Cornea Comparative Analysis of Two Femtosecond LASIK Platforms Using iTRAQ Quantitative Proteomics Sharon D’Souza,1 Andrea Petznick,2 Louis Tong,2–5 Reece C. Hall,3 Mohamad Rosman,2,3 Cordelia Chan,3 Siew Kwan Koh,2 Roger W. Beuerman,2,4,6 Lei Zhou,2,4,6 and Jodhbir S. Mehta2,3,5 1Narayana Nethralaya, Bangalore, India 2Singapore Eye Research Institute, Singapore 3Singapore National Eye Centre, Singapore 4Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 5Office of Clinical, Academic, and Faculty Affairs, Duke–National University of Singapore Graduate Medical School, Singapore 6Signature Research Program (SRP) Neuroscience and Behavioural Disorder, DUKE–National University of Singapore Graduate Medical School, Singapore Correspondence: Lei Zhou, Singa- PURPOSE. New femtosecond laser platforms may reduce ocular surface interference and LASIK- pore Eye Research Institute, 11 Third associated dry eye. This study investigated tear protein profiles in subjects who underwent Hospital Avenue, Singapore 168751; LASIK using two femtosecond lasers to assess differences in protein expression. [email protected]. Louis Tong, Singapore Eye Research METHODS. This was a randomized interventional clinical trial involving 22 patients who Institute, 11 Third Hospital Avenue, underwent femtosecond laser refractive surgery with a contralateral paired eye design. Singapore 168751; Corneal flaps of 22 subjects were created by either Visumax or Intralase laser. Tear samples [email protected]. were collected preoperatively, and at 1 week and 3 months postoperatively using Schirmer’s SD and AP contributed equally to the strips. Tear protein ratios were calculated relative to preoperative protein levels at baseline. work presented here and should The main outcome measures were the levels of a panel of dry eye protein markers analyzed therefore be regarded as equivalent using isobaric tagging for relative and absolute quantitation (iTRAQ) mass spectrometry. authors. RESULTS. A total of 824 unique proteins were quantifiable. Tear protein ratios were Submitted: February 7, 2014 differentially regulated between the eyes treated with different lasers. The secretoglobulins Accepted: April 24, 2014 Lipophilin A (1.80-fold) and Lipophilin C (1.77) were significantly upregulated (P < 0.05) at 1 Citation: D’Souza S, Petznick A, Tong week postoperatively in Visumax but not in Intralase-treated eyes. At 1 week, orosomucoid1 L, et al. Comparative analysis of two was upregulated (1.78) in Intralase but not Visumax-treated eyes. In the same eyes, lysozyme, femtosecond LASIK platforms using cathepsin B, and lipo-oxygenase were downregulated at 0.44-, 0.64-, and 0.64-folds, iTRAQ quantitative proteomics. Invest respectively. Transglutaminase-2 was downregulated in both groups of eyes. Ophthalmol Vis Sci. 2014;55:3396– 3402. DOI:10.1167/iovs.14-14113 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.) Keywords: refractive surgery, laser, tears, randomized controlled trial ASIK is the most common refractive surgery procedure. Our laboratory has used tear proteomic analysis to discover L Although it has been shown to be safe, effective, and highly biomarkers for objective diagnosis of dry eye.7 Dry-eye patients predictable,1 dry-eye complaints or tear dysfunction are major displayed an altered tear proteome profile with upregulated side effects.2 The truncation of the corneal afferent nerves inflammatory markers and decreased levels of protective during flap creation results in corneal hypoesthesia and proteins.7,10,11 Specifically, a panel consisting of a-enolase, 3 disruption of the cornea-lacrimal gland unit. This, in turn, prolactin-inducible protein (PIP), lipocalin-1, S100A9 (calgra- leads to a reduced blink rate and changes in tear quantity as nulin B) has been shown to produce a high diagnostic accuracy well as tear hyperosmolarity and ocular surface inflamma- for presence and severity of dry eye.7 In a rabbit model of tion.4,5 It has not been clear if patients with LASIK-associated Sjogren’s Syndrome–associated dry eye,12 similar dry eye dry eye have inflammatory ocular surface changes as in pathological dry eye,6,7 or if these changes are more related biomarkers such as the S100 A9 and PIP were found. Refractive to the regenerative changes in the corneal nerves, which give surgery is expected to cause major changes in the tear protein rise to only sensations of pain and sensory irritation.8 The tear profile due to disruption of the cornea-lacrimal gland functional 13,14 film is an integral part of the ocular surface and represents the unit. Previously, it has been shown that differences in extracellular matrix for ocular surface epithelial cells. Here, surgical technique can result in differential tear protein analysis of the human tear proteome can provide both levels.9,15,16 These studies are, however, limited to a few qualitative and quantitative information