August 2014
Volume 55, Issue 8
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Cornea  |   August 2014
Antioxidant and Inflammatory Cytokine in Tears of Patients With Dry Eye Syndrome Treated With Preservative-Free Versus Preserved Eye Drops
Author Affiliations & Notes
  • Donghyun Jee
    Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea
  • Sang Hee Park
    Institute of Clinical Medicine Research, Bucheon St. Mary's Hospital, Bucheon, Korea
  • Man Soo Kim
    Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea
  • Eun Chul Kim
    Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea
  • Correspondence: Eun Chul Kim, Department of Ophthalmology, Bucheon St. Mary's Hospital, 327 Sosa-ro, Wonmi-gu, Bucheon, Gyeonggi-do, 420-717, Korea; eunchol@hanmail.net
Investigative Ophthalmology & Visual Science August 2014, Vol.55, 5081-5089. doi:10.1167/iovs.14-14483
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      Donghyun Jee, Sang Hee Park, Man Soo Kim, Eun Chul Kim; Antioxidant and Inflammatory Cytokine in Tears of Patients With Dry Eye Syndrome Treated With Preservative-Free Versus Preserved Eye Drops. Invest. Ophthalmol. Vis. Sci. 2014;55(8):5081-5089. doi: 10.1167/iovs.14-14483.

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

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Abstract

Purpose.: To compare the antioxidant and inflammatory cytokine activities in tears of patients with dry eye syndrome treated with preservative-free versus preserved eye drops.

Methods.: A total of 100 patients with moderate to severe dry eye syndrome were randomly divided into two groups. Fifty patients (group 1) were treated four times with preservative-free 0.1% sodium hyaluronate and 0.1% fluorometholone eye drops in the first month and with preservative-free 0.1% sodium hyaluronate and 0.05% cyclosporine eye drops in the second and third months. Another 50 patients (group 2) were treated with preserved eye drops on the same schedule. Ocular Surface Disease Index, corneal fluorescein staining, Schirmer I test, tear film breakup time, impression cytology, and antioxidant and inflammatory cytokine activities in tears were evaluated.

Results.: Treatment with preservative-free eye drops led to significant improvements in symptoms, tear film breakup time, Schirmer I score, and impression cytologic findings compared to treatment with preserved eye drops (P < 0.05) in patients with dry eye syndrome. There was a statistically significant decrease in the IL-1β, IL-6, IL-12, and TNF-α concentrations and a statistically significant increase in the catalase, peroxiredoxin 2, superoxide dismutase 2 (SOD 2), and thioredoxin mean fluorescence intensity (MFI) of tears in the preservative-free group at 1, 2, and 3 months compared to initial values, respectively (P < 0.05).

Conclusions.: Treatment with preservative-free eye drops is effective against the dry eye syndrome. Preservative-free eye drops seem to be more effective than preserved eye drops in decreasing ocular inflammation and in increasing antioxidant contents in tears of patients with dry eye syndrome.

