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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 17  |  Issue : 3  |  Page : 109-113

Scanning electron microscopy of human corneal lenticules at variable corneal depths in small incision lenticule extraction cases


1 Department of Ophthalmology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
2 Department of Histology, Faculty of Medicine, Alexandria University, Alexandria, Egypt

Date of Submission01-Jun-2016
Date of Acceptance19-Jun-2016
Date of Web Publication6-Dec-2016

Correspondence Address:
Ihab M Osman
Department of Ophthalmology, Faculty of Medicine, Alexandria University, Alexandria 2667
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-9173.195261

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  Abstract 

Purpose
The aim of this study was to evaluate the surface quality of corneal lenticules at variable corneal depths in cases of small incision lenticule extraction using scanning electron microscopy.
Patients and methods
Forty eyes of 20 myopic patients were included. One eye was randomly assigned for lenticules at 100 µm depth (group I), whereas the other eye was assigned for lenticules at 160 µm depth (group II). The VisuMax femtosecond laser system was used for the procedure. An established scoring system and a new scoring system at higher magnification levels for surface regularity were used.
Results
The mean overall diameter of the lenticules as seen on electron microscopy was 6.54±0.17 and 6.73±0.20 mm in group I and group II, respectively (P=0.003). In both groups, around 50% of cases showed a smooth surface at ×10 magnification. The mean score was 14.90+1.74 and 13.25+2.77 in group I and group II, respectively (P=0.031).
Conclusion
The VisuMax femtosecond laser system creates predictable good-quality surface refractive corneal lenticules at superficial depth. Deeper corneal lenticules showed more irregular surfaces, especially at higher magnification levels. Energy settings still need further adjustment to be able to create deeper predictable lenticules in small incision lenticule extraction cases.

Keywords: corneal lenticules, electron microscopy, small incision lenticule extraction, variable depths


How to cite this article:
Osman IM, Madwar AY. Scanning electron microscopy of human corneal lenticules at variable corneal depths in small incision lenticule extraction cases. Delta J Ophthalmol 2016;17:109-13

How to cite this URL:
Osman IM, Madwar AY. Scanning electron microscopy of human corneal lenticules at variable corneal depths in small incision lenticule extraction cases. Delta J Ophthalmol [serial online] 2016 [cited 2017 Aug 19];17:109-13. Available from: http://www.djo.eg.net/text.asp?2016/17/3/109/195261


  Introduction Top


The femtosecond laser is an infrared, neodymium-doped yttrium aluminum garnet (Nd : YAG) laser that uses pulses of light at a very high rate to elicit a cleavage plane of tissues by creating small cavitation bubbles, thus making very accurate incisions with minimal tissue damage [1],[2].

The VisuMax femtosecond laser system was the first to develop an all-in-one laser procedure called the refractive lenticule extraction. Small incision lenticule extraction (SMILE) is the most recent type of refractive lenticule extraction surgery in which there is no flap creation; instead the femtosecond laser creates two photo disruption planes at predetermined depths from the corneal surface. The thickness between these two planes is the lenticule of the intended refractive error correction. The depth of the posterior plane determines the thickness and refractive power of the removed lenticule as the anterior plane is parallel to the corneal surface [3],[4]. The nature of the photo disruption bubbles created by the femtosecond laser mandates the presence of inter-bubble micro bridges. These bridges need to be dissected manually during the SMILE procedure to enable lenticule extraction [5],[6].

An important advantage of SMILE lies in the flapless nature of the procedure, preventing most flap-related complications and causing less postoperative dry eye. There is less inflammation at higher corrections of myopia as the same energy is used to create a lenticule irrespective of refractive error. There is also the added benefit of increased corneal biomechanical stability. As no flap is created in SMILE, the anterior stromal lamellae remain intact everywhere except at the small incision site. Therefore, the actual residual stromal thickness in SMILE is calculated as the stromal thickness below the posterior lenticule plus the stromal component of the cap [7],[8],[9],[10],[11].

The anterior 40% of the central corneal stroma was found to be the strongest region of the cornea, whereas the posterior 60% of the stroma was at least 50% weaker. This factor leads to increase in corneal biomechanical stability if SMILE is performed at deeper corneal levels, thus increasing the possibility to extend the range of myopia that can be corrected [8],[9],[10].

The aim of the current study was to evaluate the surface quality of human corneal lenticules at variable corneal depths in SMILE using scanning electron microscopy.


  Patients and methods Top


This is a prospective clinical study that included 40 eyes of 20 myopic patients. In all patients, one eye was randomly assigned for lenticules at 100 µm depth (group I), whereas the other eye was assigned for lenticules at 160 µm depth (group II). All included patients signed an informed consent form. This study was approved by the local research committee of the Faculty of Medicine, Alexandria University, Egypt.

