JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS Volume 30, Number 7, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/jop.2013.0050
Effect of Hypotonic 0.18% Sodium Hyaluronate Eyedrops on Inflammation of the Ocular Surface in Experimental Dry Eye Han Jin Oh,1 Zhengri Li,1 Soo-Hyun Park,2 and Kyung Chul Yoon1
Abstract
Purpose: To investigate the efficacy of hypotonic 0.18% sodium hyaluronate (SH) eyedrops in a mouse model of experimental dry eye (EDE). Methods: EDE was induced in C57BL/6 mice by a subcutaneous scopolamine injection and an air draft. The mice were divided into 4 groups according to topical treatment regimens: EDE control, isotonic 0.5% carboxymethycellulose (CMC), isotonic 0.1% SH, and hypotonic 0.18% SH. Tear volume, corneal smoothness, and corneal staining scores were measured at 5 and 10 days of EDE. Multiplex immunobead assay, immunohistochemistry, and flow cytometry for proinflammatory cytokines, chemokines, and inflammatory molecules were performed at 10 days of EDE. Results: The 0.18% SH group had a significantly lower corneal smoothness and staining scores than the 0.5% CMC and 0.1% SH groups at 10 days of EDE (P < 0.05). The 0.18% SH group showed significantly low levels of tumor necrosis factor-a (TNF-a), interleukin (IL)-1b, monokine induced by interferon-g, and interferon-ginducible protein 10 compared with the other groups (P < 0.05). The mean percentages of CD4 + CXCR3 +, CD40 + , and CD44 + cells in the conjunctiva were significantly lower in the 0.18% SH group than in the other groups (P < 0.05). In addition, the 0.1% SH group showed lower levels of TNF-a and IL-1b and percentages of CD40 + and CD44 + cells than the EDE and 0.5% CMC groups. Conclusions: Hypotonic 0.18% SH eyedrops are more effective in improving ocular surface irregularity and staining and decreasing inflammatory cytokines, chemokines, and cells on the ocular surface compared with isotonic 0.5% CMC or 0.1% SH eyedrops in the treatment of EDE.
Introduction
D
ry eye disease is the most common problem of the ocular surface, with a suggested prevalence of 5%– 30% in those aged 50 years and older.1 It is a multi-factorial disease of the tear and ocular surface, which results in various ocular symptoms, visual disturbance, tear film instability, and potential damage to the ocular surface.2,3 Inflammation of the ocular surface and hyperosmolarity of the tear film play a pivotal role in the pathogenesis of dry eye.4,5 Application of artificial tears is the mainstay for the treatment of dry eye disease. There are many commercially available topical lubricants, which contain various active ingredients such as sodium hyaluronate (SH) and carboxymethylcellulose (CMC). These agents can supply water
contents for aqueous inadequacy and protect the ocular surface according to each active ingredient.6–8 However, these conventional artificial tears provide only temporary symptom relief. In addition, the approach cannot resolve the causative pathologic factors of dry eye, including tear film hyperosmloarity and associated inflammation.9 This knowledge has influenced the development of a concept that hypotonic artificial tears may be beneficial for the treatment of dry eye. Until now, several studies have reported that hypotonic solutions can improve clinical symptoms and signs of dry eye disease.10,11 In addition, comparative studies between isotonic and hypotonic artificial tears have indicated that hypotonic solutions are more effective in improving the subjective symptoms, ocular surface staining, and some tear film parameters.12,13 However,
1 Department of Ophthalmology, Chonnam National University Medical School and Hospital, Center for Creative Biomedical Scientists at Chonnam National University, Gwangju, Korea. 2 Bio-Therapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea.
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previous studies on hypotonic artificial tears only evaluated the changes of clinical symptoms and signs and did not investigate the parameters associated with ocular surface inflammation. The purpose of the present study was to compare the efficacy of commercially available hypotonic 0.18% SH, isotonic 0.1% SH, and isotonic 0.5% CMC artificial tear eyedrops in a mouse model of desiccating stress-induced experimental dry eye (EDE), by evaluating the changes of tear production, corneal surface irregularity and staining, inflammatory cytokines, CD4 + T cells, and inflammatory molecules such as CD40 on the ocular surface as well as conjunctival goblet cell density. In addition, we examined the expression of CD44 on the ocular surface, because CD44 is known as a hyaluronate receptor in T cells and the hyaluronate-CD44 interaction mediates the homing and trafficking of CD4 + T cells both in vivo and in vitro.14–16
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analysis were performed. Each group consisted of 5 animals (10 eyes), and the experiments were performed on 4 different sets of mice.
Measurement of tear production Tear volume was measured with phenol red-impregnated cotton threads (Zone-Quick; Oasis, Glendora, CA) as previously described.21 The tips of the threads were placed on the peripheral conjunctiva at the lateral canthus for 20 s. The tear volume, expressed in millimeters of thread wet by the tear and turned red color, was evaluated under a microscope (SMZ 1500; Nikon, Melville, NY). The measured uptake of tear fluid in millimeters was compared with a standard curve prepared from cotton threads of a known uptake volume of a stock basic solution (1,500 mL of 0.9% saline and 5 mL of 5 N NaOH) over 20 s, with volumes in the range that would be expected in mouse tear.
Methods
Evaluation of corneal irregularity severity
This research protocol was approved by the Chonnam National University Medical School Research Institutional Animal Care and Use Committee. The maintenance of animals and all in vivo experiments were performed in accordance with institutional guidelines and the ARVO statement for the Use of Animals in Ophthalmic and Vision Research.
Reflected images of a white ring from the fiber-optic ring illuminator of the stereoscopic zoom microscope (SMZ 1500; Nikon, Tokyo, Japan) were taken without anesthesia. Corneal smoothness was evaluated by grading the distortion of a white ring reflected off the corneal epithelium in digital images. The corneal irregularity severity score was calculated using a 5-point scale (0–5) based on the number of distorted quarters in the reflected ring: 0, no distortion; 1, distortion in 1 quarter; 2, distortion in 2 quarters; 3, distortion in 3 quarters; 4, distortion in all 4 quarters; and 5, severe distortions, in which no ring could be recognized.20
Animal and EDE Eight-week-old female C57BL/6 mice were used in these experiments. EDE was induced by a subcutaneous injection of 0.5 mg/0.2 mL scopolamine hydrobromide (Sigma Aldrich, St. Louis, MO) in alternating hindquarters 4 times a day (8 am, 11 am, 2 pm, and 5 pm) with a standard desiccating environment that was created by placing the mice between 2 fans to obtain a continuous air flow (15 L/min) in a 25C room with an ambient humidity of 30% for 18 h per day, as previously described.17–21 During these experiments, the animals’ behavior, food intake, and water intake were not restricted.
Materials Commercially available artificial tear eyedrops were used. Kynex 2 (Alcon Korea, Seoul, Korea) was a hypotonic (150 mOsm/L) agent that contains SH at a concentration of 0.18%. Kynex (Alcon Korea) was an isotonic (284 mOsm/L) agent that contains 0.1% SH. Refresh plus (Allergan, Irvine, CA) was also an isotonic (318 mOsm/L) agent that contained CMC at a concentration of 0.5%. All products were preservative free.