Introduction
Dry eye syndrome is defined as a multifactorial condition of the tears and ocular surface, characterized by symptoms of discomfort, visual disturbance, and tear film instability, combined with potential damage to the ocular surface. 1 Recently, dry eye syndrome has been reported as an inflammatory disease of the lacrimal functional unit caused by multiple factors. 1 Proinflammatory cytokines and proteolytic enzymes increased by the cornea, conjunctiva, and glandular epithelial cells, as well as by the inflammatory cells, were reported previously. 2,3  
Oxidative stress plays a key role in the aging process, and the ocular surface is one of the biological systems that are most affected by this process. Lipid peroxidation increases in human tears with age. 4 Increase in the oxidative stress status in the conjunctiva of Sjogren syndrome patients appears to have a role in the pathogenesis of dry eye disease. 5 Preservatives in eye drops can induce oxidative stress and an inflammatory response in conjunctival epithelial cells and the rabbit dry eye model. 68  
Topical cyclosporine 0.05% and combined short-term treatment with topical 1% methylprednisolone acetate had the benefit of providing faster symptom relief and improvement of ocular signs. 9 However, to the best of our knowledge, no report has compared the effects of preservative-free and preserved eye drops in dry eye syndrome. 
In this study, we compared the efficacy of preservative-free 0.1% sodium hyaluronate and 0.1% fluorometholone eye drops combined with 0.05% cyclosporine with the efficacy of preserved 0.1% sodium hyaluronate and 0.1% fluorometholone eye drops combined with 0.05% cyclosporine in treating dry eye disease, based on clinical and laboratory methods. 
Methods
This report describes a randomized, parallel-group, case–control study. This study was conducted in accordance with the institutional review board regulations, informed consent regulations, sponsor and investigator obligations, and the Declaration of Helsinki. Written informed consent was obtained from all of the patients before the initiation of the study. 
Patients
The current study included 100 patients (100 eyes) who were at least 21 years of age and diagnosed as having moderate to severe dry eye syndrome. Inclusion criteria included low tear film breakup time (tBUT) (<5 seconds), low Schirmer I test (5 mm/5 min without anesthesia), and mild corneal punctate fluorescein staining (staining score ≥ 1) in either eye (scale 0–3). Exclusion criteria were history of ocular injury, infection, nondry eye ocular inflammation, trauma, or surgery within the prior 6 months and presence of uncontrolled systemic disease. Patients could be withdrawn before the completion of the study because of adverse events, protocol violations, lack of efficacy, or personal reasons. 10  
Study Population and Procedures
Subjects were divided into two groups assigned by simple randomization. Group 1 subjects (50 patients, 50 eyes) were treated with preservative-free 0.1% sodium hyaluronate (Tearin free; DHP Korea, Seoul, Korea) and 0.1% fluorometholone eye drops (Humeron, Hallym, Seoul, Korea) (four times a day) in the first month and with preservative-free 0.1% sodium hyaluronate (four times a day) and 0.05% cyclosporine (Restasis; Allergan, Inc., Irvine, CA, USA) eye drops (twice a day) in the second and third months. Group 2 subjects (50 patients, 50 eyes) were treated with preserved 0.1% sodium hyaluronate (Lacure; Samil, Seoul, Korea) and preserved 0.1% fluorometholone eye drops (Ocumetholone; Samil) (four times a day) in the first month and with preserved 0.1% sodium hyaluronate (four times a day) and 0.05% cyclosporine eye drops (Restasis) (twice a day) in the second and third months. The medication was dispensed open-label. 
Subjective symptoms scoring, tBUT, Schirmer I test (without anesthesia), corneal fluorescein staining, and conjunctival impression cytology 10 were performed by the same investigators (DJ and ECK) before treatment and at 1, 2, and 3 months after treatment. Subjective symptoms were graded on a numerical scale of zero to 4 using the Ocular Surface Disease Index (OSDI) score. 11 The sum of the scores was used in the analysis. 11 The tBUT and Schirmer I tests were performed in a dimly lit room. 10  
Corneal fluorescein staining was examined through slit-lamp evaluation with a yellow barrier filter and cobalt blue illumination. 10 Staining was graded using the Oxford Scheme 6-point scale (from 0 to 5), 12 with each investigator using the same set of photographs (provided by the study sponsor) as a guide. 
Impression cytology was performed on the lower bulbar conjunctiva by using strips of cellulose acetate filter paper (MFS membrane filters; Advantec MFS, Dublin, California, USA), according to a previously described method. 13 The specimens were fixed in 100% alcohol, stained with periodic acid–Schiff, and photographed at a magnification of 400×. 10 The degree of squamous metaplasia was graded from zero to 6 using the grading scheme of Tseng, 14 and goblet cell density was calculated as the number of cells per square millimeter. Both eyes were treated, but only the eye with worse corneal fluorescein staining score at initial enrollment was included in the analysis. 
Tear Samples
Ten-microliter polished micropipettes (Drummond, Broomall, PA, USA) were used to collect tears from the inferior marginal strip, care being taken to minimize ocular surface contact. Tear collection rate was continually monitored. Individual tear samples were collected in 10-minute aliquots; each was immediately transferred to a sterile PCR tube and immediately stored at −80°C until used for the immunoassay. 
Multiplex Bead Analysis
Cytokines, chemokines, and antioxidants were analyzed using a commercial assay system of immunoassay kits and panels (Millipore MILLIPLEX Human Cytokine/Chemokine Panel I Premixed 42 Plex [MPXHCYTO60KPMX42], Millipore MILLIPLEX Human Oxidative Stress Panel Premixed 5 Plex [H0XSTMAG-18K]; Millipore, Billerica, MA, USA) using a magnetic bead-based immunoassay kit (Luminex 200; Luminex Corp., Austin, TX, USA), as previously described. 15 The quantified cytokines, chemokines, soluble receptors, and antioxidants are detailed in Table 1. Tear samples were incubated with antibody-coated capture beads overnight at 4°C. 15 Washed beads were further incubated with biotin-labeled anti-human cytokine antibodies, followed by streptavidin–phycoerythrin incubation. 15 The standard curves of known concentrations of recombinant human cytokines/chemokines were used to convert fluorescence units to concentrations (pg/mL). 15 To calculate molecular concentrations in tear samples, we analyzed the median fluorescent intensity data using a five-parameter logistic or spline curve-fitting method. 15 Thirty microliters of tears is needed to run the multiplex bead analysis. In dry eye patients, it is impossible to get enough volume of tears per person to run the multiplex bead analysis. Thus a collection of two or three patients' tears is used to run one sample of the multiplex bead analysis. 15  
Table 1
 