A VisuMax femtosecond laser system (Carl Zeiss Meditec AG, Jena, Germany) was used for the SMILE procedure. In all patients, the same femtosecond laser parameters were used. The spot distance was 3 µm for lamellar cuts and 2 µm for side cuts. The spot energy was set to 130 nJ. The lenticule diameter was 6.5 mm, with minimum side cut thickness set to 10 µm. The cap diameter was set to 7.5 mm, with intended thickness randomly assigned either 100 or 160 µm. The lenticule side cut angle was 130° and the incision side cut angle was 70°. A small-sized cone was used in all patients. The scan mode was single with scan direction spiral-in for the lenticule and spiral-out for the cap, allowing the patient to fix at the flickering fixation target as long as possible.

The lenticules were fixed by immediate immersion in 4% formaldehyde and 1% gluteraldehyde (4F1G) with a pH of 7.2 at 4°C for 3 h. They were then postfixed in 2% osmium tetroxide (OsO4) at the same pH at 4°C for 2 h. They were washed in PBS and dehydrated at 4°C through a graded series of aqueous ethanol solution (20–100%). The lenticules were then dried by means of the critical point method using amyl acetate and carbon dioxide (CO2), mounted using carbon paste on an aluminum stub, and coated with gold up to a thickness of 400 Å in a sputter-coating unit (JFC-1100E ion sputtering device; Jeol USA Inc.). The lenticules were observed using a scanning electron microscope (JSM-5300 SEM; Jeol USA Inc.) operated between 15 and 20 KeV.

The scanning electron microscope pictures were examined by an expert at different magnification levels. The scoring system published by Wilhelm et al. [12] was used to quantify the surface irregularity of the lenticules. It allows a score range from 4 to 11 (the lower the score, the more the irregularity of the surface). [Table 1] shows the criteria of the scoring system used. A new scoring system was also used to quantify the surface irregularity of the lenticules at higher magnification levels. It allows a score range from 4 to 16 (the lower the score, the more the irregularity of the surface). It gives a score range from 0 to 4 for edge serrations and from 1 to 3 for the size of tissue fragments for each of the four quadrants separately ([Table 2]).
Table 1 The scoring system used to assess the surface regularity of the lenticules [12]

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Table 2 A new scoring system used for lenticule surface regularity assessment

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Clinical findings were statistically evaluated using Excel 2007 (Microsoft Corp.) and SPSS software, version 15.0 (SPSS Inc., Chicago, Illinois, USA). Mean and SDs were calculated. To check for normal distribution, the Kolmogorov–Smirnov test was applied. Comparisons of the means of normally distributed data were performed with the t-test. Pearson’s correlation was also used. A P value less than 0.05 was considered statistically significant.


  Results Top


Forty eyes of 20 patients were included in the study. The mean age was 24.0±3.9 years. Eleven patients (55%) were female and nine (45%) were male. The mean manifest refraction spherical equivalent for group I (depth 100 µm) was −7.66±1.37 D, with a range of −5 to −9.5 D. The mean manifest refraction spherical equivalent for group II (depth 160 µm) was −7.46±1.41 D, with a range of −5.50 to −10.0 D. There was no statistically significant difference between the two groups (t=−0.454, P=0.652).

The mean thickness of the edge of the lenticules was 11.52±1.20 µm in group I (range from 9.40 to 14.10 µm) and 12.04±0.90 µm in group II (range from 10.89 to 15.00 µm). There was no statistically significant difference between the two groups (t=−1.578, P=0.124). At higher magnification (×2000), there was a noticeable difference in the morphology of the edges in both groups, as in group I there was a tendency toward smoother edges with less fragmentation and cavitations compared with group II ([Figure 1]).
Figure 1 Electron microscopic evaluation of the edge of the lenticules: group I (a) and group II (b). Notice less cavitations and more tissue homogenicity in group I.

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On using the scoring system published by Wilhelm et al. [12], the mean score was 7.85±1.18 in group I (range from 6 to 10) and 7.50±1.28 in group II (range from 5 to 10). There was no statistically significant difference between the two groups (t=0.899, P=0.374). In both groups, around 50% of cases showed smooth surface at ×10 magnification. At ×50 magnification, most of the cases had a partially maintained regular surface.

The surface roughness appeared in two forms: one fine form of fragments of stromal corneal tissue less than 5 µm and the other coarser form of stromal corneal tissue fragments more than 10 µm in height. The fine form was present in both groups and in all quadrants of the samples, whereas the other coarser form was present only in some quadrants of the samples.

On using the new scoring system, the mean score was 14.90±1.74 in group I (range from 9 to 16) and 13.25±2.77 in group II (range from 8 to 16). There was a statistically significant difference between the two groups (t=2.254, P=0.031). The results of this scoring system correlated poorly with the results of the other score used at lower magnification levels (r=0.12 in group I and r=0.082 in group II).

