Keratocyte Density after Laser-Assisted Subepithelial Keratectomy with Mitomycin C

Keratocyte Density after Laser-Assisted Subepithelial Keratectomy with Mitomycin C LAURA DE BENITO LLOPIS, PILAR DRAKE, PILAR CAÑADAS, JOSÉ LUIS HERNÁNDEZ-VERDEJO, AND MIGUEL A. TEUS ● PURPOSE:

To study the effects of laser-assisted subepithelial keratectomy (LASEK) with mitomycin C (MMC) on the keratocyte population. ● DESIGN: Prospective, nonrandomized, interventional, comparative case series. ● METHODS: Fifty-six eyes treated at Vissum Santa Hortensia, Madrid, Spain, were included in the study. We compared 28 eyes treated with LASEK with intraoperative 0.02% MMC versus 28 non-treated eyes. Keratocyte density was measured 3 months after the surgery in the anterior, mid, and posterior stroma and was compared with the corresponding layers in the control eyes. The anterior layer in the LASEK group was compared with 2 layers in the control group: the most anterior stromal layer and the 80 ␮m-deep layer, because that was the mean ablation depth performed in eyes that underwent LASEK. ● RESULTS: We found a statistically significantly lower keratocyte population in the most anterior stromal layer after LASEK with MMC compared with both the most anterior stromal layer and the 80 ␮m-deep layer in controls. On the contrary, the treated group showed a significantly higher keratocyte density in both the mid stroma and the deep stroma. The comparison between the average densities through the entire cornea showed a significantly higher keratocyte population in the LASEK with MMC group. ● CONCLUSIONS: LASEK with MMC seems to cause a decrease in the anterior stromal cells 3 months after the surgery compared with nonoperated corneas. There seems to be a compensating proliferation of keratocytes in the deeper corneal layers, suggesting that the ability of keratocytes to repopulate the cornea is maintained after the surgical procedure. (Am J Ophthalmol 2010;150: 642– 649. © 2010 by Elsevier Inc. All rights reserved.)

Accepted for publication May 20, 2010. From Vissum Madrid, Madrid, Spain (L.d.B.-L., P.D., P.C., J.L.H.-V., M.A.T.); Universidad Europea de Madrid, Madrid, Spain (P.C.); Universidad Complutense de Madrid, Madrid, Spain (J.L.H.-V.); Universidad de Alcalá, Madrid, Spain (M.A.T.); and Hospital Universitario Príncipe de Asturias, Alcalá de Henares, Madrid, Spain (M.A.T.). Inquiries to Laura de Benito-Llopis, Vissum Madrid, Santa Hortensia 58, 28002 Madrid, Spain; e-mail: [email protected]

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M

ITOMYCIN C (MMC) HAS PLAYED A DECIDING

role in the current revival of excimer laser surface ablation techniques.1 MMC is an alkylating agent with cytotoxic and antiproliferative effects that reduces the myofibroblast repopulation after laser surface ablation, and therefore reduces the risk of postoperative corneal haze. It is used prophylactically to avoid haze after primary surface ablation2– 4 and therapeutically to treat pre-existing haze.5 The keratocytes are the target of the MMC antihaze action. It is the cytotoxic and antiproliferative effects of the MMC on the corneal stromal keratocytes that causes its capacity to reduce haze, because it inhibits its activation, proliferation, and differentiation into myofibroblasts.6–11 However, this antimitotic effect has led to fear the consequences of a possible long-term depletion of the keratocyte population.10,12,13 The few studies analyzing the postoperative keratocyte population after intraoperative MMC show contradictory results (Table 1). Therefore, we decided to study keratocyte density after laser-assisted subepithelial keratectomy (LASEK) with intraoperative MMC and to compare it with that of nonoperated healthy control eyes.