Cytokines, Chemokines, and Antioxidants for Tear Quantification in Patients With Dry Eye Syndrome
Table 1
 
Cytokines, Chemokines, and Antioxidants for Tear Quantification in Patients With Dry Eye Syndrome
Human Cytokine/Chemokine Panel I EGF, eotaxin, FGF-2, Flt-3 ligand, fractalkine, G-CSF, GM-CSF, GRO, IFN α2, IFNγ, IL-1α, IL-1β, IL-1rα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, MCP-1/CCL2/MCAF, MCP-3/CCL7, MDC/CCL22, MIP-1α, MIP-1β, PDGF-AA, PDGF-AB/BB, RANTES, sCD40L, sIL-2Rα, TGFα, TNFα, TNFβ, VEGF
Human Oxidative Stress Panel  Catalase, SOD 1, SOD 2, PRX2 (PRDX2), TRX1
Statistical Analysis
SPSS software (version 13.0; SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Pairwise comparisons of treatment group categorical variables were performed using the Mann-Whitney U test. Schirmer test, tBUT, and continuous variables were analyzed using the unpaired t-test. Wilcoxon signed rank test was used to compare within-group categorical variable changes from baseline. The repeated measures analysis of variance was used to compare within-group continuous variable changes from baseline. Repeated measures ANOVA was used to compare before and after values of inflammatory cytokines and antioxidants. A two-sided test with P < 0.05 was considered statistically significant. 
Results
The patients were treated from January 2013 to October 2013. There was no statistically significant difference between the two groups in OSDI score, tBUT, Schirmer I score, impression cytology grade, goblet cell density, or corneal staining score at baseline (Table 2). Of the total 100 patients, 46 (92%) patients in group 1 and 43 (86%) patients in group 2 completed the entire protocol. Between 8% and 14% of the patients discontinued their participation because of adverse events such as stinging eyes (6% in group 1, 10% in group 2) (Table 3). 
Table 2
 
Initial Characteristics of Each Group With Dry Eye Syndrome Before Preservative-Free or Preserved Eye Drops Were Administered
Table 2
 
Initial Characteristics of Each Group With Dry Eye Syndrome Before Preservative-Free or Preserved Eye Drops Were Administered
Group 1, O-FMLON Group 2, O-FM
Total patients (eyes) 50 (50) 50 (50)
Age 59.26 ± 6.32 56.75 ± 5.79
Sex, M:F 15:35 12:38
OSDI score 18.69 ± 7.97 19.22 ± 7.45
Tear film BUT, seconds 3.74 ± 1.65 3.47 ± 1.82
Schirmer I, mm 3.20 ± 1.53 3.67 ± 1.94
Impression cytology grade 2.32 ± 0.43 2.16 ± 0.35
Goblet cell density, cells/mm2 109.32 ± 38.64 116.28 ± 45.31
Corneal staining score 1.28 ± 0.93 1.33 ± 0.92
Table 3
 
Disposition of Patients With Dry Eye Syndrome Who Were Treated With Preservative-Free or Preserved Eye Drops at 3-Month Follow-Up
Table 3
 
Disposition of Patients With Dry Eye Syndrome Who Were Treated With Preservative-Free or Preserved Eye Drops at 3-Month Follow-Up
Group 1, O-FMLON Group 2, O-FM
Patients enrolled 50, 100% 50, 100%
Patients completed 46, 92% 43, 86%
Patients discontinued 4, 8% 7, 14%
Adverse events 3, 6% 5, 10%
Other reasons* 1, 2% 2, 4%
Ocular Surface Disease Index Score
The OSDI score was more significantly improved in the preservative-free group than in the preserved group at 2 months (−6.15 ± 0.89, −3.64 ± 0.58, respectively) and 3 months (−6.20 ± 0.76, −4.90 ± 0.62, respectively) (P < 0.05; Fig. 1). 
Figure 1
 
Change in OSDI score from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The decrease in OSDI score was statistically significant in the preservative-free group at 2 and 3 months compared to the preserved group (*P < 0.05 by Mann-Whitney U test).
Figure 1
 
Change in OSDI score from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The decrease in OSDI score was statistically significant in the preservative-free group at 2 and 3 months compared to the preserved group (*P < 0.05 by Mann-Whitney U test).
Tear Film BUT and Schirmer Tear Test
There was a statistically significant improvement in tBUT in the preservative-free group compared to the preserved group at 2 months (2.09 ± 0.27, 1.35 ± 0.21, respectively) and 3 months (2.26 ± 0.32, 1.71 ± 0.25, respectively) (P < 0.05; Fig. 2). 
Figure 2
 
