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Laboratory science

Epi-LASIK using the Amadeus II microkeratome

Evaluation of cut quality using light and electron microscopy

Kollias, Aris MD; Ulbig, Michael W. MD; Spitzlberger, Georg M. MD; Abdallat, Wajih H. MD; Froehlich, Stephan MD; Welge-Luessen, Ulrich MD; Lackerbauer, Carl-Arnold MD

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Journal of Cataract & Refractive Surgery: December 2007 - Volume 33 - Issue 12 - p 2118-2121
doi: 10.1016/j.jcrs.2007.07.038
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Abstract

Epithelial laser in situ keratomileusis (epi-LASIK), first introduced by Pallikaris et al.1 in 2003, is a new surface ablation procedure for the treatment of low to moderate ametropia. In this method, the epithelium is separated by mechanical means without previous preparation of the cornea with alcohol. A motor-driven device uses a blunt oscillating polymethylmethacrylate block to separate the epithelium from Bowman's membrane. After the underlying bed is photoablated, the epithelial flap is replaced.

Various commercial microkeratomes with a handpiece for use in epi-LASIK procedures are now available. The quality of the cleavage plane and epithelial edge has a major impact on the accuracy of corneal ablation and wound healing. The present study used light and scanning electron microscopy to investigate the quality and surface characteristics of the epithelial flap and underlying bed created by the Amadeus II microkeratome (Advanced Medical Optics) in human donor corneas.

MATERIALS AND METHODS

In this study, the Amadeus II fitted with the optional epi-LASIK unit was used. A 9.0 mm type II suction ring was chosen to perform both procedures. The following setting, as recommended by the manufacturer, was used: blade oscillating frequency, 11000 rpm; blade advantage rate, 1.5 mm/sec; hinge width, 1.0 mm; vacuum, 560 mm Hg.

The study was conducted on 2 fresh human eyes from 1 donor. The globes were obtained from the eye bank of the Department of Ophthalmology, Ludwig-Maximilians University, Munich, Germany. Approval of the ethic committee was granted. Ocular pathology and any previous ocular surgery were ruled out. Keratometric readings determined by Javal keratometry were 43.0 diopters (D) and 43.6 D. Not more than 12 hours elapsed from time of death to the cutting trial. After removal, the globes were stored in a balanced salt solution at 4°C. Before the cutting procedure, the globes were kept at room temperature for approximately 2 hours. They were positioned in a head form under an operating microscope for the procedure. After the suction unit was placed and the vacuum reached, the correct vacuum level of 560 mm Hg was confirmed with a Barraquer tonometer. Shortly before the cut was made, lubrication was applied on the applanation area of the blade holder and the cornea. After the procedure, the corneas were cut out with the attached epithelial flap. Tissues for light microscopy were fixed in formalin and processed for hematoxylin–eosin and periodic acid-Schiff reaction staining. Tissues for electron microscopy were placed in glutaraldehyde 2.5% buffered in 0.1 phosphate-buffered saline, and fixed. Tissues for scanning electron microscopy were prepared by dehydration in alcohol, critical point drying, and sputter coating. A JEOL-JSM-35CF electron microscope (JEOL) was used to examine the specimen. Tissues for transmission electron microscopy were osmicated, dehydrated in alcohol, and embedded in Epon. Ultrathin cuts of 60 to 70 nm were made and examined with a Zeiss S9 electron microscope (Carl Zeiss). Images of the central cornea as well as the superior, inferior, nasal, and temporal areas were evaluated at different magnifications.

RESULTS

Light Microscopy

Examination of all specimens showed a thoroughly separated epithelial sheet with no evident anatomical abnormalities. The cutting edge was well defined and smooth in all examined samples. Stratification of the separated epithelium and cell shape was conserved. There was minimum evidence of cell edema in isolated basal cells. The cleavage plane was located at Bowman's membrane, which had a smooth surface. There were no signs of Bowman's layer or corneal stroma in the epithelial flap (Figure 1).

Figure 1
Figure 1:
Light microscopy. Complete separation of the undamaged epithelium from Bowman's layer is achieved (original magnification ×20).

Scanning Electron Microscopy

Scanning electron microscopy showed an even cutting edge in all specimens and a very consistent transition from adherent epithelium to the denuded area (Figure 2). Bowman's layer showed a smooth surface without remains of basal lamina or basal cells (Figure 3). The bottom side of the epithelial disk had an equally even surface.