METHODS WE PERFORMED A PROSPECTIVE STUDY OF CONSECUTIVE

eyes that underwent LASEK to correct a myopic error (with or without astigmatism) and that received intraoperative MMC prophylactically because of an ablation depth of more than 50 ␮m. When evaluating for surgery, we excluded patients with unstable refraction and keratoconus suspects. We excluded from analysis those cases with previous ocular surgery (either refractive or other type) and those with a systemic disease that could alter the wound-healing process, such as diabetes or connective tissue disorders. A group of normal subjects with nonoperated healthy corneas also were analyzed and served as controls. All study patients underwent a full ophthalmologic examination before surgery that included the measurement of uncorrected visual acuity, the best spectacle-corrected visual acuity (using a Snellen chart [Nidek auto chart projector CP 670; Nidek Co, Ltd, Gamagori, Japan] and including manifest and cycloplegic refractions), slit-lamp

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0002-9394/$36.00 doi:10.1016/j.ajo.2010.05.029

Reported as nondecreased (but no data available) Nondecreased compared with eyes without MMC

● SURGICAL TECHNIQUE:

MMC ⫽ mitomycin C; min ⫽ minutes; mo ⫽ month; yrs ⫽ years.

5 yrs 2 min 0.02 Human study Midena et al.15

56

2 min Human study Gambato et al.4

36

0.02

3 yrs

Nondecreased 5 min Animal study (rabbits) Xu et al.11

20

0.02

6 mos

Time-dependent decrease Laboratory study (human corneas in vitro) Rajan et al.14

16

1 min, 2 min

1 mo

Time and dose-dependent decrease 12 s to 2 min Animal study (rabbits) Netto et al.10

182

0.002 0.02 0.02

1 mo

Decreased 2 min 0.02 Animal study (rabbits) Kim et al.8

18

Prospective, randomized, comparative study Prospective, randomized, comparative study Prospective, randomized, comparative study Prospective, randomized, comparative study Prospective, randomized, comparative study Prospective, randomized, comparative study

3 mos

Keratocyte Density at Last Follow-up Last Follow-up MMC Application Time MMC Dose (%) Type of Study Subject of Study Authors

No. of Eyes Receiving MMC

TABLE 1. Studies Reporting the Effect of Mitomycin C on the Keratocytes

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biomicroscopy, tonometry (CT-80; Topcon, Tokyo, Japan), corneal pachymetry (DGH 5100 contact pachymeter; DGH Technology, Inc, Exton, Pennsylvania, USA), keratometry and corneal topography (CSO; Compagnia Strumenti Oftalmici, Firenze, Italy), mesopic pupil measurement (Colvard pupillometer; Oasis, Glendora, California, USA), and funduscopy.

KERATOCYTE DENSITY

All procedures were performed by 2 surgeons (M.A.T.; L.d.B.-L.), using the same Esiris Schwind Excimer Laser (Schwind Eye Tech Solutions, Kleinostheim, Germany) using a PRK nomogram and conventional treatment. All surgeries were performed using topical anesthesia (lidocaine 2%). De-epithelialization was performed using 20% alcohol solution (diluted in balanced salt solution) instilled inside an 8-mm corneal marker centered on the pupil and left for 40 seconds. A cellulose sponge was used to remove the alcohol and balanced salt solution was instilled copiously to rinse the ocular surface. The edges of the flap were dried with a sponge and the epithelial flap was peeled back with a crescent blade (Alcon Surgical, Orlando, Florida, USA), leaving a hinge at the 12-o’clock position. The stromal bed was dried with a sponge, the eye tracker was set in the center of the pupil, and the ablation was performed. After laser ablation, a 7-mm round cellulose sponge soaked in MMC 0.02% was applied for 30 seconds over the ablated stroma. The programmed ablation was 10% less that the intended correction to avoid overcorrection. The stroma then was rinsed copiously with balanced salt solution, and the epithelial flap was repositioned using the same cannula. A therapeutic soft contact lens (Acuvue Oasys; Johnson & Johnson Vision Care, Inc, Jacksonville, Florida, USA) was placed carefully on the eye and antibiotic drops (ciprofloxacin 3 mg/mL) and nonsteroidal anti-inflammatory drops (ketorolac trometamol 5 mg/mL) were applied.