Change in tear film breakup time (tBUT) from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The increase in tBUT (seconds) was statistically significant in the preservative-free group at 2 and 3 months compared to the preserved group (*P < 0.05 by unpaired t-test).
Figure 2
 
Change in tear film breakup time (tBUT) from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The increase in tBUT (seconds) was statistically significant in the preservative-free group at 2 and 3 months compared to the preserved group (*P < 0.05 by unpaired t-test).
Schirmer I score was also significantly improved in the preservative-free group compared to the preserved group at 1 month (0.79 ± 0.13, 0.19 ± 0.08, respectively), 2 months (1.73 ± 0.21, 0.67 ± 0.15, respectively), and 3 months (2.70 ± 0.29, 1.71 ± 0.20, respectively) (P < 0.05; Fig. 3). 
Figure 3
 
Change in Schirmer I test value from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The increase in Schirmer I test value (mm) was statistically significant in the preservative-free group at 1, 2, and 3 months compared to the preserved group (*P < 0.05 by unpaired t-test).
Figure 3
 
Change in Schirmer I test value from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The increase in Schirmer I test value (mm) was statistically significant in the preservative-free group at 1, 2, and 3 months compared to the preserved group (*P < 0.05 by unpaired t-test).
Impression Cytology Grade and Goblet Cell Density
Impression cytology grade was significantly improved in the preservative-free group compared to the preserved group at 1 month (−0.23 ± 0.03, −0.08 ± 0.01, respectively), 2 months (−0.31 ± 0.04, −0.16 ± 0.03, respectively), and 3 months (−0.53 ± 0.07, −0.25 ± 0.03, respectively) (P < 0.05; Figs. 4, 5). 
Figure 4
 
Change in impression cytology grade from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The decrease in impression cytology grade was statistically significant in the preservative-free group at 1, 2, and 3 months compared to the preserved group (*P < 0.05 by Mann-Whitney U test).
Figure 4
 
Change in impression cytology grade from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The decrease in impression cytology grade was statistically significant in the preservative-free group at 1, 2, and 3 months compared to the preserved group (*P < 0.05 by Mann-Whitney U test).
Figure 5
 
Clinical pictures of impression cytology in each group at the initial evaluation and at 3 months (O-FMLON, preservative-free; O-FM, preserved eye drops). At 3 months, there was an increase in goblet cell density (red arrows) from the initial evaluation in the preservative-free group compared to the preserved group (400×, periodic acid-Schiff–hematoxylin stain).
Figure 5
 
Clinical pictures of impression cytology in each group at the initial evaluation and at 3 months (O-FMLON, preservative-free; O-FM, preserved eye drops). At 3 months, there was an increase in goblet cell density (red arrows) from the initial evaluation in the preservative-free group compared to the preserved group (400×, periodic acid-Schiff–hematoxylin stain).
Goblet cell density was also significantly increased in the preservative-free group compared to the preserved group at 2 months (75.28 ± 12.23, 63.56 ± 10.59 cells/mm2, respectively) and 3 months (89.53 ± 17.65, 70.59 ± 15.43, respectively) (P < 0.05; Fig. 6). 
Figure 6
 
Change in goblet cell density from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The increase in goblet cell density (cell/mm2) was statistically significant in the preservative-free group at 2 and 3 months compared to the preserved group (*P < 0.05 by unpaired t-test).
Figure 6
 
Change in goblet cell density from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The increase in goblet cell density (cell/mm2) was statistically significant in the preservative-free group at 2 and 3 months compared to the preserved group (*P < 0.05 by unpaired t-test).
Corneal Staining
There was a statistically significant decrease in the corneal staining score in the preservative-free group compared to the preserved group at 1 month (−0.84 ± 0.11, −0.36 ± 0.05, respectively), 2 months (−1.33 ± 0.26, −0.70 ± 0.12, respectively), and 3 months (−1.52 ± 17.65, −0.27 ± 0.09, respectively) (P < 0.05; Fig. 7). 
Figure 7
 
Change in corneal fluorescein staining score from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The decrease in corneal staining score was statistically significant in the preservative-free group at 1, 2, and 3 months compared to the preserved group (*P < 0.05 by Mann-Whitney U test).
Figure 7
 
