examinations. ENDOTHELIAL CELL EXAMINATION Patients were asked to fixate on the target light within the specular microscope to ensure that reproducible images of similar areas of the central corneal endothelium were obtained. A specular micrograph from each cornea was then selected for further analysis. Observations regarding qualitative changes in endothelial cells were made and recorded. For each specular micrograph, the same pre-determined area within the image was identified and all contiguous endothelial cells within this area were marked automatically. The marked cells were then automatically analysed, using the software program accompanying the specular microscope, to provide measurements of endothelial cell density (ECD), coefficient of variation (CV) of cell size and the percentage of hexagonal cells for each cornea. The CV of cell size is an index that provides a quantitative assessment of polymegathism, whereas the percentage of hexagonal cells provides a measurement of endothelial cell shape variability or pleomorphism. 21,22 The specular micrograph examinations were repeated three times each at 15 min, 1 day and 1 week post-operatively and the mean was calculated. SURGICAL TECHNIQUES During LASEK, an 8.0-mm LASEK corneal microtrephine (Kangning Medical Electronic Equipment Development Co., Wuxi, China) centred in the papillary axis was used to create an epithelial flap. An alcohol cone was placed on the corneal surface encircling the epithelial incision. The cone was filled with 20% ethanol (in distilled water) and left for 12 s. The cornea was then dried and 1485
thoroughly washed with balanced salt solution (BSS; Alcon, Freiburg, Germany). The superior hinged epithelial flap was created by lifting the epithelium at the precut margin using the sharp side of an epipeeler (Kangning Medical Electronic Equipment Development Co.) and then gently detached and folded at the hinge using the blunt, large side of the epi-peeler. In all patients, the epithelial flap was created with its hinge at the 12 o clock position. All laser ablations were performed with the MEL 80 excimer laser system (Carl Zeiss Meditec, Oberkochen, Germany), and the ablation diameter was chosen based on the scotopic pupil diameter. After laser ablation, the stromal surface was irrigated with BSS and the epithelial flap was repositioned. After surgery, the epithelial flap was secured with a 14-mm bandage contact lens (Acuvue Advance ; Johnson & Johnson, New Brunswick, NJ, USA) for 3 7 days until epithelial healing was completed. Mitomycin C was not used in the procedure. Post-operatively, patients self-administered ofloxacin 0.3% eye drops, one drop three times a day for 2 weeks, and fluorometholone 0.1% eye drops, one drop four times daily for the first 2 weeks, three times daily for the second 2 weeks, twice daily for the third 2 weeks and once a day for the last 2 weeks. STATISTICAL ANALYSIS Statistical analyses were performed using the SPSS statistical package, version 11.5 (SPSS Inc., Chicago, IL, USA) for Windows. Twotailed paired Student s t-tests were used to compare the pre-operative values for ECD, CV of cell size and percentage of hexagonal cells with the values for these parameters obtained 15 min, 1 day and 1 week following LASEK. A P-value < 0.05 was considered to be statistically significant. Results Five men and five women were included in this study; LASEK was performed in all 20 eyes. Patients ages ranged from 18 to 30 years (mean ± SD 22.1 ± 4.01 years). The degree of myopic astigmatism (in spherical equivalents) ranged from 1.50 to 7.25 dpt (mean ± SD 5.19 ± 1.55 dpt). The ablation depth ranged from 47 to 134 µm (mean ± SD 100.1 ± 23.09 µm) and the calculated residual corneal thickness ranged from 380 to 504 µm (mean ± SD 438.25 ± 33.91 µm). Mean pre- and post-operative ECD, CV of cell size and the percentage of hexagonal cells are given in Table 1. Corneal endothelial cell images obtained pre- and post-surgery at different time-points are illustrated in Fig. 1. The difference in ECD at 15 min post-surgery was statistically significant as compared with pre-operative values (P < 0.001; Table 1). Although