Confocal Microscopy of Corneal Stroma and Endothelium After LASIK and PRK

Confocal Microscopy of Corneal Stroma and Endothelium After LASIK and PRK Javad Amoozadeh, MD; Soheil Aliakbari, MD; Amir-Houshang Behesht-Nejad, MD; Mohammad-Amin Seyedian, MD; Bijan Rezvan, DDS; Hassan Hashemi, MD ABSTRACT PURPOSE: To compare with confocal microscopy the changes in stromal keratocyte density and endothelial cell count due to photorefractive keratectomy (PRK) and LASIK. METHODS: In this prospective study, 32 eyes (16 myopic patients) were examined with the NIDEK Confoscan 3 confocal microscope before and 6 months after PRK and LASIK. The preoperative mean myopia was 2.85 0.99 diopters (D) (range: 1.00 to 4.00 D) in 24 eyes that underwent PRK and 2.94 0.96 D (range: 2.00 to 4.25 D) in 8 eyes that underwent LASIK. Keratocyte density in the anterior and posterior stroma and the endothelial cell count were measured. Statistically signifi cant changes were assessed using the t test. P.05 was considered statistically signifi cant. RESULTS: Preoperative hexagonal cell percentage in the LASIK group was 52.17 11.43 and 51.33 10.98 in the PRK group. Postoperatively, the percentages were 52.96 7.55 and 53.34 10.2, respectively. Six months postoperatively, keratocyte density changed by 367.12 103.35 cells/mm 2 (34.7% reduction) in the anterior stroma (P.05) and 9.25 28.28 cells/mm 2 (1.31% reduction) in the posterior stroma (P.05) for the LASIK group. In the PRK group, these values were 319.71 83.45 cells/mm 2 (31.13% reduction) in the anterior stroma (P.05) and 0.17 38.97 cells/mm 2 (0.02% reduction) in the posterior stroma (P.05). The changes in keratocyte densities were not statistically signifi cant between groups (P.05). The mean number of keratocytes decreased by 37.2% in the retroablation zone of the LASIK group (P.05). No changes were noted in endothelial cell counts. CONCLUSIONS: A signifi cant decrease occurred in the number of stromal keratocytes in the anterior stroma. Despite differences in surgery, the change in keratocyte density and endothelial cell counts were similar between LASIK and PRK groups (P.05). [J Refract Surg. 2009;25:S963-S967.] doi:10.3928/1081597x-20090915-12 T he submicron accuracy of excimer laser ablation is an important factor in the popularity of LASIK and photorefractive keratectomy (PRK) worldwide. 1,2 Photorefractive keratectomy is a surface ablation procedure that uses the excimer laser to reshape the superficial stromal layer after removal of surface epithelium. Laser in situ keratomileusis involves the creation of a hinged corneal flap of 130- to 160-µm thickness and delivery of the excimer laser ablation to the underlying stroma. During LASIK, the anterior stroma and epithelium are preserved, which results in differences in the healing process compared to PRK. 3 Surface procedures such as PRK have been shown to be safe and predictable for correcting low and moderate refractive errors and they circumvent flap-related complications and biomechanical instability seen with LASIK. 4 Complications related to surface ablation procedures such as PRK include delayed healing, increased risk of haze, dry eye, and regression of effect. 4-6 The incorporation of mitomycin C during PRK may mitigate haze formation postoperatively. 7 Complications of LASIK include epithelial ingrowth, flap complications, keratectasia, biomechanical instability, and dry eye. 4 Despite improvements in surgical techniques and excimer laser technology, similar advances in the understanding of the cellular response following LASIK and PRK have not been made. 2 Cellular and structural changes induced by refractive surgery may aid in understanding the natural cellular processes and potential complications that occur after each type of surgery. The complex nature of tissue inter- From Farabi Eye Hospital, Tehran University of Medical Sciences (Amoozadeh, Behesht-Nejad, Hashemi); and Noor Ophthalmology Research Center, Noor Eye Hospital (Aliakbari, Seyedian, Rezvan, Hashemi), Tehran, Iran. The authors have no financial or proprietary interest in the materials presented herein. Rich Bains, consultant to NIDEK Co Ltd, assisted in the preparation of the manuscript. Portions of this article have been published previously in the Iranian Journal of Ophthalmology (2009;21:23-28). Correspondence: Soheil Aliakbari, MD, Noor Eye Hospital, No. 96, Esfandiar Blvd, Vali Asr Ave, Tehran 1968655841, Iran. Tel: 98 21 82400; Fax: 98 21 88650501; E-mail: saliakbari@hotmail.com S963