Change in corneal fluorescein staining score from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The decrease in corneal staining score was statistically significant in the preservative-free group at 1, 2, and 3 months compared to the preserved group (*P < 0.05 by Mann-Whitney U test).
Inflammatory Cytokines in Tears
In the preservative-free group, the initial concentrations of IL-1β, IL-6, IL-12, and TNF-α were 31.57 ± 1.56, 56.85 ± 2.37, 140.95 ± 8.68, and 15.63 ± 1.20 pg/mL, respectively. In the preserved group, the initial concentrations of IL-1β, IL-6, IL-12, and TNF-α were 28.48 ± 1.44, 61.48 ± 2.50, 129.83 ± 9.73, and 16.41 ± 1.58 pg/mL, respectively. Initially, there was no statistically significant difference between the two groups in the mean IL-1β, IL-6, IL-12, and TNF-α concentrations in tears. 
In the preservative-free group, there was a statistically significant decrease at 1, 2, and 3 months in the IL-1β (14.05 ± 1.13, 11.89 ± 0.93, and 9.55 ± 0.69, respectively), IL-6 (39.74 ± 2.25, 29.37 ± 1.86, and 15.07 ± 1.58, respectively), IL-12 (94.97 ± 4.57, 91.12 ± 4.64, and 76.74 ± 3.95, respectively), and TNF-α (10.16 ± 0.93, 4.77 ± 0.86, and 4.07 ± 0.78, respectively) concentrations in tears compared to initial values (P < 0.05). 
However, in the preserved group, there was no statistically significant decrease at 1, 2, and 3 months in the IL-1β (27.07 ± 1.52, 25.45 ± 1.27, and 23.96 ± 1.38, respectively), IL-6 (59.05 ± 2.87, 56.14 ± 2.45, and 53.42 ± 2.49, respectively), IL-12 (122.98 ± 6.32, 115.15 ± 5.51, and 107.94 ± 5.83, respectively), and TNF-α (14.89 ± 0.85, 14.63 ± 0.73, and 13.85 ± 0.71, respectively) concentrations in tears compared to initial values (P > 0.05) (Fig. 8). 
Figure 8
 
Change in concentrations of inflammatory cytokines in tears from baseline (O-FMLON, preservative-free; O-FM: preserved eye drops). Mean value ± standard error. The decrease in concentrations of IL-1β, IL-6, IL-12, and TNF-α in tears was statistically significant in the preservative-free group at 1, 2, and 3 months compared to initial values, respectively (*P < 0.05 by repeated measures ANOVA).
Figure 8
 
Change in concentrations of inflammatory cytokines in tears from baseline (O-FMLON, preservative-free; O-FM: preserved eye drops). Mean value ± standard error. The decrease in concentrations of IL-1β, IL-6, IL-12, and TNF-α in tears was statistically significant in the preservative-free group at 1, 2, and 3 months compared to initial values, respectively (*P < 0.05 by repeated measures ANOVA).
Antioxidants
In the preservative-free group, the initial mean fluorescence intensity (MFI) of catalase, peroxiredoxin 2, superoxide dismutase 2 (SOD 2), and thioredoxin was 8758.32 ± 412.56, 3263.32 ± 167.58, 13050.23 ± 652.29, and 9141.48 ± 432.59, respectively. In the preserved group, the initial MFI of catalase, peroxiredoxin 2, SOD 2, and thioredoxin was 9707.53 ± 495.35, 3038.63 ± 188.52, 15131.32 ± 793.28, and 7853.38 ± 412.53, respectively. Initially, there was no statistically significant difference between the two groups in the catalase, peroxiredoxin 2, SOD 2, and thioredoxin MFI of tears. 
In the preservative-free group, there was a statistically significant increase at 1, 2, and 3 months in the catalase (14285.57 ± 713.43, 16385.60 ± 853.71, and 17718.84 ± 901.53, respectively), peroxiredoxin 2 (4061.66 ± 201.56, 4091.90 ± 213.38, and 4501.82 ± 223.64, respectively), SOD 2 (19878.09 ± 938.56, 23100.84 ± 1038.52, and 34581.96 ± 1698.29, respectively), and thioredoxin (13358.86 ± 682.38, 16114.33 ± 832.47, and 22641.29 ± 1065.43, respectively) MFI in tears compared to initial values (P < 0.05). 
However, in the preserved group, there was no statistically significant increase at 1, 2, and 3 months in the catalase (10814.65 ± 532.21, 11465.88 ± 634.50, and 11949.10 ± 667.28, respectively), peroxiredoxin 2 (3265.94 ± 179.58, 3438.21 ± 203.65, and 3471.20 ± 228.51, respectively), SOD 2 (17100.88 ± 948.50, 18168.84 ± 973.26, and 19841.80 ± 1035.69, respectively), and thioredoxin (8114.91 ± 455.80, 8708.33 ± 518.64, and 8767.80 ± 532.18, respectively) MFI in tears compared to initial values (P > 0.05) (Fig. 9). 
Figure 9
 
Change in fluorescence intensity of antioxidants in tears from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. MFI, mean fluorescence intensity. The increase in fluorescence intensity of catalase, peroxiredoxin 2, superoxide dismutase 2 (SOD 2), and thioredoxin in tears was statistically significant in the preservative-free group at 1, 2, and 3 months compared to initial values, respectively (*P < 0.05 by repeated measures ANOVA).
Figure 9
 
