June 2014
Volume 55, Issue 6
Free
Cornea  |   June 2014
Comparative Analysis of Two Femtosecond LASIK Platforms Using iTRAQ Quantitative Proteomics
Author Affiliations & Notes
  • Sharon D'Souza
    Narayana Nethralaya, Bangalore, India
  • Andrea Petznick
    Singapore Eye Research Institute, Singapore
  • Louis Tong
    Singapore Eye Research Institute, Singapore
  • Reece C. Hall
    Singapore National Eye Centre, Singapore
  • Mohamad Rosman
    Singapore Eye Research Institute, Singapore
    Singapore National Eye Centre, Singapore
  • Cordelia Chan
    Singapore National Eye Centre, Singapore
  • Siew Kwan Koh
    Singapore Eye Research Institute, Singapore
  • Roger W. Beuerman
    Singapore Eye Research Institute, Singapore
    Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
    Signature Research Program (SRP) Neuroscience and Behavioural Disorder, DUKE–National University of Singapore Graduate Medical School, Singapore
  • Lei Zhou
    Singapore Eye Research Institute, Singapore
    Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
    Signature Research Program (SRP) Neuroscience and Behavioural Disorder, DUKE–National University of Singapore Graduate Medical School, Singapore
  • Jodhbir S. Mehta
    Singapore Eye Research Institute, Singapore
    Singapore National Eye Centre, Singapore
    Office of Clinical, Academic, and Faculty Affairs, Duke–National University of Singapore Graduate Medical School, Singapore
  • Correspondence: Lei Zhou, Singapore Eye Research Institute, 11 Third Hospital Avenue, Singapore 168751; zhou.lei@seri.com.sg
  • Louis Tong, Singapore Eye Research Institute, 11 Third Hospital Avenue, Singapore 168751; Louis.tong.h.t@snec.com.sg
Investigative Ophthalmology & Visual Science June 2014, Vol.55, 3396-3402. doi:10.1167/iovs.14-14113
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Sharon D'Souza, Andrea Petznick, Louis Tong, Reece C. Hall, Mohamad Rosman, Cordelia Chan, Siew Kwan Koh, Roger W. Beuerman, Lei Zhou, Jodhbir S. Mehta; Comparative Analysis of Two Femtosecond LASIK Platforms Using iTRAQ Quantitative Proteomics. Invest. Ophthalmol. Vis. Sci. 2014;55(6):3396-3402. doi: 10.1167/iovs.14-14113.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: New femtosecond laser platforms may reduce ocular surface interference and LASIK-associated dry eye. This study investigated tear protein profiles in subjects who underwent LASIK using two femtosecond lasers to assess differences in protein expression.

Methods.: This was a randomized interventional clinical trial involving 22 patients who underwent femtosecond laser refractive surgery with a contralateral paired eye design. Corneal flaps of 22 subjects were created by either Visumax or Intralase laser. Tear samples were collected preoperatively, and at 1 week and 3 months postoperatively using Schirmer's strips. Tear protein ratios were calculated relative to preoperative protein levels at baseline. The main outcome measures were the levels of a panel of dry eye protein markers analyzed using isobaric tagging for relative and absolute quantitation (iTRAQ) mass spectrometry.

Results.: A total of 824 unique proteins were quantifiable. Tear protein ratios were differentially regulated between the eyes treated with different lasers. The secretoglobulins Lipophilin A (1.80-fold) and Lipophilin C (1.77) were significantly upregulated (P < 0.05) at 1 week postoperatively in Visumax but not in Intralase-treated eyes. At 1 week, orosomucoid1 was upregulated (1.78) in Intralase but not Visumax-treated eyes. In the same eyes, lysozyme, cathepsin B, and lipo-oxygenase were downregulated at 0.44-, 0.64-, and 0.64-folds, respectively. Transglutaminase-2 was downregulated in both groups of eyes.

Conclusions.: Different laser platforms induce distinct biological responses in the cornea and ocular surface, which manifests as different levels of tear proteins. This study has implications for surgical technology and modulation of wound healing responses. (ClinicalTrials.gov number, NCT01252654.)

