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 he submicron accuracy of excimer laser ablation is an important factor in the popularity of LASIK and 1,2 PURPOSE: To compare with confocal microscopy the T photorefractive keratectomy (PRK) worldwide. changes in stromal keratocyte density and endothelial Photorefractive keratectomy is a surface ablation procedure cell count due to photorefractive keratectomy (PRK) and that uses the excimer laser to reshape the superfi cial stromal LASIK. layer after removal of surface epithelium. Laser in situ ker- atomileusis involves the creation of a hinged corneal fl ap of METHODS: In this prospective study, 32 eyes (16 130- to 160-µm thickness and delivery of the excimer laser myopic patients) were examined with the NIDEK Con- foscan 3 confocal microscope before and 6 months af- ablation to the underlying stroma. During LASIK, the ante- ter PRK and LASIK. The preoperative mean myopia was rior stroma and epithelium are preserved, which results in Ϫ2.85Ϯ0.99 diopters (D) (range: Ϫ1.00 to Ϫ4.00 D) differences in the healing process compared to PRK.3 Surface in 24 eyes that underwent PRK and Ϫ2.94Ϯ0.96 D procedures such as PRK have been shown to be safe and pre- Ϫ Ϫ (range: 2.00 to 4.25 D) in 8 eyes that underwent dictable for correcting low and moderate refractive errors and LASIK. Keratocyte density in the anterior and posterior stroma and the endothelial cell count were measured. they circumvent fl ap-related complications and biomechani- 4 Statistically signifi cant changes were assessed using the cal instability seen with LASIK. Complications related to sur- t test. PϽ.05 was considered statistically signifi cant. face ablation procedures such as PRK include delayed healing, increased risk of haze, dry eye, and regression of effect.4-6 The RESULTS: Preoperative hexagonal cell percentage in incorporation of mitomycin C during PRK may mitigate haze Ϯ Ϯ the LASIK group was 52.17 11.43 and 51.33 10.98 formation postoperatively.7 Complications of LASIK include in the PRK group. Postoperatively, the percentages were 52.96Ϯ7.55 and 53.34Ϯ10.2, respectively. Six epithelial ingrowth, fl ap complications, keratectasia, biome- 4 months postoperatively, keratocyte density changed by chanical instability, and dry eye. 367.12Ϯ103.35 cells/mm2 (34.7% reduction) in the Despite improvements in surgical techniques and ex- anterior stroma (PϽ.05) and 9.25Ϯ28.28 cells/mm2 cimer laser technology, similar advances in the understand- Ͼ (1.31% reduction) in the posterior stroma (P .05) for ing of the cellular response following LASIK and PRK have the LASIK group. In the PRK group, these values were 2 319.71Ϯ83.45 cells/mm2 (31.13% reduction) in the not been made. Cellular and structural changes induced by anterior stroma (PϽ.05) and 0.17Ϯ38.97 cells/mm2 refractive surgery may aid in understanding the natural cel- (0.02% reduction) in the posterior stroma (PϾ.05). The lular processes and potential complications that occur after changes in keratocyte densities were not statistically each type of surgery. The complex nature of tissue inter- signifi cant between groups (PϾ.05). The mean number of keratocytes decreased by 37.2% in the retroablation zone of the LASIK group (PϽ.05). No changes were From Farabi Eye Hospital, Tehran University of Medical Sciences (Amoozadeh, noted in endothelial cell counts. Behesht-Nejad, Hashemi); and Noor Ophthalmology Research Center, Noor Eye Hospital (Aliakbari, Seyedian, Rezvan, Hashemi), Tehran, Iran. CONCLUSIONS: A signifi cant decrease occurred in the number of stromal keratocytes in the anterior stroma. The authors have no financial or proprietary interest in the materials pre- sented herein. Despite differences in surgery, the change in keratocyte density and endothelial cell counts were similar be- Rich Bains, consultant to NIDEK Co Ltd, assisted in the preparation of the tween LASIK and PRK groups (PϾ.05). [J Refract Surg. manuscript. 2009;25:S963-S967.] Portions of this article have been published previously in the Iranian Journal doi:10.3928/1081597X-20090915-12 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: [email protected]