decreased ECD were also observed at 1 day and 1 week post-operatively, the differences between pre-operative and 1 day s or 1 week s post-operative mean ECDs were not statistically significant. The mean ± SD change in ECD was 236 ± 177 cells/mm 2 for the post-operative time interval 0 15 min, 101 ± 134 cells/mm 2 for the post-operative time interval 0 1 day and 71 ± 129 cells/mm 2 for the post-operative time interval 0 1 week, respectively. The changes at 15 min and 1 day post-operatively were both statistically significantly different from zero in a paired t-test (P < 0.001 and P = 0.013, respectively). In terms of the mean CV of cell size, only the difference pre-operative versus 15 min post-operative was statistically significant (P < 0.001; Table 1). The 15 min and 1 day postoperative mean percentages of hexagonal cells were both statistically significantly lower compared with the pre-operative percentage (P = 0.002 and P = 0.010, respectively; Table 1). 1486
TABLE 1: Pre- and post-operative endothelial cell measurements taken in 10 patients (20 eyes) undergoing laser-assisted subepithelial keratectomy ECD (cells/mm 2 ) CV of cell size Cell hexagonality (%) Statistical Statistical Statistical Time Mean ± SD significance a Mean ± SD significance a Mean ± SD significance a Pre-operative 2834 ± 164 41.1 ± 5.5 32.1 ± 10.1 15 min post-operatively 2598 ± 155 P < 0.001 52.0 ± 8.7 P < 0.001 22.3 ± 7.2 P = 0.002 1 day post-operatively 2733 ± 205 NS 43.2 ± 5.7 NS 24.6 ± 7.2 P = 0.010 1 week post-operatively 2763 ± 198 NS 43.3 ± 8.6 NS 29.2 ± 10.4 NS Data are mean ± SD. a Two-tailed paired Student s t-tests for post-operative versus pre-operative values. ECD, endothelial cell density; CV, coefficient of variation; NS, not statistically significant (P > 0.05). Discussion Currently, LASEK is considered the procedure of choice to reduce the risk of late flap trauma or dislocation. The smooth ablation patterns achieved with the current generation of lasers and use of the epithelial flap to decrease time to epithelialization enable patients undergoing LASEK to achieve visual recovery sooner than patients undergoing PRK. 23 Indeed, recovery levels are nearly the same as those for patients undergoing LASIK 24 which is also our experience at the Eye and ENT Hospital of Fudan University. The majority of published studies show that neither PRK nor LASIK have any harmful clinical effects on the corneal endothelium in myopic patients, even in high myopic treatments. 8 15 To date, however, the effect of LASEK on corneal endothelial cells has been unclear. The present study documented that acute endothelial changes occur, and can be considerable, on specular microscopic examination after LASEK. When taken as a whole, LASEK-treated eyes had a significant increase in post-operative CV of cell size and a significant decrease in ECD and cell hexagonality at 15 min post-operatively. These findings indicate that, immediately after LASEK, the number of cells/mm 2 decreases and the endothelial cells became much more swollen compared with their preoperative size. But these changes were transient; ECD and variations in cell area returned to near baseline (pre-operative) levels by 1 day post-operatively. An increased CV of cell size would be expected if there was a decrease in the percentage of hexagonal cells, as observed in the present study. The percentage of hexagonal cells also returned to near the baseline (pre-operative) level by 1 week post-operatively, suggesting that endothelial cell function recovered 1 week after LASEK. 1487