On higher magnifications (×2000) there was a noticable difference between the two groups. There were more islands of elevations in group II than in group I. The connecting bridges between the femtosecond cavitational bubbles seem to be more strong and to result in larger tissue bridges in group II appearing in the form of more coarse fragments on electron microscopy ([Figure 2]).
Figure 2 Electron microscopic appearance of the surface of lenticules at ×2000 magnification with group I (a) and group II (b) showing a coarser surface and more tissue bridges

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  Discussion Top


The role of electron microscopy in evaluating the SMILE lenticules is relatively novel in ophthalmology research. The anatomical changes induced by the SMILE procedure can help us correlate better with the clinical findings of this new procedure. The current study is the first to work on variable corneal depth in cases of SMILE. In addition, this study was solely conducted on lenticules extracted from human corneas, which gives a true morphological description of the effect of femtosecond laser in human eyes and not in an animal model.

Deeper corneal lenticules have the potential advantage of leaving a more superficial stronger cornea and to allow the correction of higher myopic errors. The cornea is thought to be similar to a viscoelastic material with quantifiable biomechanical properties [13],[14],[15],[16]. Keratorefractive laser surgery is expected to alter the biomechanical properties of the cornea, which is thought to play an important role in affecting treatment outcome [17],[18],[19],[20]. Palomino et al. [21] stated that, in relation to the potential impact of the flap creation on corneal hysteresis, the variation was significantly greater after laser in-situ keratomileusis with mechanical microkeratomes than after femtosecond-assisted laser in-situ keratomileusis, with better corneal biomechanics.

Heichel et al. [22] worked on porcine corneas and found that femtosecond lenticule extraction is capable of creating corneal lenticules of predictable surface quality; however, there were surface irregularities that were caused by tissue bridges, cavitation bubbles, or scratches.

Ziebarth et al. [23] assessed the quality of both the anterior and posterior surfaces of the extracted lenticules of only myopic eyes with no cylinder using the same energy level (130 nJ) but with lenticule depth of 120 μm. They used multiple magnifications (×100, ×250, and ×500) and found the lenticules to be smooth with uniform cut edges. Jagged edges were seen in several images, but were attributed to the forceps pull during extraction.

In another study Kunert et al. [24] concluded that surface irregularity decreased as pulse energy increased. Their study showed the influence of pulse energy on surface regularity in human eyes. In the 150 nJ subgroup, the mean surface regularity score was 7.5±0.75 versus 7.25±0.74 and 6.25±0.67 in the 180 and 195 nJ subgroups.

The present study used only one energy level in both depths for the surface and side cuts. This may have had an impact on the surface smoothness of deeper lenticules. It seems that, to achieve the same tissue cleavage effect in deeper layers, the laser energy should have been increased and the spot distance should have been decreased as there is always some loss of the energy effect in deeper layers.

Contrary to the fact that anterior corneal stroma is stronger, difficult tissue dissection and irregularities of surfaces were seen more commonly in the deeper lenticules. By using the new scoring system at higher magnification levels, the difference in quality between the superficial and the deeper lenticules can be clarified. Both edge serrations and surface coarse tissue fragments (>10 μm) were more evident in the deeper lenticules. Edge serrations may be caused by lenticule manipulation during dissection and extraction. Their presence indicates more difficult dissection and hence the need for higher energy levels to overcome this.

The tissue bridges were attributed to the manual dissection of the uncut corneal stroma that was present in areas where laser effect was absent. The absence of laser (skip phenomena) could be attributed to areas of meibomian secretions in the interface between the suction cone and the corneal surface acting as a barrier. Further, difficult docking and minimal saccadic eye movements while applying the laser play a definite role in producing these tiny islands of skip phenomena in which the femtosecond laser fails to induce tissue cleavage, which in turn is translated into difficult dissection and tissue bridges on electron microscopy.

However, the smaller sample size in which these events occurred hinders any significant statistical evaluation. In conclusion, the VisuMax femtosecond laser system creates predictable good-quality surface refractive corneal lenticules at superficial depth. Deeper corneal lenticules showed more irregular surfaces, especially at higher magnification levels. Further studies should focus on optimization of laser parameters as well as the surgical technique to improve the surface regularity of the corneal stroma. Energy settings still need further adjustment to be able to create deeper predictable lenticules in SMILE cases.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Blum M, Kunert KS, Engelbrecht C. Femtosecond lenticule extraction (FLEx) − results after 12 months in myopic astigmatism. Klin Monatsbl Augenheilkd 2010; 227:961–965.  Back to cited text no. 4
    
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Agca A, Ozgurhan EB, Demirok A. Comparison of corneal hysteresis and corneal resistance factor after small incision lenticule extraction and femtosecond laser-assisted LASIK: a prospective fellow eye study. Contact Lens Anterior Eye 2014; 37:77–80.  Back to cited text no. 19
    
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]



 

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