Change in fluorescence intensity of antioxidants in tears from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. MFI, mean fluorescence intensity. The increase in fluorescence intensity of catalase, peroxiredoxin 2, superoxide dismutase 2 (SOD 2), and thioredoxin in tears was statistically significant in the preservative-free group at 1, 2, and 3 months compared to initial values, respectively (*P < 0.05 by repeated measures ANOVA).
Discussion
The preservatives in most eye drops provide a level of antimicrobial activity in multiuse bottles, limiting secondary bacterial, mycotic, and amoebal ocular infections caused by contaminated solutions and prolonging the shelf life of the drug by preventing biodegradation and maintaining drug potency. 16 However, preservatives that are present in lubricating eye drops or antiseptic substances, such as quaternary ammonium salts, can alter the permeability of the ocular surface epithelium. 15 Benzalkonium chloride (BAK), a component of all multidose eye drop formulas such as those used in the treatment of glaucoma, is known to induce the lysis of cell membranes at the ocular surface, even at very low doses. 17  
Benzalkonium chloride is known to be degraded into hydrogen peroxide (H2O2), 18 which, in even amounts as low as 0.003%, is known to be an ophthalmic irritant and can cause oxidative damage to corneal and conjunctival epithelial cells. 19 It also has the potential to initiate ocular surface damage and subconjunctival inflammation. 20 Even at low concentrations, BAK induces a marked increase in the quantities of inflammatory cytokines such as IL-1, TNFα, C-reactive protein (CRP), IL-10, and IL-12 in human corneal epithelial cells. 7  
Dry eye was redefined in a report from the Dry Eye WorkShop (DEWS). 1 Increased osmolarity of the patient's tears, inflammation of the ocular surface, and potential damage to the ocular surface were clearly observed in dry eye syndrome. 18 Excessive oxidative stress can cause ocular surface epithelial damage and a decrease in aqueous secretory function. 21  
A stable rabbit dry eye model was induced by topical 0.1% BAK for 5 weeks, and after BAK removal, the signs of dry eye were sustained for 2 weeks (for the mixed type of dry eye) or for at least 3 weeks (for mucin-deficient dry eye). 8 Therefore, we hypothesized that the preservative may induce dry eye disorder by causing oxidative and inflammatory damage to the corneal and conjunctival epithelial cells. 
Topical cyclosporine A 0.05% is an effective treatment for dry eye disorder. 22 Furthermore, additional short-term use of steroids results in more rapid symptom relief and improvement of inflammatory ocular signs. 9 Jonisch et al. 23 reported that topical nonpreserved 0.01% dexamethasone could be an effective therapy for recalcitrant chronic ocular surface disease. Pianini et al. 24 reported that both the preservative-free and preserved fixed antibiotics and steroid eye drop combinations are safe and effective in controlling postoperative inflammation after cataract surgery. Astakhov et al. 25 also reported that preservative-free lacrimal substitute (Hylabak) and preserved lacrimal substitute (Systane) treatments were well tolerated and that there were no serious adverse events or discontinuations because of adverse events or other safety-related reasons, and no systemic adverse events for 3 months in patients after LASIK. 25  
However, no report has compared the effects of preservative-free and preserved lubricant and steroid eye drops in dry eye syndrome based on clinical and laboratory methods. In this study, we compared the efficacy of preservative-free 0.1% sodium hyaluronate and 0.1% fluorometholone eye drops combined with 0.05% cyclosporine with the efficacy of preserved 0.1% sodium hyaluronate and 0.1% fluorometholone eye drops combined with 0.05% cyclosporine in treating dry eye disease. Treatment with preservative-free eye drops led to significant improvements in OSDI score, tBUT, Schirmer I score, and impression cytologic findings compared to treatment with the preserved eye drops (P < 0.05) in patients with dry eye syndrome. Also, antioxidants (catalase, peroxiredoxin 2, SOD 2, and thioredoxin) activities were significantly elevated, and concentrations of anti-inflammatory cytokines (IL-1β, IL-6, IL-12, and TNF-α) were significantly decreased in the preservative-free group at 1, 2, and 3 months compared to initial values, respectively (P < 0.05). In the preserved group, however, there was no difference in antioxidant activities and concentrations of anti-inflammatory cytokines at 1, 2, and 3 months compared to initial values (P > 0.05). 
Antioxidant content in minimally stimulated tears was not significantly different from that in older subjects (398 ± 160 μmol/L) and younger subjects (348 ± 159 μmol/L; P = 0.0915). 26 Sod1 knockout mice decreased production of both stimulated and nonstimulated tears and decreased total protein secretion from the lacrimal glands. 27  
There has been no report, however, on specific antioxidant activities in tears of dry eye syndrome patients. For the first time, we report that antioxidants were increased in the tears of patients using preservative-free eye drops compared to preserved eye drops. Still, the increase of antioxidants and decrease of inflammatory response may not be a direct effect of preservative-free eye drops but secondary effects of ocular surface improvement due to preservative-free eye drops. 
In the preserved group, we did not find a significant difference in antioxidants and inflammatory cytokines because preservatives in the eye drops might affect antioxidant activities and inflammatory response in ocular surface tears in spite of anti-inflammatory and lubricant treatments. Preservatives may have bad influence on treatments for dry eye disorder by causing oxidative and inflammatory damage to the corneal and conjunctival epithelial cells. 19,20  
In conclusion, preservative-free eye drops can improve subjective symptoms, tBUT, Schirmer I score, and impression cytological findings more significantly than preserved eye drops in patients with dry eye syndrome. Preservative-free eye drops seem to be more effective in decreasing ocular inflammation and in increasing antioxidant contents in tears of patients with dry eye syndrome. However, a large multicenter trial with prolonged follow-up is needed to conclusively determine the efficacy of preservative-free eye drops. 
We used preservative-free 0.05% cyclosporine eye drops in both groups because commercial eye drops with preserved 0.05% cyclosporine were not available. However, group 1 subjects were treated four times with preservative-free 0.1% sodium hyaluronate and group 2 subjects with preserved 0.1% sodium hyaluronate in the second and third months. Twice-a-day use of preservative-free 0.05% cyclosporine eye drops, the same condition in the two groups, might not be expected to influence the groups differently. 
Acknowledgments
Supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT (Information & Communication Technology) & Future Planning (No. 2012R1A1A1038648), and the Institute of Clinical Medicine Research of Bucheon St. Mary's Hospital Research Fund (BCMC13LH03). 
Disclosure: D. Jee, None; S.H. Park, None; M.S. Kim, None; E.C. Kim, None 
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Figure 1
 