A B C D FIGURE 1: Corneal endothelial cell images after laser-assisted subepithelial keratectomy at: (A) pre-operation; (B) 15 min post-operation; (C) 1 day postoperation; and (D) 1 week post-operation Excimer laser corneal ablation has been used for surgical refractive correction for over a decade. Following such procedures, mechanical trauma from shock waves, thermal damage, actinic damage due to primary (laser beam) and secondary (fluorescence) radiation, and metabolic changes have the potential to damage the corneal endothelium. 5,25 27 Some studies have suggested a harmful effect during or shortly after refractive surgery with deep excimer laser ablations on the human corneal endothelium. 5,28 30 Pallikaris and Siganos 30 reported a mean 4.1% loss of central corneal ECD at 6 months and a lesser (2.4%) decrease from pre-operative values at 24 months after LASIK. The loss was greater in patients where higher corrections were attempted. Certainly, LASIK may increase the risk of endothelial damage compared with surface ablation techniques such as PRK and LASEK. Ablation with LASIK is closer to the endothelium than with PRK, and using a microkeratome and inducing a very high intraocular pressure may offer additional endothelial insults. Studies that have evaluated the endothelium after PRK have reported little or no endothelial change, with no clinically significant decrease in central 1488
ECD. 10 15 In LASEK, excimer laser stromal ablation is performed under a hinged flap of corneal epithelium, using diluted alcohol. Microkeratomes are not used and stromal lamellar cuts are not made. LASEK has been described as offering an excellent blend of the positive aspects of PRK and LASIK, while eliminating or reducing the complications inherent in both procedures. 24 None of the ablations performed in the present study exceeded 134 µm in depth; all left an appropriate residual corneal thickness ( 380 µm). Because the present study dealt only with LASEK, it was possible to eliminate factors inherent to LASIK and PRK procedures, such as increased intraocular pressure induced by the suction ring and epithelial removal, that could have influenced endothelial cell metabolism and viability. The fact that acute endothelial changes were observed after LASEK suggests that other factors (such as immersion in diluted alcohol and using a contact lens after surgery) may play contributory roles. There were certain limitations to the present study. First, the status of endothelial cells in the peripheral cornea was not assessed, which could have been helpful in determining the extent of acute endothelial cell damage and the contribution of peripheral cell migration to the recovery of normal central endothelial cell appearance. Secondly, a larger sample size and a longer follow-up period could have strengthened the conclusion that LASEK was safe for the corneal endothelium. This would have also provided further insight into the potential long-term consequences of the findings. Despite these limitations, the data presented revealed that immediate changes occur in the human corneal endothelium after LASEK, and that the number and morphology of corneal endothelial cells had returned to pre-operative standards by 1 week post-operation. Although the rapid resolution of these changes indicates the short-term safety of LASEK, the future consequences of this procedure are unknown and may not become evident for many years. The issue of endothelial cell safety with regard to LASEK has become extremely relevant because of the rapidly growing popularity of this procedure. Acknowledgement This study was supported by the Programme for New Century Excellent Talents in University, China (Grant NCET-07-0212). Conflicts of interest The authors had no conflicts of interest to declare in relation to this article. Received for publication 20 January 2010 Accepted subject to revision 26 January 2010 Revised accepted 16 April 2010 Copyright 2010 Field House Publishing LLP References 1 Camellin M: LASEK may offer the advantages of both LASIK and PRK. Ocul Surg News Int Edn 1999; 3: 14 15. 2 Trokel SL, Srinivasan R, Braren B: Excimer laser surgery of the cornea. Am J Ophthalmol 1983; 96: 710 715. 3 Kymionis GD, Tsiklis NS, Astyrakakis N, et al: Eleven-year follow-up of laser in situ keratomileusis. J Cataract Refract Surg 2007; 33: 191 196. 4 O Connor J, O Keeffe M, Condon PI: Twelveyear follow-up of photorefractive keratectomy for low to moderate myopia. J Refract Surg 2006; 22: 871 877. 5 Marshall J, Trokel S, Rothery S, et al: An ultrastructural study of corneal incisions induced by an excimer laser at 193 nm. Ophthalmology 1985; 92: 749 758. 6 Gaster RN, Binder PS, Coalwell K, et al: Corneal surface ablation by 193 nm excimer laser and wound healing in rabbits. Invest Ophthalmol Vis Sci 1989; 30: 90 98. 7 Hanna KD, Pouliquen Y, Waring GO 3rd, et al: 1489