● POSTOPERATIVE FOLLOW-UP: The medications consisted of topical antibiotic (ciprofloxacin 3 mg/mL) and steroid (fluorometholone alcohol 2.5 mg/mL) drops 4 times daily during the first week after surgery. The steroid drops then were tapered and stopped 1 month after surgery. The therapeutic contact lens was removed after re-epithelialization was complete, 5 to 7 days after surgery. Examinations were scheduled at 1 day, 1 week, and 1 and 3 months after surgery. Confocal microscopy was performed at the 3-month postoperative visit. ● IN VIVO CONFOCAL MICROSCOPY: Laser scanning in vivo confocal microscopy was performed using the Heidelberg Retina Tomograph II with the Rostock Cornea Module (HRTII/RCM; Heidelberg Engineering, Heidelberg, Germany). This microscope uses a 670-nm red wavelength diode laser source. A ⫻60 objective water immersion lens with a numerical aperture of 0.9 (Olympus, AFTER

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FIGURE 1. Cell count in an image of the corneal stroma obtained with the Heidelberg Retina Tomograph II with the Rostock Cornea Module (Heidelberg Engineering, Heidelberg, Germany).

FIGURE 2. Confocal image obtained with the Heidelberg Retina Tomograph II with the Rostock Cornea Module (Heidelberg Engineering, Heidelberg, Germany) of the anterior stroma of an eye 3 months after surface ablation with mitomycin C. Cell density, 20 326.79 cells/mm3.

FIGURE 3. Confocal image obtained with the Heidelberg Retina Tomograph II with the Rostock Cornea Module (Heidelberg Engineering, Heidelberg, Germany) of the posterior stroma (50 ␮m above the endothelium) of an eye 3 months after surface ablation with mitomycin C. Cell density, 39 215 cells/ mm3.

Tokyo, Japan) was used. The dimensions of the images obtained using this lens are 400 ⫻ 400 ␮m, and the manufacturer quotes the transverse resolution and optical section thickness of 1 and 4 ␮m, respectively. The RCM uses an entirely digital capture system. The device can scan 644

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automatically through 80 ␮m of the cornea, at any depth, capturing 30 frames/s, but to obtain a scan of the entire cornea, the microscope needs to be advanced manually through the full thickness of the cornea. OF

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TABLE 2. Preoperative Data of the Uncomplicated LASEK Cases Compared with the Normal Nonoperated Corneas Used as the Control Group

Age (range), yrs Sphere (range), D Cylinder (range), D

Control (n ⫽ 28)

LASEK (n ⫽ 28)

P Value

27.4 ⫾ 3.68 (24 to 34) –2.07 ⫾ 2.6 (0 to –8) –0.29 ⫾ 0.51 (0 to –2)

32.5 ⫾ 8.37 (21 to 52) –3.43 ⫾ 2.25 (0 to –9) –1.27 ⫾ 1.20 (0 to –4.5)

.004 .04 .0002

D ⫽ diopters; LASEK ⫽ laser-assisted subepithelial keratectomy; yrs ⫽ years.

TABLE 3. Comparison of Keratocyte Density in Normal Nonoperated Corneas (Control Group) and Uncomplicated Laser-Assisted Subepithelial Keratectomy Cases 3 Months after Surgery Control (n ⫽ 28)

Anterior stroma (range), cells/mm3 Stromal bed (range), cells/mm3 Mid stroma (range), cells/mm3 Posterior stroma (range), cells/mm3 Average (range), anterior-mid-posterior

LASEK (n ⫽ 28)

P Value

29 080.29 ⫾ 5788.81 (19 934.64 to 41 830.06)

16 660.53 ⫾ 7844.88 (6666.66 to 36 209.15)

.0001

20 501.86 ⫾ 2458.43 (16 470.58 to 25 228.75)

16 660.53 ⫾ 7844.88 (6666.66 to 36 209.15)

.01

18 144.25 ⫾ 1955.88 (14 509.80 to 21 111.11)

30 179.73 ⫾ 9117.64 (13 660.13 to 46 470.58)

.0001

18 076.56 ⫾ 2097.73 (13 398.69 to 22 352.94)

29 675.24 ⫾ 8158.77 (16 993.46 to 46 745.09)

.0001

21 767.04 ⫾ 2676.74 (16 383.44 to 27 886.71)

25 505.17 ⫾ 7052.47 (15 446.62 to 38 843.13)

.01

LASEK ⫽ laser-assisted subepithelial keratectomy.