Experimental design The mice were randomly divided into 4 groups according to topical regimens that were administered as follows: EDE controls (received no eyedrops), EDE + isotonic 0.5% CMC (Refresh plus), EDE + isotonic 0.1% SH (Kynex), and EDE + hypotonic 0.18% SH (Kynex2). All treatment groups received 2 mL of eyedrops 4 times a day. Tear volume, corneal smoothness score, and keratoepitheliopathy score were measured at 5 and 10 days of EDE. At 10 days, the mice were euthanized, and then multiplex immunobead assay, histology, immunohistochemistry, and flow cytometric
Evaluation of corneal staining scores (keratoepitheliopathy scores) After instillation of 1% fluorescein (1 mL volume) into the inferior conjunctival sac, punctuate staining on the corneal surface was evaluated in a masked fashion using slit-lamp biomicroscopy under a cobalt blue light. The intensity of staining was graded using a 3-point scale (0–3): 0, no staining; 1, superficial stippling micropunctate staining; 2, macropunctate staining with some coalescent areas; and 3, numerous coalescent macropunctate areas and/or patch). The cornea was divided into 5 regions: central, inferior, nasal, temporal, and superior. The scores of the 5 areas were summed to obtain a total score for each eye, ranging from 0 to 15.22
Multiplex immunobead assay The levels of proinflammatory cytokines; tumor necrosis factor-a (TNF-a) and interleukin (IL)-1b, and chemokine ligands; monokine induced by interferon-g (MIG/CXCL9) and interferon-g-inducible protein (IP-10/CXCL10), in the conjunctiva were measured using a multiplex immunobead assay (Luminex 200; Luminex Corp., Austin, TX), as previously described.20 Conjunctival tissues were obtained (4 eyes per each group) and were stored at - 70C until use. Approximately 10 mg of the tissue was transferred to a 2.0 mL microcentrifuge tube containing 100 mL of 1 · extraction reagent (Tissue extraction reagents I; BioSourceInvitrogen, San Diego, CA) that was supplemented with phenylmethanesulfonyl fluoride in ethanol as a protease inhibitor on the ice for 30 min. Then, lysing process was
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performed using TissueLyser (Qiagen, Hilden, Germany). The tissue was homogenized for 30 s until the sample was in a consistent solution and then centrifuged (4C for 10 min at 12,000 rpm). After centrifugation, the supernatant was collected and placed in a new microcentrifuge tube on ice. Protein quantification of the supernatants was performed, and 1 mg/mL of protein were added to wells containing the appropriate cytokine and chemokine-specific bead mixture that included mouse monoclonal antibodies which were specific for TNF-a, IL-1b, MIG, and IP-10 (Panomics, Santa Clara, CA) for 60 min. Serial dilutions of cytokines and chemokines were also added to wells in the sample plate as the supernatant samples to generate a standard curve. The plate was incubated for 30 min in the dark at room temperature by a biotinylated detection antibody. The reactions were detected using an analysis system (xPONET, Austin, TX) after the addition of streptavidin-phycoerythrin.
Histology Eyes and adnexa were surgically excised, fixed in 4% paraformaldehyde, and embedded in paraffin. Then, the tissue was sectioned by 6 mm and stained with periodic acidSchiff reagent. Sections from each group were examined and photographed with a microscope (Olympus, Tokyo, Japan) that was equipped with a digital camera. Goblet cell density in the superior and inferior conjunctiva was measured in 3 sections of each eye using image analysis software (Media Cybernetics, Silver Spring, MD) and was expressed as the number of goblet cells per 100 mm.
Immunohistochemistry Immunohistochemistry was performed to detect the relative expression of IL-6 in the epithelium and superficial stroma of the conjunctiva. Preparation of the tissue sections and immunohistochemistry were performed according to a previously described method.20 After being surgically excised, the conjunctival tissues from each of the groups were embedded in OTC compound (VWR, Suwanee, GA) and flash frozen in liquid nitrogen. Sagittal 8 mm sections were cut with cryostat. The cryosections were fixed in acetone at - 20C for 10 min.
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After fixation, endogenous peroxidases were quenched with 0.3% hydrogen peroxide in phosphate-buffered saline (PBS), and 20% normal serum in PBS were sequentially applied to the sections. Conjunctival sections from each group were incubated with goat monoclonal anti-mouse IL-6 antibodies (Santa Cruz Biotechnology, Heidelberg, Germany) and then, rabbit anti-goat secondary antibodies were applied. The samples were incubated with avidin-peroxidase, then incubated with 3,3¢ diaminobenzidine peroxidase substrate, and counterstained stained with Mayer’s hematocylin.
Flow cytometry Flow cytometry was performed to quantify CD4 + CXCR3 +, CD40 +, and CD44 + cells from the conjunctiva as previously described, with modifications.23 The conjunctival tissue obtained from each group (4 eyes per group) was immediately incubated with 1 mL of Dulbecco’s modified Eagle’s medium containing PBS with 1% fetal bovine serum (FBS) at 37C via a 5% CO2 incubator, and these tissues were subsequently digested in 0.5 mg/mL collagenase type D (Roche Applied Science, Indianapolis, IN). After incubation, the tissues were disrupted by gently grinding with syringe plunder and passage through a cell strainer with a pore size of 100 mm. The obtained cells were then centrifuged (for 5 min, at 1,500 rpm) and resuspended in PBS with 1% FBS. Cell suspensions were then counted, and viability was determined by a trypan blue exclusion assay. The samples were incubated with monoclonal antibodies. For detecting CD4 + CXCR3 + (double-stained) cells, the samples were incubated with fluorescein-conjugated anti-mouse CD4 antibody (0.5 mg/mL; BD Biosciences, San Jose, CA) and phycoerythrin-conjugated anti-mouse CXCR3 antibody (0.5 mg/mL; BD Biosciences), and isotype control antibody at 37C for 30 min. In addition, they were incubated with phycoerythrin-conjugated anti-mouse CD40 antibody (0.5 mg/mL; BD Biosciences) or phycoerythrinconjugated anti-mouse CD44 antibody (0.5 mg/mL; BD Biosciences). After 30 min of incubation, the cells were washed in PBS. The cells were then centrifuged thrice in 1 mL of PBS (for 5 min at 1,000 rpm) and resuspended. The
FIG. 1. Mean corneal smoothness scores (A) and representative photographs (B) in the experimental dry eye (EDE), isotonic 0.5% carboxymethylcellulose (CMC)treated, isotonic 0.1% sodium hyaluronate (SH)-treated, and hypotonic 0.18% SH-treated groups at days 5 and 10. *P < 0.05 compared with the EDE group, {P < 0.05 compared with the CMC group, and {P < 0.01 compared with the 0.1% SH group.
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FIG. 2. Mean corneal staining scores (A) and representative photographs (B) in the EDE, isotonic 0.5% CMCtreated, isotonic 0.1% SHtreated, and hypotonic 0.18% SH-treated groups at days 5 and 10. *P < 0.05 compared with the EDE group, {P < 0.05 compared with the CMC group, and {P < 0.01 compared with the 0.1% SH group.
number of stained cells was counted by an FACSCalibur cytometer with CellQuest software (BD Biosciences).
Statistical analysis Results are presented as the mean – SEM. The statistical significance of differences between the groups was determined by the 1-way analysis of variance test with Tukey post hoc analysis using the SPSS 17.0 software (SPSS, Inc., Chicago, IL). A P-value less than 0.05 was considered statistically significant.
Results Tear volume Five days after the induction of EDE, the mean tear volume was 0.023 – 0.007 mL in the EDE group, 0.024 – 0.007 mL in the CMC group (P = 0.80, vs. the EDE group), 0.024 –
FIG. 3. Concentrations of tumor necrosis factor-a (TNFa) (A), interleukin (IL)-1b (B), monokine induced by interferon-g (MIG) (C), and interferon-g-inducible protein (IP-10) (D) in the conjunctiva of the EDE, isotonic 0.5% CMCtreated, isotonic 0.1% SHtreated, and hypotonic 0.18% SH-treated groups. *P < 0.05 compared with the EDE group, {P < 0.05 compared with the CMC group, and { P < 0.01 compared with the 0.1% SH group.
0.006 mL in the 0.1% SH group (P = 0.88, vs. the EDE group; P = 0.88, vs. the CMC group), and 0.025 – 0.006 mL in the hypotonic 0.18% SH group (P = 0.57, vs. the EDE group, P = 0.96, vs. the CMC group, P = 0.57, vs. the 0.1% SH group). The findings for the mean tear volume in all groups at 10 days of EDE were similar to those at 5 days. There were no significant differences between the groups.
Corneal irregularity severity scores At 5 days of EDE, the mean corneal irregularity scores were 3.5 – 0.78 in the EDE group, 2.9 – 0.67 in the CMC group (P = 0.25, vs. the EDE group), 2.6 – 0.60 in the 0.1% SH group (P = 0.11, vs. the EDE group, P = 0.58, vs. the CMC group), and 1.8 – 0.47 in the hypotonic 0.18% SH group (P < 0.01, vs. the EDE group, P = 0.02, vs. the CMC group, P = 0.14, vs. the 0.1% SH group). At 10 days of EDE, the 0.18% SH group showed significantly lower corneal
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FIG. 4. Immunohistochemistry for IL-6 in the conjunctiva of the EDE, isotonic 0.5% CMC-treated, isotonic 0.1% SH-treated, and hypotonic 0.18% SH-treated groups.
irregularity scores compared with the EDE, CMC, and 0.1% SH groups (P < 0.05). The other 3 groups were comparable in corneal irregularities at 10 days of EDE (Fig. 1A, B).
