Histopathology of Corneal Lenticules Obtained from Small Incision Lenticule Extraction (SMILE) versus Microkeratome Excision.

Abstract

Purpose: To study the alterations on the lenticules extracted after femtosecond (Femto) small incision lenticule extraction (SMILE) versus the corneal free cap removed using a microkeratome.

Methods: The visuMax (500 kHz; laser energy: 180 nJ) was used for small-incision lenticule extraction. Free caps from human cadaveric corneas were excised by microkeratome. The collected lenticules were examined with the light and transmission electron microscope (TEM) for histological analysis, DNA fragmentation was assessed by agarose gel electrophoresis, DNA damage was evaluated using comet assay, and corneal proteins secondary structure was assessed by Fourier transform infrared spectroscopy (FTIR).

Results: Light microscopic examination showed the presence of more edematous stroma under Femto SMILE than under free cap with a percentage change of 101.6%. In the Femto SMILE group, TEM examination showed pyknotic keratocytes, disruption, and cavitation of the collagen arrays stromal area under Femto SMILE. The DNA fragmentation for the Femto SMILE group revealed one undefined band with a size of 1.1 Kbp. The comet assay analysis indicated the presence of 3% and 8.0% tailed cells for the free cap and Femto SMILE groups, respectively. The tail lengths were 1.33 $\pm$ 0.16 and 1.67 $\pm$ 0.13 µm (P $<$ 0.01), the percentage of tail DNA was 1.41 $\pm$ 0.18% (P $<$ 0.01) and 1.72 $\pm$ 0.15%, and the tail moments were 1.88 $\pm$ 0.12 AU and 2.87 $\pm$ 0.14 AU (P $<$ 0.001) for the free cap and Femto SMILE groups, respectively. FTIR spectroscopy of the Femto smile group revealed disorders in the secondary and tertiary structure of the proteins.

Conclusion: Femto SMILE technique induced more structural changes, DNA fragmentation, DNA damage, and corneal proteins secondary structure alteration than those induced by a microkeratome cutting. These changes may be attributed to the deep penetration of high energy levels to the corneal layer. These findings may highlight the potential impact of the Femto SMILE on the cornea and the necessity for managing the laser parameters used.