The method of examination was as follows: each eye was anesthetized with 1 drop of 1% tetracaine chlorohydrate (Alcon Cusí Laboratories, Barcelona, Spain). Viscotears (Carbomer 980, 0.2%; Novartis, North Ryde, Australia) was used as a coupling agent between the applanating lens cap and the cornea. During the examination, all subjects were asked to fixate on a distance target aligned to enable examination of the central cornea. The lens then was advanced manually until it contacted the cornea. The full thickness of the central cornea was scanned. The total duration of in vivo confocal examination was approximately 2 minutes per eye, and none of the subjects experienced any visual symptoms or complications as a result of the examination. Three scans through the entire cornea were recorded from each eye, and the one with fewer motion artefacts then was selected for analysis. All scans were performed by the same trained examiner (J.L.H-V.). ● KERATOCYTE DENSITY MEASUREMENT:

The digital images were loaded into an interactive computer program. One observer (P.C.) manually selected the bright objects (keratocyte nuclei) in each image. The same area (400 ⫻ 400 ␮m of cornea) was used for all images. The same examiner did the counting in all the scans. Objects that touched the bottom and left boundaries of this area were counted, whereas objects that crossed the top or right

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edges were not counted. The computer then provides the value of the keratocyte density expressed as cells per square millimeter (Figure 1). Volumetric density16 was estimated using the method published by McLaren and associates, in which the cell density was determined by the formula: D ⫽ N ⁄ A␾ , where N is the number of cells identified in the rectangular area A, and ␾ is the optical section thickness.17 This optical section thickness ␾, or depth of field, was estimated, as McLaren and associates proposed, by the distance that the scanner moved from the gradual appearance of a cell to the gradual disappearance of that cell.17 The number of frames in which a cell would have been counted was multiplied by the average distance between frames. For this purpose, we used the automatic scan mode of the HRTII/RCM to obtain 3 scans with a mean thickness of 77 ␮m, a mean frame capture of 39 frames per scan, and an average distance between frames of 1.93 ␮m. In each of those 3 automatic scans, we checked 15 keratocytes (45 keratocytes in total). This method yielded a depth of field of 15.63 ⫾ 1.82 ␮m. At each determined depth of study, 3 images were analyzed: the one that most exactly corresponded to the determined depth and the images just anterior and posterior, with AFTER

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a difference between them of less than 20 ␮m. Cells were counted in the 3 images and the mean was obtained. In the LASEK group, images were analyzed at the following depths: (1) anterior stroma (stromal bed; first good-quality image of the keratocytes under the epithelium; Figure 2), (2) mid stroma (200 ␮m above the endothelium), and (3) deep stroma (50 ␮m above the endothelium; Figure 3). In the control group, images were analyzed at the same depths and also at the layer that correlated to the depth of ablation. Taking into account the ablation performed in the LASEK group, keratocytes were measured in controls at the depth that would become anterior stroma if they received the same ablation. Because the mean programmed ablation in the LASEK group was sphere ⫺3.63 diopters and cylinder ⫺1.32 diopters, we calculated, following the parameters of our laser, the mean stromal ablation performed, which was 80 ␮m on average. Therefore, the keratocytes also were measured at a depth of 80 ␮m in control eyes. Keratocyte densities then were compared between corresponding layers. The anterior layer in the LASEK group therefore was compared with 2 layers in the control group: the most anterior stromal layer and the 80-␮m deep layer.