action postoperatively may help determine selection criteria for LASIK and PRK. Confocal microscopy has been used to investigate the cellular morphology and structure of the various corneal layers. 1-6,8-11 However, the results from these studies are contradictory. Normal keratocyte density is 1079 cells/mm 2. 12 In the present study, in vivo confocal microscopy was used to evaluate keratocyte density and endothelial cell count and to compare changes seen in eyes that underwent LASIK and PRK. PATIENTS AND METHODS STUDY POPULATION This study was a prospective, non-randomized study of 16 patients who were scheduled to undergo LASIK (LASIK group) or PRK (PRK group) from March 1 to May 15, 2007. Patients with low to moderate myopia ( 1.00 to 4.50 diopters [D]) or low to moderate myopic astigmatism ( 4.50 D sphere and up to 1.50 D astigmatism) were enrolled after signing consent forms for pre- and postoperative examination. Patient preference in consultation with the surgeon was the basis for undergoing PRK or LASIK. Eight eyes of 4 patients had LASIK and 24 eyes of 12 patients had PRK. Exclusion criteria were systemic diseases such as diabetes, traumatic or infectious complications after refractive surgery, active eye disease, and previous corneal surgery. After applying the above exclusion criteria, all patients were available for analysis. Ten patients used contact lenses prior to surgery (8 in the PRK group and 2 in the LASIK group). However, all contact lens wearers were required to discontinue use for at least 3 to 28 days (depending on contact lens type) prior to preoperative evaluation to stabilize keratometry and corneal topography. Patients were required to have normal keratometry and topography prior to undergoing refractive surgery. PRE- AND POSTOPERATIVE EXAMINATIONS Pre- and postoperative examinations included measurement of uncorrected visual acuity, best spectaclecorrected visual acuity, slit-lamp evaluation of the cornea and anterior chamber, tonometry, keratometry, topography, pachymetry, cycloplegic refraction, and confocal microscopy (Confoscan 3; NIDEK Co Ltd, Gamagori, Japan). Confocal microscopy was used to determine the number of keratocytes in the anterior and posterior stromal layers, endothelial cell count, and number of hexagonal cells. In the LASIK group, the keratocytes were counted in the retroablation zone, defined as the space 5 µm behind the flap to the endothelium. Keratocyte density was measured using the Confoscan 3 preoperatively and 6 months postoperatively. Confoscan 3 measurements were performed by an experienced ophthalmologist (H.H.) who did not perform the surgeries on this study cohort. Measurements were acquired in automatic mode using the 20 non-contact lens. Topical tetracaine 0.5% drops were applied to anesthetize the eyes. Methyl cellulose drops (Viscotears Gel; CIBA Vision, Duluth, Ga) were applied on the objective lens of the machine to provide a regular image, and the lens was aligned with the pupil center. For each eye, 350 frames, 5.0-µm apart were acquired. The cells in each layer were marked by an examiner (S.A.) (who was masked to the type of surgery) to avoid inadvertently counting the same cell twice. The number of cells was calculated by counting the number of clear and distinct cells in areas of 379 286 µm 2. The keratocyte density was studied in two layers 25 µm posterior to Bowman s layer (anterior stroma) and 40 µm anterior to Descemet s membrane (posterior stroma). The layer 25 µm posterior to Bowman s layer was chosen based on previous studies, 3-5 as the exact amount of tissue removal for each patient could not be predicted preoperatively. Postoperatively, the same 25 µm from Bowman s layer was used, although this may be the midstroma compared to preoperatively. Additionally, the Confoscan operator (S.A.) was not aware of the ablation depth of each patient postoperatively. By using Bowman s layer as the reference point, epithelial hyperplasia was ruled out as a source of error. For deep stroma, the endothelium was chosen as the reference point. SURGERIES All surgeries were performed by a single surgeon (H.H.). The surgeon did not perform confocal microscopy measurements. The Technolas 217 Planoscan ablation (Bausch & Lomb, Rochester, NY) was used for all patients. For PRK, the epithelium was manually debrided with a hockey stick spatula. At the end of the procedure, an O2 Optix bandage contact lens (CIBA Vision) was placed, with removal 3 days after surgery or upon complete re-epithelialization. Laser in situ keratomileusis was performed using the Hansatome (Bausch & Lomb) microkeratome. Postoperative drop regimen for the LASIK group was chloramphenicol drops for 3 days, betamethasone drops for 1 week, and artificial tears for 2 months postoperatively. Patients who underwent PRK were requested to instill diclofenac sodium drops every 8 hours during the first 24 hours, chloramphenicol drops for 5 days, S964 journalofrefractivesurgery.com