Change in OSDI score from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The decrease in OSDI score was statistically significant in the preservative-free group at 2 and 3 months compared to the preserved group (*P < 0.05 by Mann-Whitney U test).
Figure 1
 
Change in OSDI score from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The decrease in OSDI score was statistically significant in the preservative-free group at 2 and 3 months compared to the preserved group (*P < 0.05 by Mann-Whitney U test).
Figure 2
 
Change in tear film breakup time (tBUT) from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The increase in tBUT (seconds) was statistically significant in the preservative-free group at 2 and 3 months compared to the preserved group (*P < 0.05 by unpaired t-test).
Figure 2
 
Change in tear film breakup time (tBUT) from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The increase in tBUT (seconds) was statistically significant in the preservative-free group at 2 and 3 months compared to the preserved group (*P < 0.05 by unpaired t-test).
Figure 3
 
Change in Schirmer I test value from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The increase in Schirmer I test value (mm) was statistically significant in the preservative-free group at 1, 2, and 3 months compared to the preserved group (*P < 0.05 by unpaired t-test).
Figure 3
 
Change in Schirmer I test value from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The increase in Schirmer I test value (mm) was statistically significant in the preservative-free group at 1, 2, and 3 months compared to the preserved group (*P < 0.05 by unpaired t-test).
Figure 4
 
Change in impression cytology grade from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The decrease in impression cytology grade was statistically significant in the preservative-free group at 1, 2, and 3 months compared to the preserved group (*P < 0.05 by Mann-Whitney U test).
Figure 4
 
Change in impression cytology grade from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The decrease in impression cytology grade was statistically significant in the preservative-free group at 1, 2, and 3 months compared to the preserved group (*P < 0.05 by Mann-Whitney U test).
Figure 5
 
Clinical pictures of impression cytology in each group at the initial evaluation and at 3 months (O-FMLON, preservative-free; O-FM, preserved eye drops). At 3 months, there was an increase in goblet cell density (red arrows) from the initial evaluation in the preservative-free group compared to the preserved group (400×, periodic acid-Schiff–hematoxylin stain).
Figure 5
 
Clinical pictures of impression cytology in each group at the initial evaluation and at 3 months (O-FMLON, preservative-free; O-FM, preserved eye drops). At 3 months, there was an increase in goblet cell density (red arrows) from the initial evaluation in the preservative-free group compared to the preserved group (400×, periodic acid-Schiff–hematoxylin stain).
Figure 6
 
Change in goblet cell density from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The increase in goblet cell density (cell/mm2) was statistically significant in the preservative-free group at 2 and 3 months compared to the preserved group (*P < 0.05 by unpaired t-test).
Figure 6
 
Change in goblet cell density from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The increase in goblet cell density (cell/mm2) was statistically significant in the preservative-free group at 2 and 3 months compared to the preserved group (*P < 0.05 by unpaired t-test).
Figure 7
 