increased keratocyte density in the mid and deep stroma, resulting in a mean keratocyte density in the entire cornea higher in the treated group compared with control eyes. The different optical designs of the available confocal microscopes may lead to different measurements18; therefore, we could not compare reliably the densities obtained in our study with those of other studies, because we could not find in the literature any other study using the HRTII/RCM confocal microscope.19 Only 2 studies address the keratocyte density after surface ablation with MMC performed in human corneas in vivo (Table 1). Gambato and associates described “at early postoperative examinations, dark lacunae because of keratocyte apoptosis . . . . Anterior stromal keratocytes showed progressive increased density . . . . By 6 to 12 months after PRK in MMC-treated eyes, normalization of density of keratocytes,” but they do not report any data on the actual measurements of keratocyte density.4 The other study reported decreased keratocyte density 5 years after surface ablation compared with that before surgery, but regardless of the use of MMC, with no significant difference between the group that received MMC and the control group.15 However, the comparison in this case was performed dividing the cornea in 5 stromal layers (0% to 10%, 11% to 33%, 34% to 66%, 67% to 90%, and 91% to 100%) and comparing the preoperative and postoperative measurements. In our opinion, this method does not compare the real stromal layer from the preoperative cornea that becomes anterior after the laser ablation, which may lead to differences that may be explained by removal of the most populated anterior stromal layers with the laser, not by an actual decrease of keratocytes in the remaining stromal bed caused by the excimer laser, the MMC, or both. To avoid this possible source of error, we compared the anterior stroma in operated eyes with the 80-␮m deep stromal layer in nonoperated eyes, taking into account the mean ablation depth performed. By doing this, the difference found between the stromal bed in LASEK eyes and the corresponding layer in control eyes was much smaller, although still statistically significant, than the difference detected if the stromal bed was compared with the most anterior stromal layer in control eyes (Table 3). Among the animal or laboratory studies using intraoperative MMC, all those reporting keratocyte depletion show a short follow-up (1 to 3 months; Table 1). The only animal study with longer follow-up shows that, after an initial depletion, keratocyte density increases again, reaching preoperative levels 6 months after the application of MMC. A review of the studies that did not use MMC reveals that the long-term effect of the laser ablation itself on the stromal population is controversial: some of the studies find a similar or higher postoperative keratocyte density,20 –24 whereas others report a decreased stromal population25,26 after surface ablation with no adjunctive MMC, which needs to be taken into account when analyzing postoperative keratocyte density changes after surface ablation with

● STATISTICAL ANALYSIS:

Statview⫹Graphics software (Abacus Concept, Inc, Cupertino, California, USA) was used for data analysis. The normality of the distribution was checked using the Bonferroni test. Statistical comparisons were carried out with the unpaired 2-tailed Student t test. P ⱕ .05 was considered statistically significant. Continuous data are expressed as the mean ⫾ standard deviation.

RESULTS TWENTY-EIGHT LASEK EYES WERE INCLUDED IN THE STUDY

and were compared with 28 healthy nonoperated corneas. Table 2 shows the preoperative data of both groups. The comparison of keratocyte densities (Table 3) showed a statistically significantly lower keratocyte population in the most anterior stromal layer after LASEK compared with both the most anterior stromal layer and the 80-␮m deep layer in controls. On the contrary, it showed a significantly higher keratocyte population in both the mid stroma and the deep stroma in the LASEK plus MMC group. The comparison between the average densities (calculated as the mean between the anterior, mid, and deep stromal layer densities in each group) showed a statistically significantly higher keratocyte population in the LASEK plus MMC group.

DISCUSSION OUR STUDY SHOWS A DECREASED KERATOCYTE POPULA-

tion in the stromal bed 3 months after LASEK with intraoperative MMC compared with control eyes, but an 646

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MMC. In fact, the de-epithelialization itself causes immediate keratocyte depletion.20,27–33 Regarding the keratocytes of the posterior layers, we found an increase in the mid and deep stroma compared with nonoperated corneas. The other clinical studies using MMC reported no change in keratocyte morphologic features4 or density15 in deep corneal layers, nor did the clinical studies after surface ablation without MMC.23–26 The laboratory study performed in human corneas maintained in vitro14 described no difference of keratocyte density in the mid and deep stromal layers between the MMC and the control groups 1 month after surgery, but the study did not indicate whether the density in those layers was compared with the preoperative data. However, this increase in the keratocyte population has been described in rabbits after surface ablation34 and after cryoablation,35 and also in humans in a few clinical studies measuring keratocyte density after laser in situ keratomileusis (LASIK).36,37 This increase in the deep stromal keratocyte density may reflect the response of the deeper keratocytes to the apoptosis of the most anterior ones after surface ablation. The keratocytes have been shown to be interconnected closely, forming a network through the entire cornea.38 – 40 Rajan and associates showed that the application of MMC, with its antiproliferative effect, decreased the keratocyte repopulation of the stromal bed,14 suggesting that this repopulation is achieved with some degree of mitotic activity, not only by migration of the existing surrounding keratocytes. Our observation of a higher keratocyte density in the mid and deep stromal layers, and moreover, in the average density of the entire cornea, also may reflect either a mitotic activity in the keratocytes underlying the stromal bed to allow for the repopulation of the anterior layers or the repopulation of the stroma by cells derived from the bone marrow, which has been proposed as the source of corneal stromal cells.41–46 A longitudinal study with a longer follow-up may allow detection of a progressive decrease in the posterior layers, as has been described