betamethasone drops for 2 weeks, artificial tears for 1 month, and fluorometholone drops for 12 weeks after cessation of betamethasone drops. No patient underwent retreatment for the duration of this study. STATISTICAL ANALYSIS Data analysis was performed with SPSS 16 software (SPSS Inc, Chicago, Ill). The t test was used for analyzing the pre- to postoperative mean differences in anterior stromal cell density, posterior stromal cell density, endothelial cell count, and hexagonal cell density in both groups. P.05 was considered statistically significant. RESULTS Mean patient age was 28.25 5.75 years (range: 19 to 36 years). Mean age of the LASIK group was 28.50 5.86 years (range: 19 to 36 years) and 27.50 6.24 years (range: 20 to 35 years) in the PRK group. Preoperative mean myopia was 2.94 0.96 D (range: 2.00 to 4.25 D) and the mean astigmatism was 0.50 0.25 D (range: 0.50 to 1.00 D) in the LASIK group. Preoperative mean myopia was 2.85 0.99 D (range: 1.00 to 4.00 D) and the mean astigmatism was 0.775 0.42 D (range: 0.25 to 1.00 D) in the PRK group. The mean ablation depth was 61 17.17 µm (range: 36 to 71 µm) in the LASIK group and 62.13 15.41 µm (range: 38 to 69 µm) in the PRK group. No statistically significant difference was noted in ablation depth between groups (P.05). The mean anterior keratocyte cell density, posterior keratocyte cell density, and endothelial cell count are presented in the Table. The changes in the variables in each group were not statistically significant (P.05) (Table). Preoperative hexagonal cell percentage was 52.17 11.43 for the LASIK group and 51.33 10.98 for the PRK group. Postoperatively, the hexagonal cell percentiles were 52.96 7.55 for the LASIK group and 53.34 10.2 for the PRK group. In the LASIK group, the mean reduction in keratocyte density was 34.7% in the anterior stromal layer and 1.31% in the posterior stromal layer. The decrease in the anterior stroma was statistically significant (P.05). A 0.27% increase in endothelial cell count was noted in the LASIK group (Table). In the PRK group, the mean reduction in keratocyte density was 31.13% in the anterior stroma and 0.02% in the posterior stroma (Table). The decrease in the anterior stroma was statistically significant (P.05) Endothelial cell count increased by 1.39% in the PRK group (Table). Postoperatively, hexagonal cell density increased by 0.79% in the LASIK group, 2.01% in PRK group, and 1.57% in the study cohort. No statistically TABLE Anterior and Posterior Stromal Keratocyte Density and Endothelial Cell Count in Eyes That Underwent LASIK and PRK Mean Standard Deviation (cells/mm 2 ) Keratocyte Density Anterior Stroma Posterior Stroma Endothelial Cell Count Preop Postop Difference Preop Postop Difference Preop Postop Difference LASIK (n=24) 1058 95 690 55 368 103* 708 40 699 57 9 28 3022 224 3030 186 8 118 PRK (n=8) 1027 80 707 63 320 84* 719 51 719 45 0 38.98 2983 293 3025 404 42 167 Total (n=32) 1034.7 83.7 703.2 60.8 331.6 89.5* 716 47.7 713.5 47.9 2.4 36.4 2992.9 274.8 3026.2 359.3 33.3 154.8 *Statistically significant difference (P.05). S965

significant differences were noted between groups or changes in overall hexagonal cell density (P.05). The mean number of keratocytes in the retroablation zone was 947.13 16.78 cells/mm 2 (range: 845 to 1024 cells/mm 2 ), which decreased to 610 19.43 cells/mm 2 (range: 573 to 828 cells/mm 2 ) postoperatively (37.2% difference) (P.05). DISCUSSION The present in vivo confocal microscopy study of eyes that underwent LASIK or PRK found a mean reduction in keratocyte density 6 months postoperatively. Eyes that underwent PRK and LASIK both experienced a statistically significant reduction in anterior keratocyte density postoperatively (P.05) (Table). In the LASIK group, a 34.7% difference in anterior keratocyte reduction was noted compared to posterior keratocyte density (Table). This outcome is similar to separate investigations of PRK and LASIK. Ghirlando et al 5 evaluated 50 myopes 1 month after PRK and reported a greater number of activated keratocytes in the anterior stroma compared to the posterior stroma. McLaren et al 12 reported a statistically significant reduced number of keratocytes in the anterior stroma compared to the posterior stroma after LASIK. The magnitude of reduction found in our study (31.1%) is similar to the 40% reported at 6 months by Erie et al 3 for PRK. However, Erie et al reported lower keratocyte loss (22%) for LASIK patients compared to our results (34.7%). These differences may be due to counting errors or differences in the volume of tissue removed between studies. 