Corneal staining scores At 5 and 10 days after EDE induction, the corneal staining scores were significantly lower in the hypotonic 0.18% SH-treated group compared with the EDE, 0.5% CMC, and 0.1% SH-treated groups (P < 0.05). At 10 days of EDE, the 0.1% SH-treated group showed significantly lower corneal staining scores than those in the EDE group (P = 0.02) (Fig. 2A). Representative biomicroscopic photographs showing the degrees of corneal staining in each group at 5 and 10 days of EDE are shown in Fig. 2B.
Levels of inflammatory cytokines/chemokines in conjunctiva Multiplex immunobead assay showed that the concentrations of TNF-a, IL-1b, MIG, and IP-10 in conjunctival tissues
of the hypotonic 0.18% SH-treated group were significantly lower compared with the EDE, CMC, and 0.1% SH-treated groups (P < 0.05). In addition, the 0.1% SH-treated group showed significantly lower levels of TNF-a and IL-1b than the EDE- and CMC-treated groups (P < 0.05) (Fig. 3). However, the levels of MIG and IP-10 were comparable among the EDE-, CMC-, and 0.1% SH-treated groups. Immunohistochemical examination showed that IL-6 in the conjunctiva was strongly expressed in the EDE group. However, relatively weak staining was observed in both SH groups (Fig. 4).
Histology At 10 days after EDE induction, the conjunctival goblet cell density (cells/100 mm) was 8.00 – 2.16 in the EDE group, 9.86 – 2.41 in the 0.5% CMC-treated group (P = 0.06, vs. the EDE group), 10.86 – 2.41 in the 0.1% SH-treated group (P = 0.07, vs. the EDE group, P = 0.59, vs. the 0.5% CMC group), and 17.29 – 2.50 in the hypotonic 0.18% SH-treated group (P = 0.02, vs. the EDE group, P = 0.02, vs.
FIG. 5. Mean number of goblet cells (A) and representative photographs (B) in the EDE, isotonic 0.5% CMC, isotonic 0.1% SH, and hypotonic 0.18% SH-treated groups. *P < 0.05 compared with the EDE group, {P < 0.05 compared with the CMC group, and { P < 0.01 compared with the 0.1% SH group.
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Table 1. Mean Percentage of CD4 + CXCR3 +, CD40 +, and CD44 + Cells in the Conjunctiva of the Experimental Dry Eye, 0.5% Carboxymethylcellulose, Isotonic 0.1% Sodium Hyaluronate, and Hypotonic 0.18% SH Groups, Measured by Flow Cytometry Percentages of positive expression Marker CD4, CXCR3 CD40 CD44
EDE
0.5% CMC
0.1% SH
0.18% SH
83.75 – 12.87 57.25 – 13.77 82.00 – 9.83
78.75 – 12.99 51.00 – 5.29 79.50 – 9.15
71.00 – 11.16 44.25 – 15.71a,b 65.00 – 14.54 a,b
60.00 – 14.97a,b,c 22.75 – 10.03a,b,c 53.50 – 15.22a,b,c
Data are expressed as the mean – standard deviation. a P < 0.05 compared with the EDE group. b P < 0.05 compared with the CMC group. c P < 0.01 compared with the 0.1% SH group. CMC, carboxymethycellulose; EDE, experimental dry eye; SH, sodium hyaluronate.
the 0.5% CMC group, P = 0.02, vs. the 0.1% SH group) (Fig. 5A). Representative histologic findings showing the conjunctival goblet cell density in each group at 10 days of EDE are also shown in Fig. 5B.
Flow cytometric analysis Flow cytomeric data are summarized in Table 1. The mean percentages of CD4 + CXCR3 + cells were 83.75% – 12.87% in the EDE group, 78.75% – 12.99% in the 0.5% CMCtreated group (P = 0.34, vs. the EDE group), 71.00% – 11.16% in the 0.1% SH-treated group (P = 0.08, vs. the EDE group, P = 0.23, vs. the 0.5% CMC group), and 60.00% – 14.97% in the hypotonic 0.18% SH-treated group (P < 0.01, vs. the EDE group, P = 0.01, vs. the 0.5% CMC group, P = 0.03, vs. the 0.1% SH group). The mean percentages of CD40 + and CD44 + cells in the hypotonic 0.18% SH group were significantly lower compared with the EDE, 0.5% CMC, and 0.1% SH-treated groups (P < 0.05). Furthermore, the 0.1% SH group had
FIG. 6. Representative histograms showing percentage of CD4 + CXCR3 + doublestained T cells in the conjunctiva of the EDE (A), 0.5% CMC-treated (B), isotonic 0.1% SH-treated (C), and hypotonic 0.18% SHtreated groups (D).
lower percentages of CD40 + and CD44 + cells compared with the EDE and 0.5% CMC groups (P < 0.05). Histograms and percentages of CD4 + CXCR3 + T cells, CD40 + cells, and CD44 + cells from representative samples in the EDE, CMC, 0.1% SH, and 0.18% SH groups are presented in Figs. 6–8, respectively.
Discussion Recently, it has been widely recognized that inflammation plays a key role in the pathogenesis of dry eye disease. Both hyperosmolar stress and tear film hyperosmolarity induce a cascade of inflammatory events in the ocular surface epithelium, along with the generation of inflammatory cytokines.24,25 Consequently, these chronic inflammatory events lead to the apoptosis of ocular surface epithelium cells and goblet cells, resulting in dry eye.26–28 In addition, tear film osmolarity increased by approximately 30–40 mOsm/L in patients with dry eye.29 On the basis of the role of hyperosmolarity in the pathogenesis, hypotonic artificial tears
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FIG. 7. Representative histograms showing percentage of CD40-positive cells in the conjunctiva of the EDE (A), 0.5% CMC-treated (B), isotonic 0.1% SH-treated (C), and hypotonic 0.18% SHtreated groups (D).
have been used in an attempt to treat dry eye disease.8,10–13 Several hypotonic solutions with various brand names are currently marketed in Europe and Asia. Desiccating stress stimulated by environmental and pharmacological means has been used for the induction of dry eye in several different mouse strains, including C57BL/6 mice.17–21 Desiccating ocular stress has been known to induce auto-reactive T cells that cause Sjo¨gren’s syndrome-like inflammation in the cornea, conjunctiva, and lacrimal gland.19 We have previously found that desiccating stress stimulated the expression of inflammatory cytokines (IL-6 and TNF-a) and Th-1 chemokines (CCL3, CCL4, CCL5, CXCL9, and CXCL10) and their receptors (CCR5 and CXCR3) in the tear film and ocular surface of clinical and EDE.20,21,23,30,31 SH has good pseudoplastic and elastic properties that help stabilize tear film and enhance the residence times both during and between blinking.32 Moreover, previous experimental studies have shown that SH has wound-healing effects, including stimulation of migration, adhesion, and proliferation of the corneal epithelium.33–35 In addition, in vivo corneal abrasion models have shown the dose-
dependent ability of hyaluronate to reduce wound size.36 Clinically, Brignole et al.37 demonstrated that hypotonic 0.18% SH tended to show faster recovery in superficial keratitis and more improvement of subjective symptoms than 0.5% CMC eyedrops in patients with moderate dry eye. In the present study, we demonstrated that 0.18% SH eyedrops showed better results in corneal irregularity and staining scores compared with 0.5% CMC and 0.1% SH eyedrops. Although there is controversy about whether SH is superior to the other ingredients or which concentration of SH is more effective in improving symptoms and signs of dry eye in clinical situations, the improvement of corneal surface parameters by hypotonic 0.18% SH in our animal model can be partially explained by the intrinsic and dosedependent wound-healing property of SH itself. Both IL-1b and TNF-a are proinflammatory cytokines that are secreted on the ocular surface in response to hyperosmolar and desiccating stress in dry eye. Homing and infiltrating T cells onto the ocular surface are predominantly CD4 + T cells in dry eye disease.38,39 Th-1-related chemokine receptors such as CCR5 and CXCR3, and their ligands have an important role in the trafficking of activated CD4 +
FIG. 8. Representative histograms showing percentage of CD44-positive cells in the conjunctiva of the EDE (A), 0.5% CMC-treated (B), isotonic 0.1% SH-treated (C), and hypotonic 0.18% SHtreated groups (D).