Introduction
LASIK is the most common refractive surgery procedure. Although it has been shown to be safe, effective, and highly predictable, 1 dry-eye complaints or tear dysfunction are major side effects. 2 The truncation of the corneal afferent nerves during flap creation results in corneal hypoesthesia and disruption of the cornea-lacrimal gland unit. 3 This, in turn, leads to a reduced blink rate and changes in tear quantity as well as tear hyperosmolarity and ocular surface inflammation. 4,5 It has not been clear if patients with LASIK-associated dry eye have inflammatory ocular surface changes as in pathological dry eye, 6,7 or if these changes are more related to the regenerative changes in the corneal nerves, which give rise to only sensations of pain and sensory irritation. 8 The tear film is an integral part of the ocular surface and represents the extracellular matrix for ocular surface epithelial cells. Here, analysis of the human tear proteome can provide both qualitative and quantitative information that is useful in assessing the health of the ocular surface. 9  
Our laboratory has used tear proteomic analysis to discover biomarkers for objective diagnosis of dry eye. 7 Dry-eye patients displayed an altered tear proteome profile with upregulated inflammatory markers and decreased levels of protective proteins. 7,10,11 Specifically, a panel consisting of α-enolase, prolactin-inducible protein (PIP), lipocalin-1, S100A9 (calgranulin B) has been shown to produce a high diagnostic accuracy for presence and severity of dry eye. 7 In a rabbit model of Sjogren's Syndrome–associated dry eye, 12 similar dry eye biomarkers such as the S100 A9 and PIP were found. Refractive surgery is expected to cause major changes in the tear protein profile due to disruption of the cornea-lacrimal gland functional unit. 13,14 Previously, it has been shown that differences in surgical technique can result in differential tear protein levels. 9,15,16 These studies are, however, limited to a few proteins such as nerve growth factor, or IL-6, -8, matrix metalloproteinase 9, and epidermal growth factor only. 15,16  
Newer femtosecond lasers create smaller flaps, use higher repetition rates and lower laser pulse energies and use different corneal stabilization methods. 17 These advances may result in fewer adverse consequences for the ocular surface and may affect the expression of tear protein differently. It has been shown that different femtosecond lasers can induce different wound healing responses in rabbit corneal tissue. 18,19  
A comparison between Intralase (Abott Medical Optics, Inc., Santa Ana, CA, USA) and the newer femtosecond laser Visumax (Carl Zeiss Meditec, Jena, Germany) has shown that both platforms have similar efficacy and predictability of visual outcomes. 17,20 The Intralase laser uses a lower repetition rate, higher energy and conjunctival suction, while the Visumax laser platform uses higher repetition rate, lower energy, and corneal suction. 17 In a contralateral paired-eye study with 45 myopic patients, no statistically significant differences in corneal sensitivity, Schirmer's test, tear break-up time (TBUT), and corneal staining were identified between the Intralase and Visumax platform over a 3-month period. 20 In this comparison the clinical signs, such as Schirmer's, TBUT, corneal fluorescein staining, corneal sensitivity, and visual outcomes including contrast sensitivity, were not sensitive enough to differentiate the laser technologies. The corneal sensitivity however, revealed slightly faster recovery rates in Visumax-operated eyes as compared with Intralase-operated eyes, which may be attributable to the technical differences. 17  
We hypothesize that the tear proteome may be altered after LASIK in a different way from previously reported profiles in idiopathic dry eye, and the Visumax femtosecond laser platform with different technical specifications may induce distinct protein changes from the Intralase platform. To address these hypotheses, a proteomic study of tears was carried out with patients who underwent a randomized contralateral eye treatment using the Visumax and Intralase laser platforms and evaluated at 1 week and 3 months postoperatively. 
Methods
Subject Selection
Institutional review board approval for this study was obtained from the Singapore Eye Research Institute ethics committee. This prospective contralateral paired eye clinical study adhered to the tenets of the Declaration of Helsinki and was registered with the National Institutes of Health database (http://www.clinicaltrials.gov, record number: NCT01252654). Informed written consent was obtained from 45 consecutive subjects after explanation of the nature and possible consequences of the study. For the purpose of this publication, tear protein profiles of 22 subjects were analyzed. 
Inclusion criteria were as follows: greater or equal to 21-years old, stable myopic prescription, best corrected visual acuity of at least 20/20, minimum corneal thickness of 500 μm, preoperative Schirmer's Type I test value greater than 5.0 mm in 5 minutes, and cessation of soft contact lens wear for at least 2 weeks or rigid gas-permeable lenses for at least 3 weeks. Subjects were excluded if they had any anterior or posterior segment pathology, previous ocular surgery, metabolic or autoimmune disease, severe dry eye, connective tissue disease or significant atopic syndrome, and were undergoing chronic systemic corticosteroid or immunosuppressive therapy. 
Surgical Technique
Corneal flaps were created with a 500-kHz Visumax femtosecond laser (Carl Zeiss Meditec) or a 60-kHz Intralase femtosecond laser (Abott Medical Optics, Inc.). The targeted flap thickness was between 110 to 115 μm with a mean targeted flap diameter of 8.4 ± 0.0 mm for Visumax (nonconverted 7.9 ± 0.1 mm) and 8.9 ± 0.2 mm for Intralase. Laser parameters for Visumax flaps were: small (S) cone, superior hinge, 85°-sidecut angle, 70°-hinge angle, laser bed energy 0.16 to 0.165 μJ, spot separation 1.5 (rim) and 4.8 μm (lamellar), and line spot separation 1.5 (rim) and 4.8 μm (lamellar). Laser parameters for Intralase flaps were: standard suction, superior hinge, 70°-sidecut angle, 50°-hinge angle, laser bed energy 0.95 μJ (range 0.92–1.08 μJ), pocket enable on, 0.25-mm pocket width, 230-μm pocket start depth, 7-μm pocket tangent, and 6-μm radian spot separation. 
Stromal tissue laser ablation in all eyes was performed with the Wavelight Allegretto Eye-Q 400Hz excimer laser system (Alcon Laboratories, Inc., Fort Worth, TX, USA) using the wavefront-optimized treatment profile. The attempted optical zone was 6.50 mm and target refraction was plano spherical equivalent. 
All eyes were treated with nonpreserved moxifloxacin hydrochloride 0.5% eyedrops (Vigamox; Alcon Laboratories, Inc.) and dexamethasone 0.1% eyedrops (Maxidex; Alcon Laboratories, Inc.) four times daily for 1 week. Nonpreserved artificial tears (Tears Naturale Free; Alcon Laboratories, Inc.) were prescribed postoperatively with a dosage adjusted to subject's symptoms and needs. 
Tear Sample Collection
Tear samples were collected using a Schirmer's Type I tear test without local anesthesia, as employed in previous studies in our laboratory. 7,11,21 No other drops were administered prior to the Schirmer's test. Briefly, a precalibrated filter paper strip (Sno strips; Bausch & Lomb, Rochester, NY, USA) was gently placed in the inferior temporal fornix for 5 minutes, the eye shut, and the patient told not to move the eye under the closed lids. After collection, the wetted area of the Schirmer's strips were measured and the strip immediately frozen at −80°C until analysis. To start the analysis procedure, Schirmer's strips were cut into small pieces and soaked in 150 μL of 50 mM ammonium bicarbonate and 1.3× protease inhibitor (Pierce; Thermo Scientific, Rockford, IL, USA) for 3 hours to elute tear proteins. Total tear protein concentrations of each tear sample were measured using the RC DC Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Tear samples were lyophilized and stored at −80°C until further analysis. 
Quantitative Tear Protein Analysis
Tear samples collected at the visit prior to surgery (day 0), and then at 1 week and 3 months postoperatively were analyzed using the proteomics study design shown in Figure 1. Samples were quantitatively analyzed using isobaric tagging for relative and absolute quantitation (iTRAQ)-based technology combined with one-dimensional nano liquid chromatography (LC)-nano-electrospray ionization (ESI)-mass spectrometry (MS)/MS in triplicates. 
Figure 1
 
Design of the iTRAQ experiments for the analysis of pre- and postoperatively collected tears.
Figure 1
 