Figure 2
Figure 2:
Scanning electron microscopy. A: Low-power view of the temporal half of the handled cornea. A very consistent separation edge and a smooth surface are present. The particles on the surface are artifacts due to fixation (original magnification ×40). B and C: Higher magnification (original magnification ×400 and ×1000) shows a clear transition from adherent epithelium (E) to Bowman's layer (Bo).
Figure 3
Figure 3:
Scanning electron microscopy. High-power view of the central cornea. Bowman's layer shows a very smooth surface (original magnification ×1000).

Transmission Electron Microscopy

Examination of the epithelial disk showed a fairly even surface. The basal cells showed no signs of damage, and the surface of Bowman's layer had minor superficial irregularities. At high magnification, interruptions of the basement membrane were found in all specimens (Figure 4). The cleavage plane was posterior to the basal lamina and Bowman's layer.

Figure 4
Figure 4:
Transmission electron microscopy. A: The basal cell layer and the intercellular contacts display a normal morphology (original magnification ×1800). B: Higher magnification of a segment of A shows that the basement membrane (arrowheads) is discontinuous (original magnification ×9500).

DISCUSSION

Preliminary reports2 of epi-LASIK, a new treatment option in surface ablation, show very satisfying results. The technique has an advantage over laser-assisted subepithelial keratectomy (LASEK) in that previous preparation of the cornea with alcohol is not necessary. Alcohol has a dose-dependent toxic effect on corneal and surrounding tissue and impairs wound healing3 as well as the laser ablation rate. Since epi-LASIK is automated, results should be of constant good quality and reproducible. In addition, the technique does not require entirely new equipment except a new handpiece. For surgeons who are familiar with LASIK, epi-LASIK is easy to perform.

The state of the separated epithelial flap, underlying Bowman's layer, and separation margin has a major impact on the accuracy of the intended ablation, wound healing, and the refractive outcome. The aim of this study was to examine the cutting quality of the epi-LASIK unit of the Amadeus II in human corneas. To our knowledge, this is the first study to evaluate corneal surface characteristics using this type of microkeratome.

In our study, the separation of the epithelium was uneventful in both cases. Light microscopy showed an intact epithelial flap that was separated at the level of Bowman's layer. Basal cells did not show any alterations except light edema in isolated cells. Transmission electron microscopy at high magnification showed interruptions of the basement membrane but intact basal cells. The observed positive impact of the epithelial flap in wound healing4–6 after photorefractive keratectomy is connected with the viability of the replaced tissue. The viability of cells cannot be assessed merely by morphology.7 Therefore, further studies, including viability staining, should be conducted to support the advantages of epi-LASIK over LASEK.8

The separation edge had a consistent transition without damage to the surrounding tissue. The cleavage plane located at Bowman's layer showed no residual cells or remains of basal lamina. Its surface was very smooth and well suited for the subsequent laser ablation. In conclusion, epi-LASIK using the Amadeus II microkeratome is a very promising technology for corneal surface ablation.

REFERENCES

1. Pallikaris IG, Katsanevaki VJ, Kalyvianaki MI. Naoumidi II. Advances in subepithelial excimer refractive surgery techniques: epi-LASIK. Curr Opin Ophthalmol. 2003;14:207-212.
2. Pallikaris IG, Kalyvianaki MI, Katsanevaki VJ, Ginis HS. Epi-LASIK: preliminary clinical results of an alternative surface ablation procedure. J Cataract Refract Surg. 2005;31:879-885.
3. Pallikaris IG, Naoumidi II, Kalyvianaki MI, Katsanevaki VJ. Epi-LASIK: comparative histological evaluation of mechanical and alcohol-assisted epithelial separation. J Cataract Refract Surg. 2003;29:1496-1501.
4. Azar DT, Ang RT, Lee J-B, et al. Laser subepithelial keratomileusis: electron microscopy and visual outcomes of flap photorefractive keratectomy. Curr Opin Ophthalmol. 2001;12:323-328.
5. Netto MV, Mohan RR, Ambrósio R Jr, et al. Wound healing in the cornea; a review of refractive surgery complications and new prospects for therapy. Cornea. 2005;24:509-522.
6. Shah S, Sebai Sarhan AR, Doyle SJ, et al. The epithelial flap for photorefractive keratectomy. Br J Ophthalmol. 2001;85:393-396.
7. Chen CC, Chang J-H, Lee JB, et al. Human corneal epithelial cell viability and morphology after dilute alcohol exposure. Invest Ophthalmol Vis Sci. 2002;43:2593-2602.
8. Katsanevaki VJ, Naoumidi II, Kalyvianaki MI, Pallikaris IG. Epi-LASIK: histological findings of separated epithelial sheets 24 hours after treatment. J Refract Surg. 2006;22:151-154.
© 2007 by Lippincott Williams & Wilkins, Inc.