Journal of Refractive Surgery Volume 25 October (Suppl) 2009 Commercially Sponsored Section S963 Keratocyte Density After LASIK and PRK/Amoozadeh et al

action postoperatively may help determine selection the Confoscan 3 preoperatively and 6 months post- criteria for LASIK and PRK. operatively. Confocal microscopy has been used to investigate Confoscan 3 measurements were performed by the cellular morphology and structure of the various an experienced ophthalmologist (H.H.) who did not corneal layers.1-6,8-11 However, the results from these perform the surgeries on this study cohort. Mea- studies are contradictory. Normal keratocyte density is surements were acquired in automatic mode us- 1079 cells/mm2.12 In the present study, in vivo confo- ing the 20ϫ non-contact lens. Topical tetracaine cal microscopy was used to evaluate keratocyte den- 0.5% drops were applied to anesthetize the eyes. sity and endothelial cell count and to compare changes Methyl cellulose drops (Viscotears Gel; CIBA Vi- seen in eyes that underwent LASIK and PRK. sion, Duluth, Ga) were applied on the objective lens of the machine to provide a regular image, and the PATIENTS AND METHODS lens was aligned with the pupil center. For each eye, 350 frames, 5.0-µm apart were acquired. The STUDY POPULATION cells in each layer were marked by an examiner This study was a prospective, non-randomized study (S.A.) (who was masked to the type of surgery) to of 16 patients who were scheduled to undergo LASIK avoid inadvertently counting the same cell twice. (LASIK group) or PRK (PRK group) from March 1 to The number of cells was calculated by counting May 15, 2007. Patients with low to moderate myopia the number of clear and distinct cells in areas of (Ϫ1.00 to Ϫ4.50 diopters [D]) or low to moderate myo- 379ϫ286 µm2. The keratocyte density was studied pic astigmatism (ϽϪ4.50 D sphere and up to 1.50 D in two layers—25 µm posterior to Bowman’s layer astigmatism) were enrolled after signing consent forms (anterior stroma) and 40 µm anterior to Descemet’s for pre- and postoperative examination. Patient prefer- membrane (posterior stroma). The layer 25 µm poste- ence in consultation with the surgeon was the basis for rior to Bowman’s layer was chosen based on previous undergoing PRK or LASIK. Eight eyes of 4 patients had studies,3-5 as the exact amount of tissue removal for LASIK and 24 eyes of 12 patients had PRK. Exclusion each patient could not be predicted preoperatively. criteria were systemic diseases such as diabetes, trau- Postoperatively, the same 25 µm from Bowman’s matic or infectious complications after refractive sur- layer was used, although this may be the midstro- gery, active eye disease, and previous corneal surgery. ma compared to preoperatively. Additionally, the After applying the above exclusion criteria, all pa- Confoscan operator (S.A.) was not aware of the abla- tients were available for analysis. Ten patients used tion depth of each patient postoperatively. By using contact lenses prior to surgery (8 in the PRK group and 2 Bowman’s layer as the reference point, epithelial hy- in the LASIK group). However, all contact lens wearers perplasia was ruled out as a source of error. For deep were required to discontinue use for at least 3 to 28 days stroma, the endothelium was chosen as the reference (depending on contact lens type) prior to preoperative point. evaluation to stabilize keratometry and corneal topogra- phy. Patients were required to have normal keratometry SURGERIES and topography prior to undergoing refractive surgery. All surgeries were performed by a single surgeon (H.H.). The surgeon did not perform confocal micros- PRE- AND POSTOPERATIVE EXAMINATIONS copy measurements. The Technolas 217 Planoscan ab- Pre- and postoperative examinations included mea- lation (Bausch & Lomb, Rochester, NY) was used for surement of uncorrected visual acuity, best spectacle- all patients. For PRK, the epithelium was manually de- corrected visual acuity, slit-lamp evaluation of the cornea brided with a hockey stick spatula. At the end of the and anterior chamber, tonometry, keratometry, topogra- procedure, an O2 Optix bandage contact lens (CIBA phy, pachymetry, cycloplegic refraction, and confocal Vision) was placed, with removal 3 days after surgery microscopy (Confoscan 3; NIDEK Co Ltd, Gamagori, Ja- or upon complete re-epithelialization. pan). Laser in situ keratomileusis was performed using Confocal microscopy was used to determine the the Hansatome (Bausch & Lomb) microkeratome. Post- number of keratocytes in the anterior and posterior operative drop regimen for the LASIK group was chlor- stromal layers, endothelial cell count, and number amphenicol drops for 3 days, betamethasone drops for of hexagonal cells. In the LASIK group, the kerato- 1 week, and artifi cial tears for 2 months postopera- cytes were counted in the retroablation zone, de- tively. Patients who underwent PRK were requested to fi ned as the space 5 µm behind the fl ap to the en- instill diclofenac sodium drops every 8 hours during dothelium. Keratocyte density was measured using the fi rst 24 hours, chloramphenicol drops for 5 days,