Design of the iTRAQ experiments for the analysis of pre- and postoperatively collected tears.
Data consisted of protein ratios for the 1 week postoperative visit (preoperative to 1 week) and the 3-month postoperative visit (preoperative to 3 months). Protein ratios larger than 1.300 were considered upregulated and below 0.769 downregulated. 
Analysis of Tear Fluid Using Mass Spectrometry and Liquid Chromatography
Lyophilized tear proteins (25-μg total protein) were reconstituted, denatured, and reduced in 50 mM ammonium bicarbonate solution (filter-aided sample preparation [FASP] kit; Expedeon, Inc., Harston, UK) 2% SDS solution (Vivantis Technologies, Selangor, Malaysia), and tris(2-carboxyethyl)phosphine (TCEP) from iTRAQ kit (AB Sciex, Framingham, MA, USA) for 1 hour at 60°C. The reduced protein samples were then transferred to 30 kDa cut-off membrane cartridge (FASP kit; Expedeon, Inc.) to remove the excess reagent using 75% urea solution. The reduced samples were then alkylated using methyl methane thiosulfonate (MMTS; AB Sciex) for 20 minutes at room temperature. The alkylated-samples were further washed with 75% urea solution and 50 mM ammonium bicarbonte solution prior to trypsin digestion overnight with substrate: enzyme ratio of 1:25 at 37°C. Peptides were then eluted with 50 mM ammonium bicarbonate solution and sodium chloride (provided in FASP kit; Expedeon, Inc.). The eluted samples were lyophilized and tear samples were labeled with iTRAQ reagents for 3 hours at room temperature as follows: preoperatively collected tears with iTRAQ 114, 1 week postoperatively collected tears with iTRAQ 115 and 3 month postoperatively collected tears with iTRAQ 117 (Fig. 1). 
Labeled samples (iTRAQ 114, iTRAQ 115, iTRAQ 117) were then pooled together, dried and desalted using ultramicro spin columns (The Nest Group, Inc., Southboro, MA, USA) prior to nano-LC/MSMS analysis. 
The one dimensional nano LC-MS/MS system (Dionex Ultimate 3000 Nano LC system; Thermo Fisher Scientific, Sunnyvale, CA, USA), coupled with AB Sciex TripleTOF 5600 system (AB Sciex), was used for the proteomic analysis. A 50 cm × 75 μm (internal diameter) Dionex Acclaim PepMap RSLC C18 packed column was employed (Thermo Fisher Scientific). This column was connected to a spray tip (New Objective, Inc., Woburn, MA, USA), which was directly coupled with the nanospray interface of AB Sciex TripleTOF 5600 MS. Samples were loaded onto a trap column (Dionex Acclaim PepMap 100 C18, 2 cm × 75 μm i.d.; Thermo Fisher Scientific) at a flow rate of 5 μL/min. After a 3 minute wash with loading buffer (2/98 vol/vol of acetonitrile [ACN]/water with 0.1% formic acid) the system was switched into line with the C18 analytical capillary column. A step linear gradient of mobile phase B (2/98 vol/vol of water/ACN with 0.1% formic acid) starting from 7% to 24% for 57 minutes, to 24% to 40% for 27 minutes, to 40% to 60% for 7 minutes, and 60% to 95% for 1 minute at a flow rate of 300 nL/min was used for this analysis. The typical parameters for TripleTOF 5600-MS were as follows: ionspray voltage floating (ISVF) = 2400 V, curtain gas (CUR) = 30, ion source gas 1 (GS1) = 12, interface heater temperature (IHT) = 125°C, declustering potential (DP) = 100 V. All data was acquired using information-dependent acquisition (IDA) mode with Analyst TF 1.5 software (AB Sciex). Time-of-flight mass spectrometry (TOF-MS) scan (experiment 1) parameters were set as follows: 0.25 seconds TOF-MS accumulation time in the mass range of 350 to approximately 1250 Da followed by product ion scan (experiment 2) of 0.05 seconds accumulation time in the mass range of 100 to approximately 1500 Da. Switching criteria were set to ions greater than m/z = 350 and smaller than m/z = 1250 with charge state of 2 to 5, and an abundance threshold of greater than 120 counts per second. Former target ions were excluded for 12 seconds and former ions were excluded after one repeat. Maximum number of candidate ions per cycle was 30 spectra. Information-dependent acquisition advanced “rolling collision energy (CE)” and “adjust CE when using iTRAQ reagent” were required. 
The data was processed and searched against the IPI Human version 3.77 protein database (115194 proteins searched) using ProteinPilot software 4.1 (AB Sciex). Some important settings in ProteinPilot were configured as follows: (1) sample type: iTRAQ 4plex (peptide labeled), (2) Cys alkylation: MMTS; (3) digestion: trypsin, (4) instrument: TripleTOF 5600, (5) special factors: none, (6) identification focus: biological modifications, (7) search effort: thorough identification, and (8) bias correction and background correction were applied. Ninety-five percent confidence level was used at the peptide level. False discovery rate (FDR) analysis in the ProteinPilot software was performed and FDR less than 1% was set for protein identification. Reverse database search strategy was used to calculate FDR for peptide identification. For relative quantification, ProteinPilot software uses Pro Group algorithm to calculate the reporter ions' (iTRAQ114, 115, and 116) peak areas. Auto bias correction was applied to eliminate possible pipetting error during sample preparation. 
Statistical Analysis
Each sample was analyzed in triplicates by iTRAQ, and only data points that were within the 30% coefficient of variance value were used for the analysis, while outliers were filtered out. Data was sorted according to upregulated proteins (ratio > 1.300) and downregulated proteins (ratio < 0.769). A paired Wilcoxon test for nonparametric data was done on the log-transformed median values to identify significantly upregulated and downregulated proteins at 1 week and 3 months postoperatively for both femtosecond laser platforms using the R software (R Development Core Team, Vienna, Austria). A further comparison with the paired Wilcoxon test was performed to compare differences between the log-transformed median of tear protein ratios of Visumax-operated and Intralase-operated eyes at the 1 week and 3 month postoperative visits using the R software. The P value was adjusted for multiple comparisons. A significance level (alpha) of 0.05 was used. 
Results
Subject demographics including age, sex, and mean spherical equivalent refraction are displayed in Table 1. Twenty-two patients completed all three visits and were included in this analysis. A total of 1594 unique proteins (908 unique proteins with ≥2 peptides) with a false detection rate of less than 1% were identified in tears by iTRAQ analysis. Among them, 1229 unique proteins (824 unique proteins with ≥2 peptides) were quantifiable. The tears collected from Visumax-operated eyes showed greater number of upregulated proteins (eight proteins in Visumax versus two proteins in Intralase), while the Intralase-operated eyes had a greater number of downregulated proteins (nine proteins in Intralase versus seven proteins in Visumax; Tables 2 and 3). 
Table 1
 