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T cells.20,23,40 CD40 appeared to be co-expressed with human leukocyte antigen-DR in patients with dry eye.41 It is involved in the regulation of immune response and cell apoptosis.42 In the present study, the conjunctiva of hypotonic 0.18% SH-treated mice showed decreased expression of IL-1b, IL-6, TNF-a, MIG, and IP-10 and decreased numbers of CD4 + CXCR3 + and CD40 + compared with the other isotonic solution groups. Our results indicate that hypotonic 0.18% SH has an advantage compared with other isotonic artificial tears in that it can effectively decrease inflammatory parameters on the ocular surface. It is known that conjunctival goblet cells can be damaged by inflammation of the lacrimal functional unit. The degree of CD4 + T-cell infiltration has an inverse correlation with the conjunctival goblet cell density in desiccating stressinduced dry eye.43 IL-6 and interferon-g can promote the loss of goblet cells on the ocular surface in desiccating stress.44,45 In the present study, the use of hypotonic 0.18% SH achieved higher goblet cell density compared with other isotonic eyedrops. Decreased inflammation on the ocular surface by hypotonic eyedrops might lead to an improvement of goblet cell density. CD44, known as a hyaluronate receptor, is a widely expressed cell surface glycoprotein that serves as an adhesion molecule in cell–substrate and cell–cell interactions, including lymphocyte homing, homeopoiesis, and migration.46 In mature lymphocytes, CD44 is up-regulated in response to antigenic stimuli and participates in the effector stage of immunological response.47 A previous study demonstrated that CD44 binding with hyaluronic acid was influenced by the type of macrophages and consequent cytokines.48 CD44 is also expressed in corneal and conjunctival cells.49 The level of in vitro and in vivo expression of CD44 on lymphocytes from patients with primary Sjo¨gren’s syndrome significantly increased compared with lymphocytes from normal subjects.50 Zhu et al.51 surmised that the regulation of CD44 was closely related to corneal inflammatory reactions. According to a recent study, the use of hypotonic SH eyedrops in patients with moderate dry eye was beneficial in improving symptoms and signs of dry eye and decreasing the number of CD44 + cells on the conjunctiva.37 They demonstrated that, after hyaluron bound with CD44 on the cell surface, the CD44-hyaluron complex was internalized and degraded within lysosomes, which was evidenced by increased CD63.37 We also found that the hypotonic 0.18% SH group had the lowest number of CD44 + cells among the groups. However, it is unclear how hypotonic SH could decrease the number of CD44 + cells. Since we think that CD4 + T cells play a major role in the pathogenesis of dry eye, the nature of CD44 + cells was not determined in the present study. We suppose that the decrease of CD44 by SH might be due to not only decreased migration of various inflammatory cells but also increased lysosomal degradation after binding. Interestingly, 0.1% SH showed lower levels of TNF-a, IL-6, CD40, and CD44 compared with 0.5% CMC. However, corneal staining was tolerable between 0.1% SH and 0.5% CMC. Lower levels of CD40 and CD44 indicated that SH itself may play a role in controlling the localized inflammation, as previously reported.12,52 Despite the precise mechanism of how hypotonic SH solutions reduced inflammation, our experimental results can support the current expectation that hypotonic artificial
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tears can improve dry eye condition, where increased tear film osmolarity and consequently inflammation play a part in the pathogenesis of the ocular surface damage. Our study had limitations. Since we evaluated commercially available SH eyedrops from the same company, the concentration was not controlled. To confirm the role of hypotonic artificial tears, a comparison of isotonic and hypotonic SH eyedrops with the same concentration will be needed. Besides CD4 + T cells, antigen-presenting cells, macrophages, and B cells can also participate in the pathogenesis. Although we evaluated the efficacy of artificial tear eyedrops using a murine EDE model, clinical responses to the agents for dry eye might be variable in real situations. Further clinical studies are needed to confirm the correlation between inflammatory parameters and clinical signs and symptoms of dry eye after using hypotonic SH eyedrops. In conclusion, the use of hypotonic 0.18% SH eyedrops is more effective in improving clinical parameters and number of goblet cells and decreasing inflammatory cytokines, chemokines, and cells on the ocular surface compared with isotonic 0.5% CMC or 0.1% SH eyedrops in the treatment of EDE.
Acknowledgments This study was supported by Forest Science and Technology Projects (Project No. S121313L050100) provided by Korea Forest Service, and the Alcon Laboratory, Seoul, Korea.
Author Disclosure Statement The authors declare that there are no conflicts of interest.
References 1. Report of the Epidemiology Subcommittee of the International Dry Eye WorkShop. The epidemiology of dry eye disease. Ocul. Surf. 5:93–107, 2007. 2. Jhonson, M.E., and Murphy, P.J. Changes in the tear film and ocular surface from dry eye syndrome. Prog. Retin Eye Res. 23:449–474, 2004. 3. Report of the Definition and Classification Subcommittee of International Dry Eye WorkShop. The definition and classification of dry eye disease. Ocul. Surf. 5:75–92, 2007. 4. Pflugfelder, S.C., Jones, D., Ji, Z., Afonso, A., and Monory, D. Altered cytokines balance in the tear fluid and conjunctiva for patients with Sjo¨gren’s syndrome keratoconjunctivitis sicca. Curr. Eye Res. 19:201–211, 1999. 5. Tsubota, K., Fujihara, T., Saito, K., and Takeuchi, T. Conjunctival epithelium expression of HLA-DR in dry eye patients. Ophthalmologica. 213:16–19, 1999. 6. Grene, R.B., Lankston, P., Mordaunt, J., et al. Unpreserved carboxymethyl-cellulose artificial tears evaluated in patients with keratoconjunctivitis sicca. Cornea. 11:294–301, 1992. 7. Bruix, A., Ada´n, A., and Casaroli-Marano, R.P. Efficacy of sodium carboxymethylcellulose in the treatment of dry eye syndrome. Arch. Soc. Esp. Oftalmol. 81:85–92, 2006. 8. Johnson, M.E., Murphy, P.J., and Boulton, M. Effectiveness of sodium hyaluronate eyedrops in the treatment of dry eye. Graefes Arch. Clin. Exp. Ophthalmol. 244:109–112, 2006. 9. Report of the Management and Therapy Subcommittee of the International Dry Eye WorkShop. Management and therapy of dry eye disease. Ocul. Surf. 5:163–178, 2007.
