Purpose
To determine the effects of keratocyte loss on optical properties and vision after laser in situ keratomileusis (LASIK) with the flap created with a femtosecond laser or a mechanical microkeratome.
Design
Randomized clinical paired-eye study.
Methods
Both eyes of 21 patients received LASIK for myopia or myopic astigmatism. One eye of each patient was randomized by ocular dominance to flap creation with a femtosecond laser and the other eye to flap creation with a mechanical microkeratome. Before LASIK and at 1, 3, and 6 months and 1, 3, and 5 years after LASIK, keratocyte density was measured using confocal microscopy, and high-contrast visual acuity and anterior corneal wavefront aberrations were measured by standard methods. At each visit, all variables were compared between methods of creating the flap and to the same variable before treatment using paired tests with Bonferroni correction for multiple comparisons.
Results
Keratocyte density in the flap decreased by 20% during the first year after LASIK and remained low through 5 years ( P < .001). High-order wavefront aberrations increased and uncorrected visual acuity improved immediately after surgery, but these variables did not change further to 5 years. There were no differences in any variables between treatments.
Conclusions
A sustained reduction in keratocyte density does not affect vision or optical properties of the cornea through 5 years after LASIK. The method of creating a LASIK flap does not influence the changes in keratocyte density in the flap.
In the first year after laser in situ keratomileusis (LASIK), cell density in the stromal portion of the flap decreases considerably and does not return to presurgical densities after at least 5 years. Most of these stromal cells are keratocytes, and normally the density of these cells is highest in the anterior-most layer of the sub-basal stroma. Keratocytes maintain the health of the stroma, and although the purpose of their high concentration in the anterior region is not fully understood, their demise could lead to an eventual degradation of stromal tissue that might increase corneal haze or progressively increase anterior corneal aberrations. In spite of the known chronic changes in cell density, in clinical practice there appears to be little impact of ketatocyte loss on structural or visual function of corneas after LASIK, although delayed manifestations of these changes are unknown.
If anterior stromal cell loss is a consequence of cutting the corneal stroma, then it is also possible that cell loss could vary with the method of creating the LASIK flap. Netto and associates found that after cutting a flap with the microkeratome, cells were primarily degraded by apoptosis, while after creating incisions with a 30 kHz femtosecond laser cells were lost by necrosis, which has more expanded effects on surrounding tissue. If the hypothesis that decreased keratocyte density restricts the ability of the cornea to maintain ideal structural and optical properties is true, then optical properties of the cornea, such as surface irregularities or light scattered from the stroma, should change as cell density decreases. If methods of cutting the flap differentially affect the function of keratocytes, then these properties should also depend on the method used to cut the flap. Differences could appear sooner or later, depending on how these treatments affect the function of the keratocytes and the role that keratocytes play in maintaining these features of the cornea.
In this randomized paired-eye study, we compared cell loss between the methods of flap creation and investigated the effect of decreased cell density in the stromal flap on visual and optical properties after LASIK with the flap created either using a femtosecond laser or using a mechanical microkeratome. We previously reported comparisons of visual acuity and aberrations between flap creation methods through 3 years, and here we extend these measurements through 5 years. In addition, we include measurements of forward scatter and backscatter from 1 year through 5 years, and examine possible relationships with stromal cell density.
Methods
Participants and Randomization
This study explored the relationships between stromal cell loss and visual and optical properties for 5 years after LASIK with the flap cut by femtosecond laser or mechanical microkeratome, in a randomized paired-eye design. Twenty-one patients eligible for LASIK to treat myopia or myopic astigmatism were recruited from the refractive surgery service at Mayo Clinic, Rochester, Minnesota, between August 16, 2004 and January 10, 2005. This group has been described in detail previously. Patients were excluded if they had corneal abnormalities; had a history of ocular disease, trauma, surgery, diabetes mellitus, or other systemic diseases that affect the eye; or used any ocular or systemic medications that affect the cornea or anterior segment. The study was reviewed and approved prospectively by the Mayo Clinic Institutional Review Board and conformed to the Declaration of Helsinki. All patients provided written informed consent to participate after review and discussion of the nature and possible consequences of the study. The trial was registered at www.clinicaltrials.gov , # NCT00350246 .