Subject Data Demographics
Table 1
 
Subject Data Demographics
Mean Age ± SD, Range, y Sex (n) Eyes (n) Mean SER ± SD, D
Male Female
28.45 ± 4.45 9 13 22 (Visumax) −4.47 ± 1.71
22–39 22 (Intralase) −4.34 ± 1.70
Table 2
 
Upregulated and Downregulated Proteins in Visumax-Operated Eyes at 1 Week and 3 Months Postoperatively
Table 2
 
Upregulated and Downregulated Proteins in Visumax-Operated Eyes at 1 Week and 3 Months Postoperatively
1 wk 3 mo
Gene Symbol Median (Q1–Q3) P Value Gene Symbol Median (Q1–Q3) P Value
Upregulated proteins PIGR 3.011 (1.337–6.1) 0.001 PIGR 2.239 (1.1–3.251) 0.004
IGHM 2.599 (1.372–3.856) 0.001 IGHA1 1.706 (0.938–2.173) 0.022
IGHA1 2.403 (1.186–2.902) 0.001
CST1 2.065 (0.958–2.559) 0.012
SCGB1D1 1.804 (1.063–4.207) 0.017
IGLV3–19 1.776 (1.275–2.831) 0.001
SCGB2A1 1.765 (1.006–3.587) 0.005
CLU 1.343 (1.052–2.213) 0.002
Downregulated proteins S100A9 0.773 (0.633–1.054) 0.018 S100A8 0.78 (0.46–0.96) 0.015
ZG16B 0.756 (0.248–0.997) 0.021 S100A9 0.673 (0.362–0.904) 0.009
PSME2 0.735 (0.621–0.971) 0.004 TF 0.607 (0.2020–1.019) 0.017
TGM2 0.705 (0.479–0.914) 0.029 ALB 0.563 (0.191–0.932) 0.017
Table 3
 
Upregulated and Downregulated Proteins in Intralase-Operated Eyes at 1 Week and 3 Months Postoperatively.
Table 3
 
Upregulated and Downregulated Proteins in Intralase-Operated Eyes at 1 Week and 3 Months Postoperatively.
1 wk 3 mo
Gene Symbol Median (Q1–Q3) P Value Gene Symbol Median (Q1–Q3) P Value
Upregulated proteins IGHM 1.983 (0.938–3.192) 0.014
ORM1 1.477 (1.072–3.622) 0.048
Downregulated proteins LGALS3BP 0.692 (0.308–0.979) 0.018 HP 0.67 (0.246–0.947) 0.04
CTSB 0.643 (0.424–0.931) 0.016 SERPINB1 0.577 (0.338–1.103) 0.024
LPO 0.637 (0.231–1.078) 0.018 SCGB1D1 0.536 (0.238–1.132) 0.034
TGM2 0.617 (0.423–0.997) 0.001
ZG16B 0.452 (0.256–1.202) 0.03
LYZ 0.435 (0.131–1.159) 0.05
Tears from Visumax eyes exhibited eight upregulated proteins (protein ratio range of 1.3–3.0) after the first week of surgery, with two proteins that remained upregulated (protein ratio range of 1.7–2.2) at 3 months postsurgery (Table 2). Visumax showed four downregulated proteins (protein ratio range of 0.7–0.8) at 1 week postoperatively and four downregulated proteins (protein ratio range of 0.6–0.8) after 3 months of surgery (Table 2). In tears from Intralase-operated eyes, two upregulated proteins (protein ratio range of 1.5–2.0) were identified at 1 week postoperatively, while no proteins were found to be upregulated at 3 months postoperatively. In the same group, six proteins were downregulated (protein ratio of 0.4–0.7) at 1 week following surgery, and three proteins were downregulated (protein ratio of 0.5–0.7) at 3 months following surgery (Table 3). 
A comparison between Visumax-operated and Intralase-operated eyes was performed to detect proteins that were distinctly different between the two femtosecond laser platforms at 1 week and 3 months of surgery. The lacrimal gland protein lacritin (LACRT) had increased in tears collected from Visumax-operated eyes by more than two times, while in Intralase-operated eyes LACRT remained unchanged at 1 week postoperatively (protein ratio of 2.228 in Visumax and 0.847 in Intralase, P = 0.008; Fig. 2). However, LACRT was not significantly upregulated in Visumax-operated eyes due its relatively large variability (Fig. 2). 
Figure 2
 
Upregulation of proteins in tears collected from Visumax-operated eyes versus Intralase-operated eyes at 1 week (P < 0.05). Ratio: Ratio of tear lacritin (LACRT) at 1 week relative to baseline.
Figure 2
 
Upregulation of proteins in tears collected from Visumax-operated eyes versus Intralase-operated eyes at 1 week (P < 0.05). Ratio: Ratio of tear lacritin (LACRT) at 1 week relative to baseline.
At 3 months following surgery, the protein ratios of LACRT, Clusterin (CLU), and Pre–pro-megakaryocyte potentiating factor (MSLN) were statistically greater in Visumax-operated eyes as compared with Intralase-operated eyes (LARCT with 1.432 vs. 0.803, P = 0.019; CLU with 1.343 vs. 1.028, P = 0.008; and MSLN with 1.486 vs. 1.033, P = 0.016 for Visumax and Intralase, respectively; Fig. 3). The comparison also showed that Intralase-operated eyes had significantly lower amounts of Lactotransferrin (LTF), Alpha-2-glycoprotein 1 zinc (AZGP1), Lysozyme (LYZ), and Lactoperoxidase (LPO) than Visumax-operated eyes (LTF with 0.973 vs. 0.686, P = 0.023; AZGP1 with 1.248 vs. 1.887, P = 0.013; LYZ with 1.112 vs. 0.783, P = 0.024; and LPO with 1.225 vs. 0.964, P = 0.018 for Visumax and Intralase, respectively; Fig. 4). 
Figure 3
 