that is useful in proteins such as nerve growth factor, or IL-6, -8, matrix assessing the health of the ocular surface.9 metalloproteinase 9, and epidermal growth factor only.15,16 Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783 3396 Downloaded from tvst.arvojournals.org on 09/26/2021 Tear Protein Profile in LASIK IOVS j June 2014 j Vol. 55 j No. 6 j 3397 Newer femtosecond lasers create smaller flaps, use higher flap diameter of 8.4 6 0.0 mm for Visumax (nonconverted 7.9 repetition rates and lower laser pulse energies and use 6 0.1 mm) and 8.9 6 0.2 mm for Intralase. Laser parameters different corneal stabilization methods.17 These advances for Visumax flaps were: small (S) cone, superior hinge, 858- may result in fewer adverse consequences for the ocular sidecut angle, 708-hinge angle, laser bed energy 0.16 to 0.165 surface and may affect the expression of tear protein lJ, spot separation 1.5 (rim) and 4.8 lm (lamellar), and line differently. It has been shown that different femtosecond spot separation 1.5 (rim) and 4.8 lm (lamellar). Laser lasers can induce different wound healing responses in rabbit parameters for Intralase flaps were: standard suction, superior corneal tissue.18,19 hinge, 708-sidecut angle, 508-hinge angle, laser bed energy 0.95 A comparison between Intralase (Abott Medical Optics, lJ (range 0.92–1.08 lJ), pocket enable on, 0.25-mm pocket Inc., Santa Ana, CA, USA) and the newer femtosecond laser width, 230-lm pocket start depth, 7-lm pocket tangent, and 6- Visumax (Carl Zeiss Meditec, Jena, Germany) has shown that lm radian spot separation. both platforms have similar efficacy and predictability of visual Stromal tissue laser ablation in all eyes was performed with outcomes.17,20 The Intralase laser uses a lower repetition rate, the Wavelight Allegretto Eye-Q 400Hz excimer laser system higher energy and conjunctival suction, while the Visumax (Alcon Laboratories, Inc., Fort Worth, TX, USA) using the laser platform uses higher repetition rate, lower energy, and wavefront-optimized treatment profile. The attempted optical corneal suction.17 In a contralateral paired-eye study with 45 zone was 6.50 mm and target refraction was plano spherical myopic patients, no statistically significant differences in equivalent. corneal sensitivity, Schirmer’s test, tear break-up time (TBUT), All eyes were treated with nonpreserved moxifloxacin and corneal staining were identified between the Intralase and hydrochloride 0.5% eyedrops (Vigamox; Alcon Laboratories, Visumax platform over a 3-month period.20 In this comparison Inc.) and dexamethasone 0.1% eyedrops (Maxidex; Alcon the clinical signs, such as Schirmer’s, TBUT, corneal fluorescein Laboratories, Inc.) four times daily for 1 week. Nonpreserved staining, corneal sensitivity, and visual outcomes including artificial tears (Tears Naturale Free; Alcon Laboratories, Inc.) contrast sensitivity, were not sensitive enough to differentiate were prescribed postoperatively with a dosage adjusted to the laser technologies. The corneal sensitivity however, subject’s symptoms and needs. revealed slightly faster recovery rates in Visumax-operated eyes as compared with Intralase-operated eyes, which may be Tear Sample Collection attributable to the technical differences.17 We hypothesize that the tear proteome may be altered after Tear samples were collected using a Schirmer’s Type I tear test LASIK in a different way from previously reported profiles in without local anesthesia, as employed in previous studies in idiopathic dry eye, and the Visumax femtosecond laser our laboratory.7,11,21 No other drops were administered prior platform with different technical specifications may induce to the Schirmer’s test. Briefly, a precalibrated filter paper strip distinct protein changes from the Intralase platform. To address (Sno strips; Bausch & Lomb, Rochester, NY, USA) was gently these hypotheses, a proteomic study of tears was carried out placed in the inferior temporal fornix for 5 minutes, the eye with patients who underwent a randomized contralateral eye shut, and the patient told not to move the eye under the closed treatment using the Visumax and Intralase laser platforms and lids. After collection, the wetted area of the Schirmer’s strips evaluated at 1 week and 3 months postoperatively. were measured and the strip immediately frozen at À808C until analysis. To start the analysis procedure, Schirmer’s strips were cut into small pieces and soaked in 150 Lof50mM METHODS l ammonium bicarbonate and 1.33 protease inhibitor (Pierce; Subject Selection Thermo Scientific, Rockford, IL, USA) for 3 hours to elute tear proteins. Total tear protein concentrations of each tear sample Institutional review board approval for this study was obtained were measured using the RC DC Protein Assay (Bio-Rad from the Singapore Eye Research Institute ethics committee. Laboratories, Hercules, CA, USA).