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10. Gilbard, J.P., and Kenyon, K.R. Tear diluents in the treatment of keratoconjunctivitis sicca. Ophthalmology. 92:646–650, 1985. 11. Vogel, R., Crockett, R.S., Oden, N., Laliberte, T.W., and Molina, L. Demonstration of efficacy in the treatment of dry eye disease with 0.18% sodium hyaluronate ophthalmic solution (Vismed, Rejena). Am. J. Ophthalmol. 149:594– 601, 2010. 12. Aragona, P., Di Stefano, G., Ferreri, F., Spinella, R., and Stilo, A. Sodium hyaluronate eye drops of different osmolarity for treatment of dry eye in Sjogren syndrome patients. Br. J. Ophthalmol. 86:879–884, 2002. 13. Troiano, P., and Monaco, G. Effect of hypotonic 0.4% hyaluronic acid drops in dry eye patients; a cross-over study. Cornea. 27:1126–1130, 2008. 14. Clark, R.A., Alon, R., and Springer, T.A. CD44 and hyaluronan-dependent rolling interactions of lymphocytes on tonsillar stroma. J. Cell. Biol. 134:1075–1087, 1996. 15. Bonder, C.S., Clark, S.R., Norman, M.U., et al. Use of CD44 by CD4 + Th1 and Th2 lymphocytes to roll and adhere. Blood. 107:4798–4806, 2006. 16. Ohmichi, Y., Hirakawa, J., Imai, Y., Fukuda, M., and Kawashima, H. Essential role of peripheral node addressin in lymphocyte homing to nasal-associated lymphoid tissues and allergic immune responses. J. Exp. Med. 208:1015–1025, 2011. 17. Dursun, D., Wang, M., Monroy, D., et al. A mouse model of keratoconjunctivitis sicca. Invest. Ophthalmol. Vis. Sci. 43:632–638, 2002. 18. De Pavia, C.S., Corrales, R.M., Villarreal, A.L., et al. Corticosteroid and doxycline suppress MMP-9 and inflammatory cytokines expression, MAPK activation in the corneal epithelium in experimental dry eye. Exp. Eye. Res. 83:526–535, 2006. 19. Niederkorn, J.Y., Stern, M.E., Pflugfelder, S.C., et al. Desiccating stress induces T cell-mediated Sjogren’s syndrome-like lacrimal keratoconjunctivitis. J. Immunol. 176:3950–3957, 2006. 20. Yoon, K.C., De Paiva, C.S., Qi, H., et al. Expression of Th1 chemokines and chemokine receptors on the ocular surface of C57BL/6 mice; effects of desiccating stress. Invest. Ophthalmol. Vis. Sci. 48:2561–2569, 2007. 21. Yoon, K.C., Ahn, K.Y., Choi, W., et al. Tear production and ocular surface changes in experimental dry eye after elimination of desiccating stress. Invest. Ophthalmol. Vis. Sic. 52:7267–7273, 2011. 22. Lemp, M.A. Report of the National Eye Institute/Industry workshop on clinical trials in dry eyes. CLAO J. 21:221– 228, 1995. 23. Yoon, K.C., Park, C.S., You, I.C., et al. Expression of CXCL-9, -10, -11, and CXCR3 in the tear film and ocular surface of patients with dry eye syndrome. Invest. Ophthalmol. Vis. Sci. 51:643–650, 2010. 24. Li, D.Q., Chen, Z., Song, X.J., Luo, L., and Pflugfelder, S.C. Stimulation of matrix metalloproteinases by hyperosmolarity via a JNK pathway in human conjunctival epithelial cells. Invest. Ophthalmol. Vis. Sci. 45:4302–4311, 2004. 25. Luo, L., Li, D.Q., Corrales, R.M., and Pflugfelder, S.C. Hyperosmolar saline is proinflammatory stress on the mouse ocular surface. Eye Contact Lens. 31:186–193, 2005. 26. Brignole, F., Pisella, P.J., Goldschild, M., et al. Flow cytometric analysis of inflammatory markers in the conjunctiva of patients with dry eyes. Invest. Ophthalmol. Vis. Sci. 41:1356–1363, 2000. 27. Kunert, K.S., Tisdale, A.S., and Gipson, I.K. Goblet cell numbers and epithelial proliferation in the conjunctiva of
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patients with dry eye syndrome treated with cyclosporine. Arch. Ophthalmol. 120:330–337, 2002. Yeh, S., Song, X.J., Farley, W., et al. Apoptosis of ocular surface cells in experimentally induced dry eye. Invest. Ophthalmol. Vis. Sci. 44:124–129, 2003. Lemp, M.A., Bron, A.J., Baudouin, C., et al. Tear osmolarity in the diagnosis and management of dry eye disease. Am. J. Ophthalmol. 151:792–798, 2011. Yoon, K.C., Jeong, I.Y., Park, Y.G., and Yang, S.Y. Interleukin-6 and tumor necrosis factor-alpha levels in tears of patients with dry eye syndrome. Cornea. 26:431–437, 2007. Choi, W., Li, Z., Oh, H.J., et al. Expression of CCR5 and its ligands CCL3, - 4, and - 5 in the tear film and ocular surface of patients with dry eye disease. Curr. Eye Res. 37:12–17, 2012. Tiffany, J.M. Viscoelastic properties of human tears and polymer solution. Adv. Exp. Med. Biol. 350:267–270, 1994. Stuart, J.C., and Linn, J.G. Dilute sodium hyaluronate (Healon) in the treatment of ocular surface disorders. Ann. Ophthalmol. 17:190–192, 1985. Nishida, T., Nakamura, M., Mishima, H., and Otori, T. Hyaluronan stimulates corneal epithelial migration. Exp. Eye Res. 53:753–758, 1991. Inoue, M., and Katakami, C. The effect of hyaluronic acid on corneal epithelial cell proliferation. Invest. Ophthalmol. Vis. Sci. 34:13–15, 1993. Nakamura, M., Hikida, M., and Nakano, T. Concentration and molecular weight dependency of rabbit corneal epithelial wound healing on hyaluronan. Curr. Eye Res. 11:981–986, 1992. Brignole, F., Pisella, P.J., Dupas, B., Baeyens, V., and Baudouin, C. Efficacy and safety of 0.18% sodium hyaluronate in patients with moderate dry eye syndrome and superficial keratitis. Grafes Arch. Clin. Exp. Ophthalmol. 243:531–538, 2005. Stern, M.E., Gao, J., Schwalb, T.A., et al. Conjunctival Tcell subpopulations in Sjo¨gren’s and non-Sjo¨gren’s patients with dry eye. Invest. Ophthalmol. Vis. Sci. 43:2609–2614, 2002. Yoon, K.C., De Paiva, C.S., Qi, H., et al. Desiccating environmental stress exacerbate autoimmune lacrimal keratoconjunctivitis in non-obese mice. J. Autoimmun. 4:212– 221, 2008. Pflugfelder, S.C., and Stern, M.E. Symposium participants. Immunoregulation on the ocular surface; 2nd Cullen Symposium. Ocul. Surf. 7:67–77, 2009. Bourcier, T., De Saint-Jean, M., Brignole, F., Goguel, A., and Baudouin, C. Expression of CD40 and CD40 ligand in the human conjunctival epithelium. Invest. Ophthlamol. Vis. Sci. 41:120–126, 2000. Van Kooten, C., and Banchereau, J. CD40-CD40 lignad: a multifunctional receptor-ligand pair. Adv. Immunol. 61:1– 77, 1996. De Paiva, C.S., Corrales, R.M., Villarreal, A.L., et al. Dry eye-induced conjunctival epithelial squamous metaplasia is modulated by interferon-gamma. Invest. Ophthalmol. Vis. Sci. 48:2552–2560, 2007. Zhang, X., Chen, W., De Paiva, C.S., et al. Interferon-g exacerbates dry eye-induced apoptosis in conjunctiva through dual apoptotic pathways. Invest. Ophthalmol. Vis. Sci. 31:6279–6285, 2011. De Paiva, C.S., Schwartz, C.E., Gjo¨rstrup, P., et al. Resolvin E1 (RX-10001) reduces corneal epithelial barrier disruption and protects against goblet cell loss in a murine model of dry eye. Cornea. 31:1299–1303, 2012.
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46. Mackay, C.R., Terpe, H.J., Stauder, R., et al. Expression and modulation of CD44 variant isoforms in human. J. Cell. Biol. 124:71–82, 1994. 47. Lesley, J., Hyman, R., and Kincade, P.W. CD44 and its interaction with extracellular matrix. Adv. Immunol. 54:271–335, 1993. 48. Ruffell, B., Poon, G.F., Lee, S.S., et al. Differential use of chondroitin sulfate to regulate hyaluronan binding by receptor CD44 in Inflammatory and Interleukin 4-activated macrophages. J. Biol. Chem. 286:19179–19190, 2011. 49. Lerner, L.E., Schawrtz, D.M., Hwang, D.G., Howes, E.L., and Stern, R. Hyaluronan and CD44 in the human cornea and limbal conjunctiva. Exp. Eye Res. 67:481– 484, 1998. 50. Aziz, K.E., and Wakefield, D. In vivo and in vitro expression of adhesion molecules by peripheral blood lymphocytes from patients with primary Sjogren’s syndrome: culture-associated enhancement of LECAM-1 and CD44. Rheumatol. Int. 15:69–74, 1995.
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51. Zhu, S.N., No¨lle, B., and Duncker, G. Expression of adhesion molecule CD44 on human corneas. Br. J. Ophthalmol. 81:80–84, 1997. 52. Stern, M.E., Beuerman, R.W., Fox, R.I., et al. The pathology of dry eye: the interaction between the ocular surface and lacrimal gland. Cornea. 17:584–589, 1998.