Upregulation of proteins in tears collected from Visumax-operated eyes versus Intralase-operated eyes at 3 months (P < 0.05).
Figure 3
 
Upregulation of proteins in tears collected from Visumax-operated eyes versus Intralase-operated eyes at 3 months (P < 0.05).
Figure 4
 
Downregulated proteins in tears collected from Intralase-operated eyes versus Visumax-operated eyes at 3 months postoperatively (P < 0.05).
Figure 4
 
Downregulated proteins in tears collected from Intralase-operated eyes versus Visumax-operated eyes at 3 months postoperatively (P < 0.05).
Discussion
This study carried out detailed tear protein profiling following LASIK using two different femtosecond platforms, Visumax and Intralase and identified differences in the relative quantity of proteins between the two femtosecond laser technologies. Numerous proteins were up- and downregulated in both platforms. However, the profile of dysregulated proteins was dissimilar between the two platforms, and somewhat different from some proteins reported previously in dry eye. 7  
The iTRAQ tagging technology coupled with nano-LC/MS has been shown previously to be useful for documenting changes in tear proteins in disease. 7,9,11,21 Unlike previous studies, we employed iTRAQ to compare three different samples, one collected prior and two following LASIK surgery with either Visumax and Intralase lasers. This enabled a comparison of the two femtosecond laser platforms and their impact on selected dry-eye proteins at two time points. To our knowledge, no previous studies that evaluated changes in tear proteins after LASIK have been published. The present study identified upregulated tear proteins such as the inflammatory protein alpha1-acid glycoprotein 1 (ORM1) and the cystatin SN precursor (CST1) that had previously been found to be dysregulated in dry eye. We also detected downregulation of proteins LYZ, TF, and secretoglobin family 1D member 1 (SCGB1D1). Furthermore, the current tear analysis detected novel changes of ALB, calgranulin A (S100A8), S100A9, and mammaglobin B precursor (SCGB2A1). Tear LACRT was significantly higher in the Visumax group compared with the Intralase groups at 1 week and 3 months postoperatively. 
We identified upregulation of CST1 1 week postoperatively in the Visumax-operated eyes. Cystatin SN precursor is supportive of the innate immunity system, 22 and has antimicrobial functions. 23 It also has a protective function, acting as a natural inhibitor of the highly abundant cysteine proteinases. Another cystatin, CST4, has been detected in elevated levels in dry eye and meibomian gland dysfunction. 24 The CST1 protein was downregulated in tears collected from patients with fungal keratitis 25 and keratoconus. 26 We observed other evidence of activated immune system, such as the elevation of immunoglobulins, polymeric immunoglobulin, Ig mu chain C region IGHM protein (IGHM), and IG alpha 1 protein (IGHA1) in the Visumax-operated eyes. 
A higher level of ORM1 was noted in Intralase-operated eyes at 1 week postoperatively. The inflammatory protein alpha1-acid glycoprotein 1 is a constitutively expressed binding/transport protein that modulates inflammatory responses, with highest levels found during acute inflammation. 27 It is secreted by hepatocytes, other epithelial cells, endothelial cells, macrophages, and polymorphonuclear leukocytes. The degree of ORM1 fucosylation has been identified as a possible prognostic measure. 27 In the present study, the upregulation of ORM1 probably indicates an increased early inflammatory response to the Intralase procedure. 
Following 3 months of surgery, the plasma-derived proteins serum serotransferrin (SF) albumin (ALB) were downregulated in tears of Visumax-operated eyes. Serum serotransferrin is an iron-binding transport protein that is secreted by the liver and epithelia, including lacrimal gland acini. Reduced levels of SF were detected in mild to moderate dry eye patients. 28 The lacrimal gland secreted protein lysozyme (LYZ) was also downregulated at 1 week postoperatively. This may suggest a greater disruption of the corneal-lacrimal gland unit as a result of a slightly larger flap diameter of the Intralase platform consequently damaging a larger number of corneal nerves. Similarly, the protein SCGB1D1 (lipophilin A) was lowered at 3 months postoperatively, perhaps reflecting an alteration of lacrimal secretion. Albumin is considered an indirect sign of subclinical inflammation and is usually detected at low levels in normal tears. 28 In dry eye, however, serum albumin is significantly increased. 28 The calcium-binding proteins S100A8 and S100A9 (also called calgranulins), are often found as heterodimers and have mainly been considered to be pro-inflammatory proteins. 29 Previous studies have reported that S100A8 and S100A9 were detected with increased levels in the tears of patients with inflammatory ocular surface conditions, such as dry eye, 7 meibomian gland dysfunction, 11 the use of long-term glaucoma medication, 21 and pterygium. 30 Since these proteins may also play a protective role 31,32 and because LASIK induces reactive oxygen radical formation, 33 a reduction of S100A8 and S100A9 in Visumax-operated eyes may signify immune dysfunction and impaired ocular surface protection. 
Lacritin, a lacrimal functional unit specific protein, has been previously found to be downregulated in dry eye. 34 It is mainly produced in the lacrimal gland, moderately in the conjunctiva and the meibomian gland, and only weakly in corneal epithelium. In the current study, the increase in LACRT postoperatively may be a compensatory measure. Since it has a role in the maintenance of the ocular surface, 35 the response in the Visumax patients may be more beneficial than that in the Intralase patients. Some proteins downregulated in dry eye have not been found to be affected post refractive surgery in our study: tear lipocalin-1 (LIPOC-1) and zinc-alpha-2-glycoprotein (ZAG-2). 36  