after LASIK,36,37 accompanied by a progressive increase in the anterior stromal population. Qazi and associates reported that late dense haze may develop 17 months after uncomplicated primary surface ablation with prophylactic MMC, suggesting that stromal cellularity is not affected significantly by a permanent MMC effect and that the keratocytes seem to keep their capacity to activate and proliferate.47 Our study further supports the fact that excimer laser ablation surgery with intraoperative MMC does not lead to permanent keratocyte depletion, but to a temporary decrease in the anterior keratocytes, compensated with an increase in the underlying layers. Our study may be flawed by the intrinsic limitations of using confocal microscopy to measure keratocyte density, mainly because of 2 factors: the possibility of anteroposterior movement of the subject and the subjectivity of the manual cell counting. We tried to decrease the first source of error by performing 3 scans in each eye and selecting the one with the least motion.18 The subjectivity in cell counting was decreased by the fact that all the scans were assessed by the same person within a few days. A study comparing the results of keratocyte density measurements performed manually and with an automated program showed that, despite differences in the final cell densities, the conclusions regarding the change of cell densities after surgery would have been the same.48 The fact that the images obtained with the HRTII/ RCM show a high contrast18 also allows for a lower variability in the manual assessment.48 In conclusion, surface ablation with intraoperative MMC seems to cause a decrease in the anterior stromal cells 3 months after the surgery compared with nonoperated corneas. There seems to be a compensating proliferation of keratocytes in the deeper corneal layers, despite the use of MMC, suggesting that the ability of the keratocytes to repopulate the cornea is maintained after the surgical procedure.

THE AUTHORS INDICATE NO FINANCIAL SUPPORT OR FINANCIAL CONFLICT OF INTEREST. INVOLVED IN DESIGN AND conduct of study (L.d.B.L., M.A.T.); Collection of data (P.D., P.C., J.L.H.-V.); Analysis and interpretation of data (M.A.T., L.d.B.L.); Preparation of the manuscript and literature search (L.d.B.L.); Review of the manuscript (L.d.B.L., P.C., P.D., J.L.H.-V., M.A.T.); and Final approval of the manuscript (L.d.B.L., P.C., P.D., J.L.H.-V., M.A.T.). Institutional review board approval from the “Comité de Ética e Investigación Clínica” of Vissum Corporación Oftalmológica, Alicante, Spain, and patient informed consent for surgery and for participation in this research were obtained.

4. Gambato C, Ghirlando A, Moretto E, Busato F, Midena E. Mitomycin C modulation of corneal wound healing after photorefractive keratectomy in highly myopic eyes. Ophthalmology 2005;112(2):208 –219. 5. Majmudar PA, Forstot SL, Dennis RF, et al. Topical mitomycin C for subepithelial fibrosis after refractive corneal surgery. Ophthalmology 2000;107(1):89 –94. 6. Chang SW. Corneal keratocyte apoptosis following topical intraoperative mitomycin C in rabbits. J Refract Surg 2005;21(5):446– 453. 7. Kim TI, Lee SY, Pak JH, Tchah H, Kook MS. Mitomycin C, ceramide, and 5-fluorouracil inhibit corneal haze and apoptosis after PRK. Cornea 2006;25(1):55– 60.

REFERENCES 1. Teus MA, de Benito-Llopis L, Alió JL. Mitomycin C in corneal refractive surgery. Surv Ophthalmol 2009;54(4): 487–502. 2. Carones F, Vigo L, Scandola E, Vacchini L. Evaluation of the prophylactic use of mitomycin-C to inhibit haze formation after photorefractive keratectomy. J Cataract Refract Surg 2002;28(12):2088 –2095. 3. Argento C, Cosentino MJ, Ganly M. Comparison of laser epithelial keratomileusis with and without the use of mitomycin C. J Refract Surg 2006;22(8):782–786.