Received: March 7, 2013 Accepted: March 21, 2014 Address correspondence to: Dr. Kyung Chul Yoon Department of Ophthalmology Chonnam National University Medical School and Hospital 8 Hakdong, Donggu Gwangju 501-757 Korea E-mail: [email protected]
Effect of Hypotonic 0.18% Sodium Hyaluronate Eyedrops on Inflammation of the Ocular Surface in Experimental Dry Eye Han Jin Oh,1 Zhengri Li,1 Soo-Hyun Park,2 and Kyung Chul Yoon1
Abstract
Purpose: To investigate the efficacy of hypotonic 0.18% sodium hyaluronate (SH) eyedrops in a mouse model of experimental dry eye (EDE). Methods: EDE was induced in C57BL/6 mice by a subcutaneous scopolamine injection and an air draft. The mice were divided into 4 groups according to topical treatment regimens: EDE control, isotonic 0.5% carboxymethycellulose (CMC), isotonic 0.1% SH, and hypotonic 0.18% SH. Tear volume, corneal smoothness, and corneal staining scores were measured at 5 and 10 days of EDE. Multiplex immunobead assay, immunohistochemistry, and flow cytometry for proinflammatory cytokines, chemokines, and inflammatory molecules were performed at 10 days of EDE. Results: The 0.18% SH group had a significantly lower corneal smoothness and staining scores than the 0.5% CMC and 0.1% SH groups at 10 days of EDE (P < 0.05). The 0.18% SH group showed significantly low levels of tumor necrosis factor-a (TNF-a), interleukin (IL)-1b, monokine induced by interferon-g, and interferon-ginducible protein 10 compared with the other groups (P < 0.05). The mean percentages of CD4 + CXCR3 +, CD40 + , and CD44 + cells in the conjunctiva were significantly lower in the 0.18% SH group than in the other groups (P < 0.05). In addition, the 0.1% SH group showed lower levels of TNF-a and IL-1b and percentages of CD40 + and CD44 + cells than the EDE and 0.5% CMC groups. Conclusions: Hypotonic 0.18% SH eyedrops are more effective in improving ocular surface irregularity and staining and decreasing inflammatory cytokines, chemokines, and cells on the ocular surface compared with isotonic 0.5% CMC or 0.1% SH eyedrops in the treatment of EDE.
Introduction
D
ry eye disease is the most common problem of the ocular surface, with a suggested prevalence of 5%– 30% in those aged 50 years and older.1 It is a multi-factorial disease of the tear and ocular surface, which results in various ocular symptoms, visual disturbance, tear film instability, and potential damage to the ocular surface.2,3 Inflammation of the ocular surface and hyperosmolarity of the tear film play a pivotal role in the pathogenesis of dry eye.4,5 Application of artificial tears is the mainstay for the treatment of dry eye disease. There are many commercially available topical lubricants, which contain various active ingredients such as sodium hyaluronate (SH) and carboxymethylcellulose (CMC). These agents can supply water
contents for aqueous inadequacy and protect the ocular surface according to each active ingredient.6–8 However, these conventional artificial tears provide only temporary symptom relief. In addition, the approach cannot resolve the causative pathologic factors of dry eye, including tear film hyperosmloarity and associated inflammation.9 This knowledge has influenced the development of a concept that hypotonic artificial tears may be beneficial for the treatment of dry eye. Until now, several studies have reported that hypotonic solutions can improve clinical symptoms and signs of dry eye disease.10,11 In addition, comparative studies between isotonic and hypotonic artificial tears have indicated that hypotonic solutions are more effective in improving the subjective symptoms, ocular surface staining, and some tear film parameters.12,13 However,
1 Department of Ophthalmology, Chonnam National University Medical School and Hospital, Center for Creative Biomedical Scientists at Chonnam National University, Gwangju, Korea. 2 Bio-Therapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea.
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previous studies on hypotonic artificial tears only evaluated the changes of clinical symptoms and signs and did not investigate the parameters associated with ocular surface inflammation. The purpose of the present study was to compare the efficacy of commercially available hypotonic 0.18% SH, isotonic 0.1% SH, and isotonic 0.5% CMC artificial tear eyedrops in a mouse model of desiccating stress-induced experimental dry eye (EDE), by evaluating the changes of tear production, corneal surface irregularity and staining, inflammatory cytokines, CD4 + T cells, and inflammatory molecules such as CD40 on the ocular surface as well as conjunctival goblet cell density. In addition, we examined the expression of CD44 on the ocular surface, because CD44 is known as a hyaluronate receptor in T cells and the hyaluronate-CD44 interaction mediates the homing and trafficking of CD4 + T cells both in vivo and in vitro.14–16
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analysis were performed. Each group consisted of 5 animals (10 eyes), and the experiments were performed on 4 different sets of mice.
Measurement of tear production Tear volume was measured with phenol red-impregnated cotton threads (Zone-Quick; Oasis, Glendora, CA) as previously described.21 The tips of the threads were placed on the peripheral conjunctiva at the lateral canthus for 20 s. The tear volume, expressed in millimeters of thread wet by the tear and turned red color, was evaluated under a microscope (SMZ 1500; Nikon, Melville, NY). The measured uptake of tear fluid in millimeters was compared with a standard curve prepared from cotton threads of a known uptake volume of a stock basic solution (1,500 mL of 0.9% saline and 5 mL of 5 N NaOH) over 20 s, with volumes in the range that would be expected in mouse tear.
Methods
Evaluation of corneal irregularity severity
This research protocol was approved by the Chonnam National University Medical School Research Institutional Animal Care and Use Committee. The maintenance of animals and all in vivo experiments were performed in accordance with institutional guidelines and the ARVO statement for the Use of Animals in Ophthalmic and Vision Research.
Reflected images of a white ring from the fiber-optic ring illuminator of the stereoscopic zoom microscope (SMZ 1500; Nikon, Tokyo, Japan) were taken without anesthesia. Corneal smoothness was evaluated by grading the distortion of a white ring reflected off the corneal epithelium in digital images. The corneal irregularity severity score was calculated using a 5-point scale (0–5) based on the number of distorted quarters in the reflected ring: 0, no distortion; 1, distortion in 1 quarter; 2, distortion in 2 quarters; 3, distortion in 3 quarters; 4, distortion in all 4 quarters; and 5, severe distortions, in which no ring could be recognized.20
Animal and EDE Eight-week-old female C57BL/6 mice were used in these experiments. EDE was induced by a subcutaneous injection of 0.5 mg/0.2 mL scopolamine hydrobromide (Sigma Aldrich, St. Louis, MO) in alternating hindquarters 4 times a day (8 am, 11 am, 2 pm, and 5 pm) with a standard desiccating environment that was created by placing the mice between 2 fans to obtain a continuous air flow (15 L/min) in a 25C room with an ambient humidity of 30% for 18 h per day, as previously described.17–21 During these experiments, the animals’ behavior, food intake, and water intake were not restricted.
Materials Commercially available artificial tear eyedrops were used. Kynex 2 (Alcon Korea, Seoul, Korea) was a hypotonic (150 mOsm/L) agent that contains SH at a concentration of 0.18%. Kynex (Alcon Korea) was an isotonic (284 mOsm/L) agent that contains 0.1% SH. Refresh plus (Allergan, Irvine, CA) was also an isotonic (318 mOsm/L) agent that contained CMC at a concentration of 0.5%. All products were preservative free.