3,13 The reduction in anterior keratocyte density was similar in LASIK and PRK in our study. Despite surface versus mid-stromal ablation, the difference between reductions was not statistically significant (P.05). However, others have reported differences between LASIK and PRK, which are attributed to the deeper ablation depth in LASIK. 3 In the present study, the ablation depth was similar in LASIK and PRK. Whether ablation depth or location of ablation delivery (surface versus mid-stroma) is a contributing factor to keratocyte density remains a topic for future study. One limitation of confocal microscopy is the limited area under observation, which is overcome by observing a larger number of samples. The other limitation is the lack of registration of images between followups, as the same area is not imaged from examination to examination. This limitation is the purview of confocal manufacturers who can develop software algorithms that allow registration of a landmark based on contrast ratios of image pixels. Another limitation of confocal microscopy and this study is the subjective nature of the measurements. Interobserver variability in cell counts can approach 8% 13 yet experienced observers have shown differences of 3%. 3 Additionally, the keratocytes may not all be in the same plane, which can also cause counting errors. Other limitations of this study are the unequal number of patients in the PRK and LASIK groups and the lack of a control group. These limitations could not be avoided due to the small sample size. Keratocytes function to maintain the health and clarity of the corneal stroma. 13 Compared to corneal endothelial and epithelial cells, keratocytes have moderate regenerative capacity. Epithelial cells are readily regenerated with complete re-epithelialization after injury; however, endothelial cells do not regenerate. 14 Measuring the changes in keratocyte density after refractive surgery will help develop an understanding of the impact of surgery on the cornea. Vesaluoma et al 10 postulated that anterior stromal cell loss begins 6 months after LASIK. They attribute this observation to the loss of neural input to the keratocytes due to severing of the nerves during LASIK, but to a lesser degree in PRK. 10 Although the main cause of keratocyte loss after refractive surgery remains ambiguous, keratocyte necrosis and apoptosis have been suggested as possible causes. 10 Apoptosis and necrosis are the results of cell death due to exposure to lethal stresses. Thermal effects due to the ablation, mechanical debridement, and microkeratome cut all represent such stresses. In the present study, keratocyte density in the retroablation zone decreased and was found to be statistically significant (P.0001) 6 months after LASIK. Compared to the anterior stroma, we believe this higher decrease can be attributed to the direct effect of laser ablation on the retroablation zone. The decreases in corneal cellular density have also been attributed to the lack of a robust cellular proliferative response to cell loss. 14 Keratocyte density anterior to the retroablation zone remained reduced by 42% up to 5 years after LASIK and PRK. 9 Regions of keratocyte apoptosis at the flap edge may never be replaced by new keratocytes. 3 One study reported that the decrease in keratocyte density after LASIK has both an acute and a chronic phase. 11 The question remains whether the loss of cells following PRK or LASIK as reported in the present study reduces the health and function of the cornea. To date, clinical studies have shown that this reduction in cell count does not affect the overall health of the cornea. 14 Even various long-term studies provide evidence of visual improvement after refractive surgery, 15,16 possibly indicating that there are few clinical consequences to the reduction of keratocyte density. S966 journalofrefractivesurgery.com