Are there differences in the Visumax and Intralase femtosecond lasers that may translate into distinctive effects to the ocular surface and tear protein composition? The Visumax laser uses a higher repetition rate and lower laser bed energy, while the Intralase laser employs a lower repetition rate and higher laser bed energy. Another major difference between the two platforms lays in the suction system, which positions the eyeball and facilitates creation of a flap. Visumax generates suction on the cornea via a curved contact glass, in contrast to Intralase, which applies suction on the conjunctiva and sclera via a suction ring. If suction is placed directly onto the conjunctival epithelium and goblet cells, there may be more damage to these tear producing elements and to the conjunctiva nerves. 3739 and these may result in tear film instability aqueous-deficiency and increased tear hyperosmolarity. 4,40  
Femtosecond laser flaps sever the sensory corneal nerves, which form the afferent branch of a reflex arc at the flap margin. 41 An anticipated effect of this corneal nerve truncation would be a reduced expression of lacrimal gland proteins into tears. 4244 A downregulation of lacrimal gland proteins has been noted in studies in dry eye with lower levels of LYZ and TF in patients with more severe dry eye. 6,7,45 It is possible that Intralase has a greater impact on the neural arc reflex than Visumax, 6,42 since Intralase flaps have a 0.5-mm larger diameter leading to greater nerve damage. 
Femtosecond lasers also induce inflammation 46 via necrotic cell death 46,47 and infiltration of inflammatory cells into the corneal stroma. 46,47 The degree of necrosis is directly related to energy levels needed for flap creation. Intralase lasers with higher pulse energies stimulated a far greater level of keratocyte cell death, inflammation, and healing responses at the flap margins than laser settings with lower pulse energies. 47 This may explain the inflammatory proteins in Intralase-operated eyes. 
The present study established relative quantitative ratios but not actual concentrations. The methodological approach excludes important proteins such as cytokines involved in the inflammatory/healing cascade that are expressed at lower levels. Furthermore, a global control that would have allowed us to perform direct comparisons with other eye conditions, such as idiopathic dry-eye, was not incorporated into the proteome analysis. Our laboratory is currently developing standards to establish absolute quantitative concentrations to enable comparisons of disease conditions in the future. 9  
In summary, tear protein comparison between two platforms revealed that femtosecond laser surgery induce changes of tear proteins with a profile different from idiopathic dry eye. Higher levels of inflammation may be encountered in the Intralase eyes compared with Visumax-treated eyes, as shown by the relatively higher levels of ORM1 in the Intralase eyes. Additionally, lower levels of lacrimal gland proteins in Intralase suggested that a recovery of the neural arc reflex with innervation of the lacrimal gland may require more time as compared with Visumax-operated eye. 
Acknowledgments
The authors thank Tian Dechao for organizing the data, Hla Myint Htoon, MD, PhD, for statistical advice, and Sharon Yeo for editorial and proofreading work. 
Supported by grants from the National Medical Research Council, National Research Foundation's Translational Clinical Research Program for Translational Research Innovations in Ocular Surgery, SingHealth Foundation, and National Medical Research Council Centre Grant 2010, R738, NMRC\CSA\045\2012. 
Disclosure: S. D'Souza, None; A. Petznick, None; L. Tong, None; R.C. Hall, None; M. Rosman, None; C. Chan, None; S.K. Koh, None; R.W. Beuerman, None; L. Zhou, None; J.S. Mehta, None 
References
Yuen LH Chan WK Koh J Mehta JS Tan DTA. 10-year prospective audit of LASIK outcomes for myopia in 37,932 eyes at a single institution in Asia. Ophthalmology . 2010; 117: 1236–1244, e1231. [CrossRef] [PubMed]
Solomon KD Fernandez de Castro LE Sandoval HP LASIK world literature review: quality of life and patient satisfaction. Ophthalmology . 2009; 116: 691–701. [CrossRef] [PubMed]
Lee BH McLaren JW Erie JC Hodge DO Bourne WM. Reinnervation in the cornea after LASIK. Invest Ophthalmol Vis Sci . 2002; 43: 3660–3664. [PubMed]
Dooley I D'Arcy F O'Keefe M. Comparison of dry-eye disease severity after laser in situ keratomileusis and laser-assisted subepithelial keratectomy. J Cataract Refract Surg . 2012; 38: 1058–1064. [CrossRef] [PubMed]
Sambursky R O'Brien TP. MMP-9 and the perioperative management of LASIK surgery. Curr Opin Ophthalmol . 2011; 22: 294–303. [CrossRef] [PubMed]
Srinivasan S Thangavelu M Zhang L Green KB Nichols KK. iTRAQ quantitative proteomics in the analysis of tears in dry eye patients. Invest Ophthalmol Vis Sci . 2012; 53: 5052–5059. [CrossRef] [PubMed]
Zhou L Beuerman RW Chan CM Identification of tear fluid biomarkers in dry eye syndrome using iTRAQ quantitative proteomics. J Proteome Res . 2009; 8: 4889–4905. [CrossRef] [PubMed]
Wilson SE. Laser in situ keratomileusis-induced (presumed) neurotrophic epitheliopathy. Ophthalmology . 2001; 108: 1082–1087. [CrossRef] [PubMed]
Zhou L Beuerman RW. Tear analysis in ocular surface diseases. Prog Retin Eye Res . 2012; 31: 527–550. [CrossRef] [PubMed]
Grus FH Podust VN Bruns K SELDI-TOF-MS ProteinChip array profiling of tears from patients with dry eye. Invest Ophthalmol Vis Sci . 2005; 46: 863–876. [CrossRef] [PubMed]
Tong L Zhou L Beuerman RW Zhao SZ Li XR. Association of tear proteins with Meibomian gland disease and dry eye symptoms. Br J Ophthalmol . 2011; 95: 848–852. [CrossRef] [PubMed]
Zhou L Wei R Zhao P Koh SK Beuerman RW Ding C. Proteomic analysis revealed the altered tear protein profile in a rabbit model of Sjogren's syndrome-associated dry eye. Proteomics . 2013; 13: 2469–2481. [CrossRef] [PubMed]
Stern ME Beuerman RW Fox RI Gao J Mircheff AK Pflugfelder SC. The pathology of dry eye: the interaction between the ocular surface and lacrimal glands. Cornea . 1998; 17: 584–589. [CrossRef] [PubMed]
Stern ME Beuerman RW Fox RI Gao J Mircheff AK Pflugfelder SC. A unified theory of the role of the ocular surface in dry eye. Adv Exp Med Biol . 1998; 438: 643–651. [PubMed]
Lee HK Lee KS Kim HC Lee SH Kim EK. Nerve growth factor concentration and implications in photorefractive keratectomy vs laser in situ keratomileusis. Am J Ophthalmol . 2005; 139: 965–971. [CrossRef] [PubMed]