Experimental design The mice were randomly divided into 4 groups according to topical regimens that were administered as follows: EDE controls (received no eyedrops), EDE + isotonic 0.5% CMC (Refresh plus), EDE + isotonic 0.1% SH (Kynex), and EDE + hypotonic 0.18% SH (Kynex2). All treatment groups received 2 mL of eyedrops 4 times a day. Tear volume, corneal smoothness score, and keratoepitheliopathy score were measured at 5 and 10 days of EDE. At 10 days, the mice were euthanized, and then multiplex immunobead assay, histology, immunohistochemistry, and flow cytometric
Evaluation of corneal staining scores (keratoepitheliopathy scores) After instillation of 1% fluorescein (1 mL volume) into the inferior conjunctival sac, punctuate staining on the corneal surface was evaluated in a masked fashion using slit-lamp biomicroscopy under a cobalt blue light. The intensity of staining was graded using a 3-point scale (0–3): 0, no staining; 1, superficial stippling micropunctate staining; 2, macropunctate staining with some coalescent areas; and 3, numerous coalescent macropunctate areas and/or patch). The cornea was divided into 5 regions: central, inferior, nasal, temporal, and superior. The scores of the 5 areas were summed to obtain a total score for each eye, ranging from 0 to 15.22
Multiplex immunobead assay The levels of proinflammatory cytokines; tumor necrosis factor-a (TNF-a) and interleukin (IL)-1b, and chemokine ligands; monokine induced by interferon-g (MIG/CXCL9) and interferon-g-inducible protein (IP-10/CXCL10), in the conjunctiva were measured using a multiplex immunobead assay (Luminex 200; Luminex Corp., Austin, TX), as previously described.20 Conjunctival tissues were obtained (4 eyes per each group) and were stored at - 70C until use. Approximately 10 mg of the tissue was transferred to a 2.0 mL microcentrifuge tube containing 100 mL of 1 · extraction reagent (Tissue extraction reagents I; BioSourceInvitrogen, San Diego, CA) that was supplemented with phenylmethanesulfonyl fluoride in ethanol as a protease inhibitor on the ice for 30 min. Then, lysing process was
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performed using TissueLyser (Qiagen, Hilden, Germany). The tissue was homogenized for 30 s until the sample was in a consistent solution and then centrifuged (4C for 10 min at 12,000 rpm). After centrifugation, the supernatant was collected and placed in a new microcentrifuge tube on ice. Protein quantification of the supernatants was performed, and 1 mg/mL of protein were added to wells containing the appropriate cytokine and chemokine-specific bead mixture that included mouse monoclonal antibodies which were specific for TNF-a, IL-1b, MIG, and IP-10 (Panomics, Santa Clara, CA) for 60 min. Serial dilutions of cytokines and chemokines were also added to wells in the sample plate as the supernatant samples to generate a standard curve. The plate was incubated for 30 min in the dark at room temperature by a biotinylated detection antibody. The reactions were detected using an analysis system (xPONET, Austin, TX) after the addition of streptavidin-phycoerythrin.
Histology Eyes and adnexa were surgically excised, fixed in 4% paraformaldehyde, and embedded in paraffin. Then, the tissue was sectioned by 6 mm and stained with periodic acidSchiff reagent. Sections from each group were examined and photographed with a microscope (Olympus, Tokyo, Japan) that was equipped with a digital camera. Goblet cell density in the superior and inferior conjunctiva was measured in 3 sections of each eye using image analysis software (Media Cybernetics, Silver Spring, MD) and was expressed as the number of goblet cells per 100 mm.
Immunohistochemistry Immunohistochemistry was performed to detect the relative expression of IL-6 in the epithelium and superficial stroma of the conjunctiva. Preparation of the tissue sections and immunohistochemistry were performed according to a previously described method.20 After being surgically excised, the conjunctival tissues from each of the groups were embedded in OTC compound (VWR, Suwanee, GA) and flash frozen in liquid nitrogen. Sagittal 8 mm sections were cut with cryostat. The cryosections were fixed in acetone at - 20C for 10 min.
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After fixation, endogenous peroxidases were quenched with 0.3% hydrogen peroxide in phosphate-buffered saline (PBS), and 20% normal serum in PBS were sequentially applied to the sections. Conjunctival sections from each group were incubated with goat monoclonal anti-mouse IL-6 antibodies (Santa Cruz Biotechnology, Heidelberg, Germany) and then, rabbit anti-goat secondary antibodies were applied. The samples were incubated with avidin-peroxidase, then incubated with 3,3¢ diaminobenzidine peroxidase substrate, and counterstained stained with Mayer’s hematocylin.
Flow cytometry Flow cytometry was performed to quantify CD4 + CXCR3 +, CD40 +, and CD44 + cells from the conjunctiva as previously described, with modifications.23 The conjunctival tissue obtained from each group (4 eyes per group) was immediately incubated with 1 mL of Dulbecco’s modified Eagle’s medium containing PBS with 1% fetal bovine serum (FBS) at 37C via a 5% CO2 incubator, and these tissues were subsequently digested in 0.5 mg/mL collagenase type D (Roche Applied Science, Indianapolis, IN). After incubation, the tissues were disrupted by gently grinding with syringe plunder and passage through a cell strainer with a pore size of 100 mm. The obtained cells were then centrifuged (for 5 min, at 1,500 rpm) and resuspended in PBS with 1% FBS. Cell suspensions were then counted, and viability was determined by a trypan blue exclusion assay. The samples were incubated with monoclonal antibodies. For detecting CD4 + CXCR3 + (double-stained) cells, the samples were incubated with fluorescein-conjugated anti-mouse CD4 antibody (0.5 mg/mL; BD Biosciences, San Jose, CA) and phycoerythrin-conjugated anti-mouse CXCR3 antibody (0.5 mg/mL; BD Biosciences), and isotype control antibody at 37C for 30 min. In addition, they were incubated with phycoerythrin-conjugated anti-mouse CD40 antibody (0.5 mg/mL; BD Biosciences) or phycoerythrinconjugated anti-mouse CD44 antibody (0.5 mg/mL; BD Biosciences). After 30 min of incubation, the cells were washed in PBS. The cells were then centrifuged thrice in 1 mL of PBS (for 5 min at 1,000 rpm) and resuspended. The
FIG. 1. Mean corneal smoothness scores (A) and representative photographs (B) in the experimental dry eye (EDE), isotonic 0.5% carboxymethylcellulose (CMC)treated, isotonic 0.1% sodium hyaluronate (SH)-treated, and hypotonic 0.18% SH-treated groups at days 5 and 10. *P < 0.05 compared with the EDE group, {P < 0.05 compared with the CMC group, and {P < 0.01 compared with the 0.1% SH group.
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FIG. 2. Mean corneal staining scores (A) and representative photographs (B) in the EDE, isotonic 0.5% CMCtreated, isotonic 0.1% SHtreated, and hypotonic 0.18% SH-treated groups at days 5 and 10. *P < 0.05 compared with the EDE group, {P < 0.05 compared with the CMC group, and {P < 0.01 compared with the 0.1% SH group.
number of stained cells was counted by an FACSCalibur cytometer with CellQuest software (BD Biosciences).
Statistical analysis Results are presented as the mean – SEM. The statistical significance of differences between the groups was determined by the 1-way analysis of variance test with Tukey post hoc analysis using the SPSS 17.0 software (SPSS, Inc., Chicago, IL). A P-value less than 0.05 was considered statistically significant.
Results Tear volume Five days after the induction of EDE, the mean tear volume was 0.023 – 0.007 mL in the EDE group, 0.024 – 0.007 mL in the CMC group (P = 0.80, vs. the EDE group), 0.024 –
FIG. 3. Concentrations of tumor necrosis factor-a (TNFa) (A), interleukin (IL)-1b (B), monokine induced by interferon-g (MIG) (C), and interferon-g-inducible protein (IP-10) (D) in the conjunctiva of the EDE, isotonic 0.5% CMCtreated, isotonic 0.1% SHtreated, and hypotonic 0.18% SH-treated groups. *P < 0.05 compared with the EDE group, {P < 0.05 compared with the CMC group, and { P < 0.01 compared with the 0.1% SH group.
0.006 mL in the 0.1% SH group (P = 0.88, vs. the EDE group; P = 0.88, vs. the CMC group), and 0.025 – 0.006 mL in the hypotonic 0.18% SH group (P = 0.57, vs. the EDE group, P = 0.96, vs. the CMC group, P = 0.57, vs. the 0.1% SH group). The findings for the mean tear volume in all groups at 10 days of EDE were similar to those at 5 days. There were no significant differences between the groups.
Corneal irregularity severity scores At 5 days of EDE, the mean corneal irregularity scores were 3.5 – 0.78 in the EDE group, 2.9 – 0.67 in the CMC group (P = 0.25, vs. the EDE group), 2.6 – 0.60 in the 0.1% SH group (P = 0.11, vs. the EDE group, P = 0.58, vs. the CMC group), and 1.8 – 0.47 in the hypotonic 0.18% SH group (P < 0.01, vs. the EDE group, P = 0.02, vs. the CMC group, P = 0.14, vs. the 0.1% SH group). At 10 days of EDE, the 0.18% SH group showed significantly lower corneal
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FIG. 4. Immunohistochemistry for IL-6 in the conjunctiva of the EDE, isotonic 0.5% CMC-treated, isotonic 0.1% SH-treated, and hypotonic 0.18% SH-treated groups.
irregularity scores compared with the EDE, CMC, and 0.1% SH groups (P < 0.05). The other 3 groups were comparable in corneal irregularities at 10 days of EDE (Fig. 1A, B).