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8. Kim TI, Pak JH, Lee SY, Tchah H. Mitomycin C-induced reduction of keratocytes and fibroblasts after photorefractive keratectomy. Invest Ophthalmol Vis Sci 2004;45(9): 2978 –2984. 9. Lai YH, Wang HZ, Lin CP, Chang SJ. Mitomycin C alters corneal stromal wound healing and corneal haze in rabbits after argon-fluoride excimer laser photorefractive keratectomy. J Ocul Pharmacol Ther 2004;20(2):129 –138. 10. Netto MV, Mohan RR, Sinha S, Sharma A, Gupta PC, Wilson SE. Effect of prophylactic and therapeutic mitomycin C on corneal apoptosis, cellular proliferation, haze, and long-term keratocyte density in rabbits. J Refract Surg 2006;22(6):562–574. 11. Xu H, Liu S, Xia X, Huang P, Wang P, Wu X. Mitomycin C reduces haze formation in rabbits after excimer laser photorefractive keratectomy. J Refract Surg 2001;17(3):342–349. 12. Dupps WJ, Wilson SE. Biomechanics and wound healing in the cornea. Exp Eye Res 2006;83(4):709 –720. 13. Netto MV, Mohan RR, Ambrosio R, Hutcheon AE, Zieske JD, Wilson SE. Wound healing in the cornea. A review of refractive surgery complications and new prospects for therapy. Cornea 2005;24(5):509 –522. 14. 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(10):1741– 1747. 15. Midena E, Gambato C, Miotto S, Cortese M, Salvi R, Ghirlando A. Long-term effects on corneal keratocytes of mitomycin C during photorefractive keratectomy: a randomized contralateral eye confocal microscopy study. J Refract Surg 2007;23(9 Suppl):S1011–S1014. 16. Weibel ER. Stereological Methods. Practical Methods for Biological Morphometry. Vol 1. London: Academic Press, 1979. 17. McLaren JW, Nau CB, Kitzmann AS, Bourne WM. Keratocyte density. Comparison of two confocal microscopes. Eye Contact Lens 2005;31(1):28 –33. 18. McLaren JW, Bourne WM, Patel SV. Automated assessment of keratocyte density in stromal images from the Confoscan 4 confocal microscope. Invest Ophthalmol Vis Sci 2010; 51(4):1918 –1926. 19. Patel DV, McGhee CN. Contemporary in vivo confocal microscopy of the living human corneal using white light and laser scanning techniques: a major review. Clin Exp Ophthalm 2007;35(1):71– 88. 20. 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(9):1789 –1801. 21. Dawson DG, Edelhauser HF, Grossniklaus HE. Long-term histopathologic findings in human corneal wounds after refractive surgical procedures. Am J Ophthalmol 2005; 139(1):168 –178. 22. Frueh BE, Cadez R, Böhnke M. In vivo confocal microscopy after photorefractive keratectomy in humans. A prospective, long-term study. Arch Ophthalmol 1998;116(11):1425–1431. 23. Lee SJ, Kim JK, Seo KY, Kim EK, Lee JK. Comparison of corneal nerve regeneration and sensitivity between LASIK and laser epithelial keratomileusis (LASEK). Am J Ophthalmol 2006;141(6):1009 –1015.