Corneal stromal wound healing in rabbits after 193-nm excimer laser surface ablation. Arch Ophthalmol 1989; 107: 895 901. 8 Cennamo G, Rosa N, Del Prete A, et al: The corneal endothelium 12 months after photorefractive keratectomy in high myopia. Acta Ophthalmol Scand 1997; 75: 128 130. 9 Rosa N, Cennamo G, Del Prete A, et al: Effects on the corneal endothelium six months following photorefractive keratectomy. Ophthalmologica 1995; 209: 17 20. 10 Rosa N, Cennamo G, Del Prete A, et al: Endothelial cells evaluation two years after photorefractive keratectomy. Ophthalmologica 1997; 211: 32 39. 11 Stulting RD, Thompson KP, Waring GO 3rd, et al: The effect of photorefractive keratectomy on the corneal endothelium. Ophthalmology 1996; 103: 1357 1365. 12 Spadea L, Dragani T, Blasi MA, et al: Specular microscopy of the corneal endothelium after excimer laser photorefractive keratectomy. J Cataract Refract Surg 1996; 22: 188 193. 13 Mardelli PG, Piebenga LW, Matta CS, et al: Corneal endothelial status 12 to 55 months after excimer laser photorefractive keratectomy. Ophthalmology 1995; 102: 544 549. 14 Perez-Santonja JJ, Meza J, Moreno E, et al: Short-term corneal endothelial changes after photorefractive keratectomy. J Refract Corneal Surg 1994; 10(2 suppl): S194 S198. 15 Shimizu K, Amano S, Tanaka S: Photorefractive keratectomy for myopia: one-year follow-up in 97 eyes. J Refract Corneal Surg 1994; 10(2 suppl): S178 S187. 16 Azar DT, Ang RT, Lee JB, et al: Laser subepithelial keratomileusis: electron microscopy and visual outcomes of flap photorefractive keratectomy. Curr Opin Ophthalmol 2001; 12: 323 328. 17 Browning AC, Shah S, Dua HS, et al: Alcohol debridement of the corneal epithelium in PRK and LASEK: an electron microscopic study. Invest Ophthalmol Vis Sci 2003; 44: 510 513. 18 Espana EM, Grueterich M, Mateo A, et al: Cleavage of corneal basement membrane components by ethanol exposure in laserassisted subepithelial keratectomy. J Cataract Refract Surg 2003; 29: 1192 1197. 19 Chen CC, Chang JH, Lee JB, et al: Human corneal epithelial cell viability and morphology after dilute alcohol exposure. Invest Ophthalmol Vis Sci 2002; 43: 2593 2602. 20 Gabler B, Winkler von Mohrenfels C, Dreiss AK, et al: Vitality of epithelial cells after alcohol exposure during laser-assisted subepithelial keratectomy flap preparation. J Cataract Refract Surg 2002; 28: 1841 1846. 21 Rao GN, Waldron WR, Aquavella JV: Morphology of graft endothelium and donor age. Br J Ophthalmol 1980; 64: 523 527. 22 MacRae SM, Matsuda M, Rich LF: The effect of radial keratotomy on the corneal endothelium. Am J Ophthalmol 1985; 100: 538 542. 23 Lee JB, Seong JG, Lee JH, et al: Comparison of laser epithelial keratomileusis and photorefractive keratectomy for low to moderate myopia. J Cataract Refract Surg 2001; 27: 565 570. 24 Ambrósio R Jr, Wilson S: LASIK vs LASEK vs PRK: advantages and indications. Semin Ophthalmol 2003; 18: 2 10. 25 Seiler T, Bende T, Winckler K, et al: Side effects in excimer corneal surgery. DNA damage as a result of 193 nm excimer laser radiation. Graefes Arch Clin Exp Ophthalmol 1988; 226: 273 276. 26 Seiler T, McDonnell PJ: Excimer laser photorefractive keratectomy. Surv Ophthalmol 1995; 40: 89 118. 27 Costagliola C, Balestrieri P, Fioretti F, et al: ArF 193 nm excimer laser corneal surgery as a possible risk factor in cataractogenesis. Exp Eye Res 1994; 58: 453 457. 28 Pallikaris IG, Siganos DS: Excimer laser in situ keratomileusis and photorefractive keratectomy for correction of high myopia. J Refract Corneal Surg 1994; 10: 498 510. 29 Isager P, Guo S, Hjortdal JO, et al: Endothelial cell loss after photorefractive keratectomy for myopia. Acta Ophthalmol Scand 1998; 76: 304 307. 30 Pallikaris IG, Siganos DS: Laser in situ keratomileusis to treat myopia: early experience. J Cataract Refract Surg 1997; 23: 39 49. Author s address for correspondence Dr Xingtao Zhou Myopia Key Lab of Health Ministry, Eye and ENT Hospital of Fudan University, 83 Fenyang Road, Shanghai 200031, China. E-mail: jitong0512@163.com 1490