In the present study, endothelial cell count showed an increase of 0.27% and 1.39% after LASIK and PRK, respectively. The hexagonal cells also increased in both groups after surgery. However, this increase was not statistically significant (P.05). Because endothelial cells do not divide and laser ablation does not affect endothelial cells substantially, this slight increase in number can be attributed to counting errors that are byproducts of different cross-sections imaged before and after surgery. Using confocal microscopy, reductions in keratocyte density were noted in the anterior stroma, which were statistically significant, and the posterior stroma after PRK and LASIK. No significant differences were noted between PRK and LASIK in terms of changes in endothelial cell count in the different layers of the stroma and endothelium. The effects of different mechanical or laser ablation stresses on cell density postoperatively warrant further investigation. AUTHOR CONTRIBUTIONS Study concept and design (J.A., S.A., A.B., M.S., B.R., H.H.); data collection (S.A.); interpretation and analysis of data (S.A.); drafting of the manuscript (S.A., M.S., B.R.); critical revision of the manuscript (J.A., S.A., A.B., H.H.); administrative, technical, or material support (H.H.); supervision (J.A., A.B., M.S., B.R., H.H.) REFERENCES 1. Dawson DG, Edelhauser HF, Grossniklaus HE. Long-term histopathologic findings in human corneal wounds after refractive surgical procedures. Am J Ophthalmol. 2005;139:168-178. 2. Rajan MS, Watters W, Patmore A, Marshall J. In vitro human corneal model to investigate stromal epithelial interactions following refractive surgery. J Cataract Refract Surg. 2005;31:1789-1801. 3. Erie JC, Patel SV, McLaren JW, Hodge DO, Bourne WM. Corneal keratocyte deficits after photorefractive keratectomy and laser in situ keratomileusis. Am J Ophthalmol. 2006;141:799-809. 4. Esquenazi S, He J, Li N, Bazan NG, Esquenazi I, Bazan HE. Comparative in vivo high-resolution confocal microscopy of corneal epithelium, sub-basal nerves and stromal cells in mice with and without dry eye after photorefractive keratectomy. Clin Experiment Ophthalmol. 2007;35:545-549. 5. Ghirlando A, Gambato C, Midena E. LASEK and photorefractive keratectomy for myopia: clinical and confocal microscopy comparison. J Refract Surg. 2007;23:694-702. 6. Moilanen JA, Vesaluoma MH, Müller LJ, Tervo TM. Long-term corneal morphology after PRK by in vivo confocal microscopy. Invest Ophthalmol Vis Sci. 2003;44:1064-1069. 7. Rajan MS, O Brart DP, Patmore A, Marshall J. Cellular effects of mitomycin-c on human corneas after photorefractive keratectomy. J Cataract Refract Surg. 2006;32:1741-1747. 8. Patel S, McLaren J, Hodge D, Bourne W. Normal human keratocyte density and corneal thickness measurement by using confocal microscopy in vivo. Invest Ophthalmol Vis Sci. 2001;42:333-339. 9. Erie JC, Nau CB, McLaren JW, Hodge DO, Bourne WM. Longterm keratocyte deficits in the corneal stroma after LASIK. Ophthalmology. 2004;111:1356-1361. 10. Vesaluoma M, Pérez-Santonja J, Petroll WM, Linna T, Alió J, Tervo T. Corneal stromal changes induced by myopic LASIK. Invest Ophthalmol Vis Sci. 2000;41:369-376. Erratum in: Invest Ophthalmol Vis Sci. 2000;41:2027. 11. Dawson DG, Holley GP, Geroski DH, Waring GO III, Grossniklaus HE, Edelhauser HF. Ex vivo confocal microscopy of human LASIK corneas with histologic and ultrastructural correlation. Ophthalmology. 2005;112:634-644. 12. Ku JY, Niederer RL, Patel DV, Sherwin T, McGhee CN. Laser scanning in vivo confocal analysis of keratocyte density in keratoconus. Ophthalmology. 2008;115:845-850. 13. McLaren JW, Patel SV, Nau CB, Bourne WM. Automated assessment of keratocyte density in clinical confocal microscopy of the corneal stroma. J Microsc. 2008;229:21-31. 14. Dawson DG, O Brien TP, Edelhauser HF. Long-term corneal keratocyte deficits after PRK and LASIK: in vivo evidence of stress-induced premature cellular senescence. Am J Ophthalmol. 2006;141:918-920. 15. Alió JL, Muftuoglu O, Ortiz D, Pérez-Santonja JJ, Artola A, Ayala MJ, Garcia MJ, de Luna GC. Ten-year follow-up of laser in situ keratomileusis for myopia of up to -10 diopters. Am J Ophthalmol. 2008;145:46-54. 16. Alió JL, Muftuoglu O, Ortiz D, Artola A, Pérez-Santonja JJ, de Luna GC, Abu-Mustafa SK, Garcia MJ. Ten-year follow-up of photorefractive keratectomy for myopia of more than -6 diopters. Am J Ophthalmol. 2008;145:37-45. S967