Gonzalez-Perez J Villa-Collar C Gonzalez-Meijome JM Porta NG Parafita MA. Long-term changes in corneal structure and tear inflammatory mediators after orthokeratology and LASIK. Invest Ophthalmol Vis Sci . 2012; 53: 5301–5311. [CrossRef] [PubMed]
Rosman M Hall RC Chan C Comparison of efficacy and safety of laser in situ keratomileusis using 2 femtosecond laser platforms in contralateral eyes. J Cataract Refract Surg . 2013; 39: 1066–1073. [CrossRef] [PubMed]
Angunawela RI Riau A Chaurasia SS Tan DT Mehta JS. Manual suction versus femtosecond laser trephination for penetrating keratoplasty: intraocular pressure, endothelial cell damage, incision geometry, and wound healing responses. Invest Ophthalmol Vis Sci . 2012; 53: 2571–2579. [CrossRef] [PubMed]
Riau AK Angunawela RI Chaurasia SS Tan DT Mehta JS. Effect of different femtosecond laser-firing patterns on collagen disruption during refractive lenticule extraction. J Cataract Refract Surg . 2012; 38: 1467–1475. [CrossRef] [PubMed]
Petznick A Chew A Hall RC Comparison of corneal sensitivity, tear function and corneal staining following laser in situ keratomileusis with two femtosecond laser platforms. Clin Ophthalmol . 2013; 7: 591–598. [CrossRef] [PubMed]
Wong TT Zhou L Li J Proteomic profiling of inflammatory signaling molecules in the tears of patients on chronic glaucoma medication. Invest Ophthalmol Vis Sci . 2011; 52: 7385–7391. [CrossRef] [PubMed]
Fabian TK Hermann P Beck A Fejerdy P Fabian G. Salivary defense proteins: their network and role in innate and acquired oral immunity. Int J Mol Sci . 2012; 13: 4295–4320. [CrossRef] [PubMed]
Choi HM Lee YA Yang HI Yoo MC Kim KS. Increased levels of thymosin beta4 in synovial fluid of patients with rheumatoid arthritis: association of thymosin beta4 with other factors that are involved in inflammation and bone erosion in joints. Int J Rheum Dis . 2011; 14: 320–324. [CrossRef] [PubMed]
Soria J Duran JA Etxebarria J Tear proteome and protein network analyses reveal a novel pentamarker panel for tear film characterization in dry eye and meibomian gland dysfunction. J Proteomics . 2013; 78: 94–112. [CrossRef] [PubMed]
Ananthi S Chitra T Bini R Prajna NV Lalitha P Dharmalingam K. Comparative analysis of the tear protein profile in mycotic keratitis patients. Mol Vis . 2008; 14: 500–507. [PubMed]
Acera A Vecino E Rodriguez-Agirretxe I Changes in tear protein profile in keratoconus disease. Eye (Lond) . 2011; 25: 1225–1233. [CrossRef] [PubMed]
Ceciliani F Pocacqua V. The acute phase protein alpha1-acid glycoprotein: a model for altered glycosylation during diseases. Curr Protein Peptide Sci . 2007; 8: 91–108. [CrossRef]
Versura P Bavelloni A Grillini M Fresina M Campos EC. Diagnostic performance of a tear protein panel in early dry eye. Mol Vis . 2013; 19: 1247–1257. [PubMed]
Foell D Wittkowski H Vogl T Roth J. S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules. J Leukoc Biol . 2007; 81: 28–37. [CrossRef] [PubMed]
Zhou L Beuerman RW Ang LP Elevation of human alpha-defensins and S100 calcium-binding proteins A8 and A9 in tear fluid of patients with pterygium. Invest Ophthalmol Vis Sci . 2009; 50: 2077–2086. [CrossRef] [PubMed]
Gomes LH Raftery MJ Yan WX Goyette JD Thomas PS Geczy CL. S100A8 and S100A9-oxidant scavengers in inflammation. Free Radic Biol Med . 2013; 58: 170–186. [CrossRef] [PubMed]
Sun Y Lu Y Engeland CG Gordon SC Sroussi HY. The anti-oxidative, anti-inflammatory, and protective effect of S100A8 in endotoxemic mice. Mol Immunol . 2013; 53: 443–449. [CrossRef] [PubMed]
Bilgihan K Bilgihan A Adiguzel U Keratocyte apoptosis and corneal antioxidant enzyme activities after refractive corneal surgery. Eye . 2002; 16: 63–68. [CrossRef] [PubMed]
McKown RL Wang N Raab RW Lacritin and other new proteins of the lacrimal functional unit. Exp Eye Res . 2009; 88: 848–858. [CrossRef] [PubMed]
Nakajima T Walkup RD Tochigi A Shearer TR Azuma M. Establishment of an appropriate animal model for lacritin studies: cloning and characterization of lacritin in monkey eyes. Exp Eye Res . 2007; 85: 651–658. [CrossRef] [PubMed]
Versura P Bavelloni A Blalock W Fresina M Campos EC. A rapid standardized quantitative microfluidic system approach for evaluating human tear proteins. Mol Vis . 2012; 18: 2526–2537. [PubMed]
Jumblatt MM McKenzie RW Jumblatt JE. MUC5AC mucin is a component of the human precorneal tear film. Invest Ophthalmol Vis Sci . 1999; 40: 43–49. [PubMed]
Diebold Y Rios JD Hodges RR Rawe I Dartt DA. Presence of nerves and their receptors in mouse and human conjunctival goblet cells. Invest Ophthalmol Vis Sci . 2001; 42: 2270–2282. [PubMed]
Rodriguez AE Rodriguez-Prats JL Hamdi IM Galal A Awadalla M Alio JL. Comparison of goblet cell density after femtosecond laser and mechanical microkeratome in LASIK. Invest Ophthalmol Vis Sci . 2007; 48: 2570–2575. [CrossRef] [PubMed]
Lee JB Ryu CH Kim J Kim EK Kim HB. Comparison of tear secretion and tear film instability after photorefractive keratectomy and laser in situ keratomileusis. J Cataract Refract Surg . 2000; 26: 1326–1331. [CrossRef] [PubMed]
Dartt DA. Dysfunctional neural regulation of lacrimal gland secretion and its role in the pathogenesis of dry eye syndromes. Ocul Surf . 2004; 2: 76–91. [CrossRef] [PubMed]
Dartt DA. Neural regulation of lacrimal gland secretory processes: relevance in dry eye diseases. Prog Retin Eye Res . 2009; 28: 155–177. [CrossRef] [PubMed]
Salvatore MF Pedroza L Beuerman RW. Denervation of rabbit lacrimal gland increases levels of transferrin and unidentified tear proteins of 44 and 36 kDa. Curr Eye Res . 1999; 18: 455–466. [CrossRef] [PubMed]
Glasgow BJ Gasymov OK. Focus on molecules: tear lipocalin. Exp Eye Res . 2011; 92: 242–243. [CrossRef] [PubMed]
Versura P Nanni P Bavelloni A Tear proteomics in evaporative dry eye disease. Eye (Lond) . 2010; 24: 1396–1402. [CrossRef] [PubMed]
Netto MV Mohan RR Medeiros FW Femtosecond laser and microkeratome corneal flaps: comparison of stromal wound healing and inflammation. J Refract Surg . 2007; 23: 667–676. [PubMed]
de Medeiros FW Kaur H Agrawal V Effect of femtosecond laser energy level on corneal stromal cell death and inflammation. J Refract Surg . 2009; 25: 869–874. [CrossRef] [PubMed]
Footnotes
 SD and AP contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Figure 1
 