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24. Moller-Pedersen T, Cavanagh HD, Petroll WM, Jester JV. Stromal wound healing explains refractive instability and haze development after photorefractive keratectomy. A 1-year confocal microscopic study. Ophthalmology 2000; 107(7):1235–1245. 25. Erie JC, Patel SV, McLaren JW, Hodge DO, Bourne WM. Corneal keratocyte deficits after photorefractive keratectomy and laser in situ keratomileusis. Am J Opththalmol 2006; 141(5):799 – 809. 26. Herrman WA, Muecke M, Koller M, Gabel VP, Lohmann CP. Keratocyte density in the retroablation area after LASEK for the correction of myopia. Graefes Arch Clin Exp Ophthalmol 2007;245(3):426 – 430. 27. Nakayasu K. Stromal changes following removal of epithelium in rat cornea. Jpn J Ophthalmol 1988;32(2):113–125. 28. Dohlman CH, Gasset AR, Rose J. The effect of the absence of corneal epithelium or endothelium on stromal keratocytes. Invest Ophthalmol Vis Sci 1968;7(5):520 –534. 29. Campos M, Szerenyi K, Lee M, McDonnell JM, McDonnell PJ. Keratocyte loss after corneal deepithelialization in primates and rabbits. Arch Ophthalmol 1994;112(2):254 –260. 30. Wilson SE, He Y-G, Weng J, et al. Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp Eye Res 1996;62(4):325–338. 31. Helena MC, Baerveldt F, Kim WJ, Wilson SE. Keratocyte apoptosis after corneal surgery. Invest Ophthalmol Vis Sci 1998;39(2):276 –283. 32. Campos M, Raman S, Lee M, McDonnell PJ. Keratocyte loss after different methods of de-epithelialization. Ophthalmology 1994;101(5):890 – 894. 33. Wilson SE, Kim WJ. Keratocyte apoptosis: implications on corneal wound healing, tissue organization, and disease. Invest Ophthalmol Vis Sci 1998;39(2):220 –226. 34. Moller-Pedersen T, Li HF, Petroll WM, Cavanagh HD, Jester JV. Confocal microscopic characterization of wound repair after photorefractive keratectomy. Invest Ophthalm Vis Sci 1998;39(3):487–501. 35. Chew SJ, Beuerman RW, Kaufman HE. Real-time confocal microscopy of keratocyte activity in wound healing after cryoablation in rabbit corneas. Scanning 1994;16(5):269 – 274. 36. Pisella PJ, Auzerie O, Bokobza Y, Debbasch C, Baudouin C. Evaluation of corneal stromal changes in vivo after laser in situ keratomileusis with confocal microscopy. Ophthalmology 2001;108(10):1744 –1750. 37. Perez-Gomez I, Efron N. Change to corneal morphology after refractive surgery (myopic laser in situ keratomileusis) as viewed with a confocal microscope. Optom Vis Sci 2003; 80(10):690 – 697. 38. Watsky M. Keratocyte gap junctional communication in normal and wounded rabbit corneas and human corneas. Invest Ophthalmol Vis Sci 1995;36(13):2568 –2576. 39. Müller LJ, Pels L, Vrensen GF. Novel aspects of the ultrastructural organization of human corneal keratocytes. Invest Ophthalmol Vis Sci 1995;36(13):2557–2567. 40. Poole CA, Brookes NH, Clover GM. Confocal imaging of the human keratocyte network using the vital dye 5-chloromethylfluorescein diacetate. Clin Experiment Ophthalmol 2003;31(2):147–154. OF

OPHTHALMOLOGY

NOVEMBER 2010

41. Ebihara N, Yamagami S, Yokoo S, Amano S, Murakami A. Involvement of C-C chemokine ligan 2-CCR2 interaction in monocyte-lineage cell recruitment of normal human corneal stroma. J Immunol 2007;178(5):3288 –3292. 42. Shimura S, Kawakita T. Accessory cell populations in the cornea. Ocul Surf 2006;4(2):74 – 80. 43. West-Mays JA, Dwivedi DJ. The keratocyte: corneal stromal cell with variable repair phenotypes. Int J Biochem Cell Biol 2006;38(10):1625–1631. 44. Sosnová M, Bradl M, Forrester JV. CD34⫹ corneal stromal cells are bone marrow-derived and express hemopoietic stem cell markers. Stem Cells 2005;23(4):507–515. 45. Wilson SE, Mohan RR, Netto M, et al. RANK, RANKL, OPG, and M-CSF expression in stromal cells during corneal

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wound healing. Invest Ophthalmol Vis Sci 2004;45(7): 2201–2211. 46. Hori J, Streilein JW. Dynamics of donor cell persistence and recipient cell replacement in orthotopic corneal allografts in mice. Invest Ophthalmol Vis Sci 2001;42(8):1820 –1828. 47. Qazi MA, Johnson TW, Pepose JS. Development of late-onset subepithelial corneal haze after laser-assisted subepithelial keratectomy with prophylactic intraoperative mitomycin-C. Case report and literature review. J Cataract Refract Surg 2006;32(9): 1573–1578. 48. 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(1): 21–31.

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Biosketch Laura de Benito-Llopis, MD, graduated summa cum laude in 2000 at Valencia University School of Medicine, Spain. She did her Ophthalmology residency at the Gregorio Marañón Hospital (Madrid, Spain). She obtained her PhD in laser surface ablation surgery with cum laude at the Universidad Complutense, Madrid, and worked as a consultant at the Cornea and Refractive Surgery department at Vissum Madrid. She is currently doing a Cornea fellowship at Moorfields Eye Hospital, London.

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