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betamethasone drops for 2 weeks, artifi cial tears for 1 month, and fl uorometholone drops for 12 weeks after 167 154.8 cessation of betamethasone drops. 118 Ϯ Ϯ Ϯ 8

No patient underwent retreatment for the duration 42 Ϫ Ϫ of this study. 33.3 Ϫ

STATISTICAL ANALYSIS 186 404 Data analysis was performed with SPSS 16 software 359.3 Ϯ Ϯ (SPSS Inc, Chicago, Ill). The t test was used for analyz- Ϯ ing the pre- to postoperative mean differences in anterior stromal cell density, posterior stromal cell density, en- dothelial cell count, and hexagonal cell density in both Ͻ 224 3030 293 3025 groups. P .05 was considered statistically signifi cant. 274.8 3026.2 Ϯ Ϯ Ϯ RESULTS Mean patient age was 28.25Ϯ5.75 years (range: 19 to 36 years). Mean age of the LASIK group was 28.50Ϯ5.86 years (range: 19 to 36 years) and 27.50Ϯ6.24 years (range: ) 20 to 35 years) in the PRK group. Preoperative mean 2 28 3022 36.4 2992.9 38.98 2983 Ϯ myopia was 2.94Ϯ0.96 D (range: Ϫ2.00 to Ϫ4.25 D) and Ϯ Ϯ the mean astigmatism was Ϫ0.50Ϯ0.25 D (range: Ϫ0.50 to Ϫ1.00 D) in the LASIK group. Preoperative mean myopia was Ϫ2.85Ϯ0.99 D (range: Ϫ1.00 to Ϫ4.00 D) 57 9 45 0 Ϫ Ϯ 47.9 2.4 Ϯ Ϯ and the mean astigmatism was 0.775 0.42 D (range: Ϯ Ϫ0.25 to Ϫ1.00 D) in the PRK group. The mean abla- tion depth was 61Ϯ17.17 µm (range: 36 to 71 µm) in the LASIK group and 62.13Ϯ15.41 µm (range: 38 to 69 TABLE µm) in the PRK group. No statistically signifi cant dif- Standard Deviation (cells/mm Ϯ 40 699 51 719 47.7 713.5 Ϯ Ϯ

ference was noted in ablation depth between groups Ϯ

(PϾ.05). The mean anterior keratocyte cell density, Mean posterior keratocyte cell density, and endothelial cell count are presented in the Table. The changes in the variables in each group were not statistically signifi - Ͼ That Underwent LASIK and PRK 84* 719 cant (P .05) (Table). 89.5* 716 103* 708 Ϯ Ϯ Ϯ

Preoperative hexagonal cell percentage was Density Keratocyte 52.17Ϯ11.43 for the LASIK group and 51.33Ϯ10.98 for the PRK group. Postoperatively, the hexagonal cell percentiles were 52.96Ϯ7.55 for the LASIK group and 53.34Ϯ10.2 for the PRK group. In the LASIK group, 55 368 63 320 60.8 331.6 Ϯ Ϯ the mean reduction in keratocyte density was 34.7% Ϯ in the anterior stromal layer and 1.31% in the posterior

stromal layer. The decrease in the anterior stroma was Anterior Stroma Stroma Posterior Endothelial Cell Count .05).

Ͻ Ͻ

statistically signifi cant (P .05). A 0.27% increase in P endothelial cell count was noted in the LASIK group 95 690 80 707 83.7 703.2 Ϯ Ϯ (Table). Ϯ

In the PRK group, the mean reduction in keratocyte Preop Postop Difference Preop Postop Difference Preop Postop Difference density was 31.13% in the anterior stroma and 0.02% in the posterior stroma (Table). The decrease in the an- Density and Endothelial Cell Count in Eyes Stromal Keratocyte Anterior and Posterior terior stroma was statistically signifi cant (PϽ.05) Endothelial cell count increased by 1.39% in the PRK group (Table). Postoperatively, hexagonal cell density Statistically significant difference ( * LASIK (n=24) 1058 PRK (n=8) 1027 increased by 0.79% in the LASIK group, 2.01% in PRK (n=32)Total 1034.7 group, and 1.57% in the study cohort. No statistically