Each patient was randomized to treatment with the flap created using a femtosecond laser in either the dominant eye or the nondominant eye and with the flap created using a mechanical microkeratome in the fellow eye. An equal number of patients had the femtosecond laser incision in the dominant eye as had it in the nondominant eye. Ocular dominance was determined by observing the eye used by the patient to view a distant object through a frame made with the forefinger and thumb of both hands. Patients were examined before LASIK and at 1, 3, and 6 months and 1, 3, and 5 years after LASIK.
Twenty participants with normal eyes were also prospectively examined as a control group. These subjects met the same inclusion criteria as the LASIK patients except that they did not have LASIK. One eye of each participant was randomly selected to compare with the eye treated with the femtosecond laser in the LASIK group (femtosecond laser control eye) and the other eye was compared with the mechanical microkeratome eye (mechanical microkeratome control eye). Participants were examined at an initial visit and 1 month and 1, 3, and 5 years after the initial visit. At each visit, the same variables were measured in both eyes of the control group as were measured in eyes in the LASIK group.
Surgical Treatment
The surgical procedures for LASIK have been described. Briefly, bladeless flaps were created using a 15 kHz femtosecond laser (Intralase FS; Intralase Corp, Irvine, Calilfornia, USA). All flaps had an intended thickness of 120 μm and superior hinge. The energy of each laser pulse was 2.3 μJ in the raster cut and 2.5 μJ in the side cuts. Raster line and spot separation were 9 and 11 μm, respectively. In the contralateral eye, the flap was created by a mechanical microkeratome (Hansatome; Bausch & Lomb, Rochester, New York) with an intended thickness of 180 μm and superior hinge. All eyes received a non-wavefront-guided stromal ablation with an excimer laser (Star S4, Visx; Abbott Medical Optics, Abbott Park, Illinois) to a depth required by the refractive correction.
Emmetropia was attempted in all eyes using an ablation zone from 6.5 mm × 6.5 mm for spherical correction to 6.5 mm × 5.0 mm for spherocylindrical correction. Both eyes of all patients had the same postoperative treatment: ciprofloxacin ophthalmic solution 4 times per day for 5 days and fluorometholone 0.1% 4–8 times per day with a taper over 3 weeks.
Keratocyte Density
At each visit, both corneas of all participants were examined by clinical confocal microscopy with a Tandem Scanning confocal microscope (Tandem Scanning, Reston, Virginia, USA), as described previously. From images of the full-thickness cornea, 2 frames were selected from each of 6 layers of stroma, the anterior and posterior halves of the flap, the anterior and posterior halves of the stroma between the interface and 100 μm deep to the interface (retroablation zone), 66%–90% of stromal depth, and the posterior 10% of stroma. Cell nuclei were identified and counted in each selected frame using an automated program and cell density was expressed as volumetric density (cells/mm 3 ).
Assessment of Visual Acuity and High-Order Aberrations
High-order aberrations and visual acuity had been measured through 36 months after surgery, as reported earlier. In this study, corneal topography in all subjects was reexamined by using a Humphrey Atlas corneal topography system (Humphrey Systems, Pleasanton, California, USA) at 60 months. Wavefront aberrations were expressed as Zernike polynomials (VOLCT; Sarver and Associates, Carbondale, Illinois) and the root mean square (RMS) wavefront error from Zernike orders 3–6, as well as spherical aberration, coma, and trefoil, were calculated as described by Calvo and associates. High-contrast best spectacle-corrected visual acuity (BCVA) and uncorrected visual acuity (UCVA) were determined by the electronic Early Treatment of Diabetic Retinopathy Study (ETDRS) protocol. Visual acuity was recorded as letter scores and converted to logarithm of the minimal angle of resolution (logMAR).
Backscatter and Straylight
The original study design did not include estimates of backscatter or straylight (forward scatter) because we did not have the instruments to make these measurements at inception. During the first year of the study we obtained a ConfoScan 4 confocal microscope (Nidek Technologies, Inc, Fremont, California, USA), which allowed us to record confocal image brightness as a measure of corneal backscatter. We also obtained a C-Quant straylight meter (Oculus, Lynnwood, Washington, USA), which allowed us to estimate forward scatter. We report these data at 12, 36, and 60 months.
The cornea was scanned through its full depth using the ConfoScan 4 clinical confocal microscope equipped with a z-ring adapter to stabilize the eye and report depth in the cornea of each frame. Backscatter was the mean brightness in the center of the confocal images standardized to a scatter standard (Amco Clear; GSF Chemicals, Columbus, Ohio, USA), as described previously. Depth in the cornea was expressed as distance from the stromal surface scaled as a percentage of stromal thickness. After LASIK, depth was also scaled so the surgical interface appeared at the mean interface depth, expressed as a percent of stromal thickness.