Design of the iTRAQ experiments for the analysis of pre- and postoperatively collected tears.
Figure 1
 
Design of the iTRAQ experiments for the analysis of pre- and postoperatively collected tears.
Figure 2
 
Upregulation of proteins in tears collected from Visumax-operated eyes versus Intralase-operated eyes at 1 week (P < 0.05). Ratio: Ratio of tear lacritin (LACRT) at 1 week relative to baseline.
Figure 2
 
Upregulation of proteins in tears collected from Visumax-operated eyes versus Intralase-operated eyes at 1 week (P < 0.05). Ratio: Ratio of tear lacritin (LACRT) at 1 week relative to baseline.
Figure 3
 
Upregulation of proteins in tears collected from Visumax-operated eyes versus Intralase-operated eyes at 3 months (P < 0.05).
Figure 3
 
Upregulation of proteins in tears collected from Visumax-operated eyes versus Intralase-operated eyes at 3 months (P < 0.05).
Figure 4
 
Downregulated proteins in tears collected from Intralase-operated eyes versus Visumax-operated eyes at 3 months postoperatively (P < 0.05).
Figure 4
 
Downregulated proteins in tears collected from Intralase-operated eyes versus Visumax-operated eyes at 3 months postoperatively (P < 0.05).
Table 1
 
Subject Data Demographics
Table 1
 
Subject Data Demographics
Mean Age ± SD, Range, y Sex (n) Eyes (n) Mean SER ± SD, D
Male Female
28.45 ± 4.45 9 13 22 (Visumax) −4.47 ± 1.71
22–39 22 (Intralase) −4.34 ± 1.70
Table 2
 
Upregulated and Downregulated Proteins in Visumax-Operated Eyes at 1 Week and 3 Months Postoperatively
Table 2
 
Upregulated and Downregulated Proteins in Visumax-Operated Eyes at 1 Week and 3 Months Postoperatively
1 wk 3 mo
Gene Symbol Median (Q1–Q3) P Value Gene Symbol Median (Q1–Q3) P Value
Upregulated proteins PIGR 3.011 (1.337–6.1) 0.001 PIGR 2.239 (1.1–3.251) 0.004
IGHM 2.599 (1.372–3.856) 0.001 IGHA1 1.706 (0.938–2.173) 0.022
IGHA1 2.403 (1.186–2.902) 0.001
CST1 2.065 (0.958–2.559) 0.012
SCGB1D1 1.804 (1.063–4.207) 0.017
IGLV3–19 1.776 (1.275–2.831) 0.001
SCGB2A1 1.765 (1.006–3.587) 0.005
CLU 1.343 (1.052–2.213) 0.002
Downregulated proteins S100A9 0.773 (0.633–1.054) 0.018 S100A8 0.78 (0.46–0.96) 0.015
ZG16B 0.756 (0.248–0.997) 0.021 S100A9 0.673 (0.362–0.904) 0.009
PSME2 0.735 (0.621–0.971) 0.004 TF 0.607 (0.2020–1.019) 0.017
TGM2 0.705 (0.479–0.914) 0.029 ALB 0.563 (0.191–0.932) 0.017
Table 3
 
Upregulated and Downregulated Proteins in Intralase-Operated Eyes at 1 Week and 3 Months Postoperatively.
Table 3
 
Upregulated and Downregulated Proteins in Intralase-Operated Eyes at 1 Week and 3 Months Postoperatively.
1 wk 3 mo
Gene Symbol Median (Q1–Q3) P Value Gene Symbol Median (Q1–Q3) P Value
Upregulated proteins IGHM 1.983 (0.938–3.192) 0.014
ORM1 1.477 (1.072–3.622) 0.048
Downregulated proteins LGALS3BP 0.692 (0.308–0.979) 0.018 HP 0.67 (0.246–0.947) 0.04
CTSB 0.643 (0.424–0.931) 0.016 SERPINB1 0.577 (0.338–1.103) 0.024
LPO 0.637 (0.231–1.078) 0.018 SCGB1D1 0.536 (0.238–1.132) 0.034
TGM2 0.617 (0.423–0.997) 0.001
ZG16B 0.452 (0.256–1.202) 0.03
LYZ 0.435 (0.131–1.159) 0.05
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×