Journal of Refractive Surgery Volume 25 October (Suppl) 2009 Commercially Sponsored Section S965 Keratocyte Density After LASIK and PRK/Amoozadeh et al

signifi cant differences were noted between groups or in cell counts can approach 8%13 yet experienced ob- changes in overall hexagonal cell density (PϾ.05). servers have shown differences of Ͻ3%.3 Addition- The mean number of keratocytes in the retroablation ally, the keratocytes may not all be in the same plane, zone was 947.13Ϯ16.78 cells/mm2 (range: 845 to 1024 which can also cause counting errors. Other limita- cells/mm2), which decreased to 610Ϯ19.43 cells/mm2 tions of this study are the unequal number of patients (range: 573 to 828 cells/mm2) postoperatively (37.2% in the PRK and LASIK groups and the lack of a control difference) (PϽ.05). group. These limitations could not be avoided due to the small sample size. DISCUSSION Keratocytes function to maintain the health and The present in vivo confocal microscopy study of clarity of the corneal stroma.13 Compared to corneal eyes that underwent LASIK or PRK found a mean re- endothelial and epithelial cells, keratocytes have mod- duction in keratocyte density 6 months postoperatively. erate regenerative capacity. Epithelial cells are read- Eyes that underwent PRK and LASIK both experienced ily regenerated with complete re-epithelialization after a statistically signifi cant reduction in anterior keratocyte injury; however, endothelial cells do not regenerate.14 density postoperatively (PϽ.05) (Table). In the LASIK Measuring the changes in keratocyte density after re- group, a 34.7% difference in anterior keratocyte reduc- fractive surgery will help develop an understanding of tion was noted compared to posterior keratocyte density the impact of surgery on the cornea. (Table). This outcome is similar to separate investiga- Vesaluoma et al10 postulated that anterior stromal tions of PRK and LASIK. Ghirlando et al5 evaluated 50 cell loss begins 6 months after LASIK. They attribute myopes 1 month after PRK and reported a greater num- this observation to the loss of neural input to the ker- ber of activated keratocytes in the anterior stroma com- atocytes due to severing of the nerves during LASIK, pared to the posterior stroma. McLaren et al12 reported but to a lesser degree in PRK.10 Although the main a statistically signifi cant reduced number of keratocytes cause of keratocyte loss after refractive surgery re- in the anterior stroma compared to the posterior stro- mains ambiguous, keratocyte necrosis and apoptosis ma after LASIK. The magnitude of reduction found in have been suggested as possible causes.10 Apoptosis our study (31.1%) is similar to the 40% reported at 6 and necrosis are the results of cell death due to ex- months by Erie et al3 for PRK. However, Erie et al re- posure to lethal stresses. Thermal effects due to the ported lower keratocyte loss (22%) for LASIK patients ablation, mechanical debridement, and microkera- compared to our results (34.7%). These differences may tome cut all represent such stresses. In the present be due to counting errors or differences in the volume of study, keratocyte density in the retroablation zone tissue removed between studies.3,13 decreased and was found to be statistically signifi - The reduction in anterior keratocyte density was cant (PϽ.0001) 6 months after LASIK. Compared to similar in LASIK and PRK in our study. Despite surface the anterior stroma, we believe this higher decrease versus mid-stromal ablation, the difference between can be attributed to the direct effect of laser ablation reductions was not statistically signifi cant (PϾ.05). on the retroablation zone. However, others have reported differences between The decreases in corneal cellular density have also LASIK and PRK, which are attributed to the deeper been attributed to the lack of a robust cellular prolifera- ablation depth in LASIK.3 In the present study, the ab- tive response to cell loss.14 Keratocyte density anterior lation depth was similar in LASIK and PRK. Whether to the retroablation zone remained reduced by 42% up ablation depth or location of ablation delivery (surface to 5 years after LASIK and PRK.9 Regions of keratocyte versus mid-stroma) is a contributing factor to kerato- apoptosis at the fl ap edge may never be replaced by cyte density remains a topic for future study. new keratocytes.3 One study reported that the decrease One limitation of confocal microscopy is the limited in keratocyte density after LASIK has both an acute area under observation, which is overcome by observ- and a chronic phase.11 ing a larger number of samples. The other limitation The question remains whether the loss of cells fol- is the lack of registration of images between follow- lowing PRK or LASIK as reported in the present study ups, as the same area is not imaged from examination reduces the health and function of the cornea. To date, to examination. This limitation is the purview of con- clinical studies have shown that this reduction in cell focal manufacturers who can develop software algo- count does not affect the overall health of the cornea.14 rithms that allow registration of a landmark based on Even various long-term studies provide evidence of contrast ratios of image pixels. Another limitation of visual improvement after refractive surgery,15,16 possi- confocal microscopy and this study is the subjective bly indicating that there are few clinical consequences nature of the measurements. Interobserver variability to the reduction of keratocyte density.

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

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