Retinal straylight (forward scatter) was estimated using the compensation comparison method implemented with the straylight meter, as described in detail elsewhere. Forward light scatter was expressed as the log of the straylight parameter determined after each subject completed a 2-alternative forced-choice protocol that was under computer control.
Statistical Analysis
Differences between treatments and differences between preoperative and postoperative variables within treatments at each visit were examined using 2-tailed paired t tests if the data were distributed normally and Wilcoxon signed rank tests if they were not. Variables in the LASIK patients were compared to those in the control subjects using nonpaired tests. P values were adjusted for multiple comparisons using the Bonferroni method; P < .05 was considered significant.
Results
Keratocyte Density, Laser In Situ Keratomileusis Patients
Before LASIK, cell densities were not different between eyes destined for flap creation by the femtosecond laser or by the mechanical microkeratome ( P > .9; Bonferroni-adjusted for 7 comparisons, Figures 1 and 2 , Table 1 ). During the first year after LASIK, cell density in the flap gradually decreased after both procedures ( P < .032, Bonferroni-adjusted for 6 comparisons). There were no other consistent significant changes in cell density in layers deep to the surgical interface. Cell density in the anterior stroma did not recover, but remained low after both treatments to 5 years ( P < .001). Cell densities in the flap were generally lower than cell densities in analogous regions of the anterior stroma in the control subjects ( Table 1 ).
| Visit | LASIK Patients | Control | ||
|---|---|---|---|---|
| Mechanical | Femtosecond | Randomized to Mechanical | Randomized to Femtosecond | |
| Pre-LASIK | 47 147 ± 6071 | 46 093 ± 6038 | 43 348 ± 7124 | 45 054 ± 9332 |
| 1 month | 42 557 ± 7277 | 44 709 ± 5488 | 43 463 ± 8271 | 45 515 ± 7935 |
| 3 months | 41 108 ± 6823 b | 41 349 ± 7092 | – | – |
| 6 months | 41 349 ± 7286 a | 41 305 ± 7026 a | – | – |
| 1 year | 41 393 ± 5970 b | 40 295 ± 4905 b , d | 40 848 ± 8334 | 44 998 ± 5507 d |
| 3 years | 37 407 ± 5022 c , e | 35 406 ± 7457 c , e | 46 022 ± 9473 e | 45 054 ± 8192 e |
| 5 years | 36 105 ± 7897 c | 36 635 ± 5017 c , e | 43 194 ± 7667 | 42 694 ± 5811 e |
a P < .05, compared to pre-LASIK.
b P < .04, compared to pre-LASIK.
c P < .001, compared to pre-LASIK.
d P < .04, compared to control.
Cell density was not different between treatments at any time in any region ( P > .059). The average minimum detectable difference in the flap was 6400 cells/mm 3 and in layers deep to the interface was 4540 cells/mm 3 (α = 0.05, β = 0.20, Bonferroni adjusted for 7 comparisons).
Keratocyte Density, Control Subjects
In control subjects, there were no consistent changes in cell densities in all 6 layers through 5 years ( Figures 1 and 2 ). There were no differences in keratocyte density between corneas randomized for comparison to the femtosecond laser–created flaps and the fellow eyes ( P > .19).
Visual Acuity and Refractive Error
Before LASIK and at 1 month and 5 years, mean UCVA was not different between treatments ( P > .34, Bonferroni-adjusted for 3 comparisons); the minimum detectable difference was 0.10 logMAR, or 5 ETDRS letters (α = 0.05, β = 0.20). At 5 years, mean uncorrected visual acuity was 0.01 ± 0.13 logMAR and 0.04 ± 0.15 logMAR in the eyes treated with the femtosecond laser and mechanical microkeratome, respectively ( P = .30). These were not different from the visual acuity at 1 month of 0.01 ± 0.14 logMAR and 0.00 ± 0.13 logMAR after these treatments, respectively ( P > .43).
