1. University of Ulsan College of Medicine
Using lower raster energy to make a corneal flap with a 60 KHz IntraLase FS laser increases flap inflammation, but strengthens flap adhesion
Jae Yong Kim
Kyoung-Hoon Kim
Hee-Kyung Lee
Hungwon Tchah
Asan Medical Center
University of Ulsan College of Medicine
The authors have no financial disclosure regarding this subject.

2. INTRODUCTION
Kim et al. reported that a 15 kHz femtosecond (FS) laser created a stronger flap than a mechanical microkeratome because it induced more inflammation in the flaps.1
After the introduction of 60 kHz FS laser, Netto et al. noted that postoperative flap edema and diffuse lamellar keratitis in the eyes of patients who underwent LASIK with the 60 kHz lasers were markedly reduced compared with those with 15 kHz lasers.2

3. PURPOSE
We aimed to compare the early inflammatory cell infiltration and adhesion strength of rabbit corneal flaps made with a 60 KHz FS laser using different raster energy levels.

4. METHODS
90 New Zealand white rabbits (male, 2.5kg) were used to make a corneal flap with a 60 KHz FS laser (IntraLase Corporation, Irvine, CA, USA).
Initial settings were a planned flap of 9.0mm, a planned flap thickness of 110 ㎛, a beam separation of 8 X 8 ㎛ and side cut energy of 1.1 μJ.
They were divided according to the used raster energy: 0.7 μJ in group I (n=30), 1.1 μJ in group 2 (n=30), and 2.4 μJ in group 3 (n=30).
Experiment I: no flap lifting (NLF) - 10 subject per group for H&E and TUNEL at 4 h and 24h after creating a flap.
Experiment II: flap lifting (FL) was performed -20 subject per group for H&E and TUNEL at 4 h and 24h and adhesion strength at 1 m and 3 m after creating a flap.

5. METHODS Measurement of flap inflammation
The rabbits were sacrificed at 4 h and 24 h after making a flap. Eyes were immediately enucleated and fixed in 4% paraformaldehyde for 24 h.
The corneas were excised, bisected, and embedded in paraffin wax and sectioned at 5 ㎛.
After H&E staining, the number of inflammatory cells in the corneal stroma around the flap was counted in three fields of three tissue slides on each specimen by a single observer.
Kruskal-Wallis test was performed to compare the difference among groups

6. METHODS Measurement of flap adhesion
Adhesion strength was measured with a tension meter 1 m and 3 m after creating a flap.
We attempted to find the edge of the flap and separate at least a portion of it so as to allow it to be gripped by a curved mosquito clamp for 6-0 prolene loop suturing under microscopy.
A tension meter (Attonic, Aichi, Japan) was used to measure the adhesion strength and flap movement was observed under microscopy.

7. RESULTS
Figure 1. Quantitation of inflammatory cells in the central cornea and TUNEL-positive cells in the central cornea at 4 hours with (FL) or without flap lifting (NFL). In FL experiment, a lower energy flap creation had more inflammation, but not in NFL experiment. TUNEL-positive cells were decreased in FL experiment, but were increased in NFL experiment, as the raster energy was increased. ***, **, * Significant differences between those groups at a particular raster energy (P < 0.05).

8. RESULTS
Figure 2. Quantitation of inflammatory cells in the central cornea and TUNEL-positive cells in the central cornea at 24 hours with (FL) or without flap lifting (NFL). In FL experiment, a lower energy flap creation had more inflammation, but not in NFL experiment. TUNEL-positive cells were decreased in FL experiment, but were increased in NFL experiment, as the raster energy was increased. ***, **, * Significant differences between those groups at a particular raster energy (P < 0.05).

9. RESULTS
Figure 3. The H&E staining of central cornea 4h and 24h after making a flap in flap lifting (FL) experiment. the inflammatory cell infiltration of central cornea (black arrow) was found at high power field (X400) under light microscopy

10. Figure 4. TUNEL assay at 4 and 24 hours after making a flap in flap lifting (FL) experiment. The TUNEL-positive cells (red arrow) were scattered in the area anterior and posterior to the lamellar interface created by a FS laser.

11. RESULTS
Figure 5. Measurements of adhesion strength. Adhesion in the 2.4uJ group was significantly stronger than that in the 0.7uJ group at 3 months (P < 0.05). **, * Significant differences between those groups at a raster energy.

12. CONCLUSION
Using lower raster energy to make a corneal flap with a 60 KHz IntraLase FS laser increased flap inflammation only after flap lifting, but decreased without flap lifting.
Flap lifting might have significant effects on flap inflammation and flap apoptosis.
Using lower raster energy may strengthen the flap adhesion only within one month after a flap creation.
Conversely, higher raster energy can increase the adhesion strength at three months.
Further study was needed to understand the wound healing process after a flap creation with 60 kHz FS laser.