Change in corneal fluorescein staining score from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The decrease in corneal staining score was statistically significant in the preservative-free group at 1, 2, and 3 months compared to the preserved group (*P < 0.05 by Mann-Whitney U test).
Figure 7
 
Change in corneal fluorescein staining score from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. The decrease in corneal staining score was statistically significant in the preservative-free group at 1, 2, and 3 months compared to the preserved group (*P < 0.05 by Mann-Whitney U test).
Figure 8
 
Change in concentrations of inflammatory cytokines in tears from baseline (O-FMLON, preservative-free; O-FM: preserved eye drops). Mean value ± standard error. The decrease in concentrations of IL-1β, IL-6, IL-12, and TNF-α in tears was statistically significant in the preservative-free group at 1, 2, and 3 months compared to initial values, respectively (*P < 0.05 by repeated measures ANOVA).
Figure 8
 
Change in concentrations of inflammatory cytokines in tears from baseline (O-FMLON, preservative-free; O-FM: preserved eye drops). Mean value ± standard error. The decrease in concentrations of IL-1β, IL-6, IL-12, and TNF-α in tears was statistically significant in the preservative-free group at 1, 2, and 3 months compared to initial values, respectively (*P < 0.05 by repeated measures ANOVA).
Figure 9
 
Change in fluorescence intensity of antioxidants in tears from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. MFI, mean fluorescence intensity. The increase in fluorescence intensity of catalase, peroxiredoxin 2, superoxide dismutase 2 (SOD 2), and thioredoxin in tears was statistically significant in the preservative-free group at 1, 2, and 3 months compared to initial values, respectively (*P < 0.05 by repeated measures ANOVA).
Figure 9
 
Change in fluorescence intensity of antioxidants in tears from baseline (O-FMLON, preservative-free; O-FM, preserved eye drops). Mean value ± standard error. MFI, mean fluorescence intensity. The increase in fluorescence intensity of catalase, peroxiredoxin 2, superoxide dismutase 2 (SOD 2), and thioredoxin in tears was statistically significant in the preservative-free group at 1, 2, and 3 months compared to initial values, respectively (*P < 0.05 by repeated measures ANOVA).
Table 1
 
Cytokines, Chemokines, and Antioxidants for Tear Quantification in Patients With Dry Eye Syndrome
Table 1
 
Cytokines, Chemokines, and Antioxidants for Tear Quantification in Patients With Dry Eye Syndrome
Human Cytokine/Chemokine Panel I EGF, eotaxin, FGF-2, Flt-3 ligand, fractalkine, G-CSF, GM-CSF, GRO, IFN α2, IFNγ, IL-1α, IL-1β, IL-1rα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, MCP-1/CCL2/MCAF, MCP-3/CCL7, MDC/CCL22, MIP-1α, MIP-1β, PDGF-AA, PDGF-AB/BB, RANTES, sCD40L, sIL-2Rα, TGFα, TNFα, TNFβ, VEGF
Human Oxidative Stress Panel  Catalase, SOD 1, SOD 2, PRX2 (PRDX2), TRX1
Table 2
 
Initial Characteristics of Each Group With Dry Eye Syndrome Before Preservative-Free or Preserved Eye Drops Were Administered
Table 2
 
Initial Characteristics of Each Group With Dry Eye Syndrome Before Preservative-Free or Preserved Eye Drops Were Administered
Group 1, O-FMLON Group 2, O-FM
Total patients (eyes) 50 (50) 50 (50)
Age 59.26 ± 6.32 56.75 ± 5.79
Sex, M:F 15:35 12:38
OSDI score 18.69 ± 7.97 19.22 ± 7.45
Tear film BUT, seconds 3.74 ± 1.65 3.47 ± 1.82
Schirmer I, mm 3.20 ± 1.53 3.67 ± 1.94
Impression cytology grade 2.32 ± 0.43 2.16 ± 0.35
Goblet cell density, cells/mm2 109.32 ± 38.64 116.28 ± 45.31
Corneal staining score 1.28 ± 0.93 1.33 ± 0.92
Table 3
 
Disposition of Patients With Dry Eye Syndrome Who Were Treated With Preservative-Free or Preserved Eye Drops at 3-Month Follow-Up
Table 3
 
Disposition of Patients With Dry Eye Syndrome Who Were Treated With Preservative-Free or Preserved Eye Drops at 3-Month Follow-Up
Group 1, O-FMLON Group 2, O-FM
Patients enrolled 50, 100% 50, 100%
Patients completed 46, 92% 43, 86%
Patients discontinued 4, 8% 7, 14%
Adverse events 3, 6% 5, 10%
Other reasons* 1, 2% 2, 4%
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