Mean spherical equivalent before and at 1 month and 5 years after LASIK were not different between treatments ( P > .6, Bonferroni-adjusted for 3 comparisons); the minimum detectable difference was 0.50 diopter (D) before LASIK and 0.25 D after LASIK (α = 0.05, β = 0.20). At 5 years mean spherical equivalent was −0.18 ± 0.37 D and −0.15 ± 0.29 D in eyes treated with the femtosecond laser and mechanical microkeratome, respectively ( P = .65). These had not changed from −0.09 ± 0.28 D and −0.17 ± 0.27 D at 1 month after these treatments, respectively ( P > .30). Best-corrected visual acuity before and at 1 month and 5 years was not different between treatments ( P > .13), and the minimum detectable difference was 0.06 logMAR, or 3 ETDRS letters. At 5 years, BCVA was −0.08 ± 0.06 logMAR and −0.04 ± 0.08 logMAR in the eyes treated with the femtosecond laser and mechanical microkeratome, respectively, and was not different from BCVA of −0.05 ± 0.08 logMAR and −0.05 ± 0.09 logMAR after these treatments, respectively, at 1 month ( P > .09). All measurements other than at 5 years were from Calvo and associates.
High-Order Aberrations
Before LASIK and at 1 month and 5 years, mean total high-order aberrations, spherical aberrations, coma, and trefoil were not different between treatments ( P > .12) at any pupil diameter, except that coma was slightly higher at 1 month after the femtosecond laser treatment at a 5 mm pupil ( P = .035; Bonferroni-adjusted for 3 comparisons, Table 2 ). At 5 years these aberrations were not different from high-order aberrations at 1 month at any pupil diameter ( P > .08), except that in the mechanical microkeratome group spherical aberration increased slightly across a 5 mm pupil ( P = .044) and 6 mm pupil ( P = .008, Table 2 ). These changes were not considered to be clinically important.
| All High | Sphere | Coma | Trefoil | |||||
|---|---|---|---|---|---|---|---|---|
| Mechanical | Femtosecond | Mechanical | Femtosecond | Mechanical | Femtosecond | Mechanical | Femtosecond | |
| 4 mm pupil | ||||||||
| Pre-LASIK | 0.13 ± 0.04 | 0.14 ± 0.07 | 0.04 ± 0.02 | 0.04 ± 0.02 | 0.06 ± 0.03 | 0.07 ± 0.06 | 0.06 ± 0.03 | 0.06 ± 0.05 |
| 1 month | 0.17 ± 0.04 c | 0.19 ± 0.06 a | 0.06 ± 0.03 | 0.07 ± 0.04 a | 0.08 ± 0.04 a | 0.11 ± 0.06 | 0.08 ± 0.04 | 0.07 ± 0.04 |
| 5 years | 0.19 ± 0.05 c | 0.18 ± 0.05 | 0.06 ± 0.04 a | 0.08 ± 0.03 c | 0.10 ± 0.04 c | 0.11 ± 0.06 | 0.09 ± 0.05 a | 0.07 ± 0.04 |
| 5 mm pupil | ||||||||
| Pre-LASIK | 0.26 ± 0.09 | 0.26 ± 0.09 | 0.12 ± 0.05 | 0.12 ± 0.04 | 0.14 ± 0.10 | 0.14 ± 0.11 | 0.11 ± 0.06 | 0.10 ± 0.06 |
| 1 month | 0.33 ± 0.09 b | 0.38 ± 0.11 c | 0.16 ± 0.06 b | 0.19 ± 0.06 c | 0.18 ± 0.11 f , a | 0.23 ± 0.12 f , a | 0.13 ± 0.06 | 0.12 ± 0.10 |
| 5 years | 0.37 ± 0.13 b | 0.37 ± 0.09 b | 0.19 ± 0.08 d , b | 0.20 ± 0.07 b | 0.20 ± 0.11 a | 0.23 ± 0.12 a | 0.16 ± 0.11 | 0.12 ± 0.08 |
| 6 mm pupil | ||||||||
| Pre-LASIK | 0.47 ± 0.22 | 0.46 ± 0.18 | 0.26 ± 0.09 | 0.26 ± 0.09 | 0.27 ± 0.23 | 0.26 ± 0.20 | 0.18 ± 0.10 | 0.16 ± 0.09 |
| 1 month | 0.66 ± 0.21 c | 0.76 ± 0.25 c | 0.40 ± 0.15 c | 0.43 ± 0.16 c | 0.37 ± 0.26 | 0.46 ± 0.26 b | 0.20 ± 0.10 | 0.22 ± 0.20 |
| 5 years | 0.70 ± 0.19 c | 0.75 ± 0.22 c | 0.44 ± 0.15 e , c | 0.46 ± 0.13 c | 0.35 ± 0.22 | 0.44 ± 0.26 a | 0.23 ± 0.16 | 0.21 ± 0.17 |
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