Corneal staining scores At 5 and 10 days after EDE induction, the corneal staining scores were significantly lower in the hypotonic 0.18% SH-treated group compared with the EDE, 0.5% CMC, and 0.1% SH-treated groups (P < 0.05). At 10 days of EDE, the 0.1% SH-treated group showed significantly lower corneal staining scores than those in the EDE group (P = 0.02) (Fig. 2A). Representative biomicroscopic photographs showing the degrees of corneal staining in each group at 5 and 10 days of EDE are shown in Fig. 2B.
Levels of inflammatory cytokines/chemokines in conjunctiva Multiplex immunobead assay showed that the concentrations of TNF-a, IL-1b, MIG, and IP-10 in conjunctival tissues
of the hypotonic 0.18% SH-treated group were significantly lower compared with the EDE, CMC, and 0.1% SH-treated groups (P < 0.05). In addition, the 0.1% SH-treated group showed significantly lower levels of TNF-a and IL-1b than the EDE- and CMC-treated groups (P < 0.05) (Fig. 3). However, the levels of MIG and IP-10 were comparable among the EDE-, CMC-, and 0.1% SH-treated groups. Immunohistochemical examination showed that IL-6 in the conjunctiva was strongly expressed in the EDE group. However, relatively weak staining was observed in both SH groups (Fig. 4).
Histology At 10 days after EDE induction, the conjunctival goblet cell density (cells/100 mm) was 8.00 – 2.16 in the EDE group, 9.86 – 2.41 in the 0.5% CMC-treated group (P = 0.06, vs. the EDE group), 10.86 – 2.41 in the 0.1% SH-treated group (P = 0.07, vs. the EDE group, P = 0.59, vs. the 0.5% CMC group), and 17.29 – 2.50 in the hypotonic 0.18% SH-treated group (P = 0.02, vs. the EDE group, P = 0.02, vs.
FIG. 5. Mean number of goblet cells (A) and representative photographs (B) in the EDE, isotonic 0.5% CMC, isotonic 0.1% SH, and hypotonic 0.18% SH-treated groups. *P < 0.05 compared with the EDE group, {P < 0.05 compared with the CMC group, and { P < 0.01 compared with the 0.1% SH group.
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Table 1. Mean Percentage of CD4 + CXCR3 +, CD40 +, and CD44 + Cells in the Conjunctiva of the Experimental Dry Eye, 0.5% Carboxymethylcellulose, Isotonic 0.1% Sodium Hyaluronate, and Hypotonic 0.18% SH Groups, Measured by Flow Cytometry Percentages of positive expression Marker CD4, CXCR3 CD40 CD44
EDE
0.5% CMC
0.1% SH
0.18% SH
83.75 – 12.87 57.25 – 13.77 82.00 – 9.83
78.75 – 12.99 51.00 – 5.29 79.50 – 9.15
71.00 – 11.16 44.25 – 15.71a,b 65.00 – 14.54 a,b
60.00 – 14.97a,b,c 22.75 – 10.03a,b,c 53.50 – 15.22a,b,c
Data are expressed as the mean – standard deviation. a P < 0.05 compared with the EDE group. b P < 0.05 compared with the CMC group. c P < 0.01 compared with the 0.1% SH group. CMC, carboxymethycellulose; EDE, experimental dry eye; SH, sodium hyaluronate.
the 0.5% CMC group, P = 0.02, vs. the 0.1% SH group) (Fig. 5A). Representative histologic findings showing the conjunctival goblet cell density in each group at 10 days of EDE are also shown in Fig. 5B.
Flow cytometric analysis Flow cytomeric data are summarized in Table 1. The mean percentages of CD4 + CXCR3 + cells were 83.75% – 12.87% in the EDE group, 78.75% – 12.99% in the 0.5% CMCtreated group (P = 0.34, vs. the EDE group), 71.00% – 11.16% in the 0.1% SH-treated group (P = 0.08, vs. the EDE group, P = 0.23, vs. the 0.5% CMC group), and 60.00% – 14.97% in the hypotonic 0.18% SH-treated group (P < 0.01, vs. the EDE group, P = 0.01, vs. the 0.5% CMC group, P = 0.03, vs. the 0.1% SH group). The mean percentages of CD40 + and CD44 + cells in the hypotonic 0.18% SH group were significantly lower compared with the EDE, 0.5% CMC, and 0.1% SH-treated groups (P < 0.05). Furthermore, the 0.1% SH group had
FIG. 6. Representative histograms showing percentage of CD4 + CXCR3 + doublestained T cells in the conjunctiva of the EDE (A), 0.5% CMC-treated (B), isotonic 0.1% SH-treated (C), and hypotonic 0.18% SHtreated groups (D).
lower percentages of CD40 + and CD44 + cells compared with the EDE and 0.5% CMC groups (P < 0.05). Histograms and percentages of CD4 + CXCR3 + T cells, CD40 + cells, and CD44 + cells from representative samples in the EDE, CMC, 0.1% SH, and 0.18% SH groups are presented in Figs. 6–8, respectively.
Discussion Recently, it has been widely recognized that inflammation plays a key role in the pathogenesis of dry eye disease. Both hyperosmolar stress and tear film hyperosmolarity induce a cascade of inflammatory events in the ocular surface epithelium, along with the generation of inflammatory cytokines.24,25 Consequently, these chronic inflammatory events lead to the apoptosis of ocular surface epithelium cells and goblet cells, resulting in dry eye.26–28 In addition, tear film osmolarity increased by approximately 30–40 mOsm/L in patients with dry eye.29 On the basis of the role of hyperosmolarity in the pathogenesis, hypotonic artificial tears
HYPOTONIC SH IN EDE
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FIG. 7. Representative histograms showing percentage of CD40-positive cells in the conjunctiva of the EDE (A), 0.5% CMC-treated (B), isotonic 0.1% SH-treated (C), and hypotonic 0.18% SHtreated groups (D).
have been used in an attempt to treat dry eye disease.8,10–13 Several hypotonic solutions with various brand names are currently marketed in Europe and Asia. Desiccating stress stimulated by environmental and pharmacological means has been used for the induction of dry eye in several different mouse strains, including C57BL/6 mice.17–21 Desiccating ocular stress has been known to induce auto-reactive T cells that cause Sjo¨gren’s syndrome-like inflammation in the cornea, conjunctiva, and lacrimal gland.19 We have previously found that desiccating stress stimulated the expression of inflammatory cytokines (IL-6 and TNF-a) and Th-1 chemokines (CCL3, CCL4, CCL5, CXCL9, and CXCL10) and their receptors (CCR5 and CXCR3) in the tear film and ocular surface of clinical and EDE.20,21,23,30,31 SH has good pseudoplastic and elastic properties that help stabilize tear film and enhance the residence times both during and between blinking.32 Moreover, previous experimental studies have shown that SH has wound-healing effects, including stimulation of migration, adhesion, and proliferation of the corneal epithelium.33–35 In addition, in vivo corneal abrasion models have shown the dose-
dependent ability of hyaluronate to reduce wound size.36 Clinically, Brignole et al.37 demonstrated that hypotonic 0.18% SH tended to show faster recovery in superficial keratitis and more improvement of subjective symptoms than 0.5% CMC eyedrops in patients with moderate dry eye. In the present study, we demonstrated that 0.18% SH eyedrops showed better results in corneal irregularity and staining scores compared with 0.5% CMC and 0.1% SH eyedrops. Although there is controversy about whether SH is superior to the other ingredients or which concentration of SH is more effective in improving symptoms and signs of dry eye in clinical situations, the improvement of corneal surface parameters by hypotonic 0.18% SH in our animal model can be partially explained by the intrinsic and dosedependent wound-healing property of SH itself. Both IL-1b and TNF-a are proinflammatory cytokines that are secreted on the ocular surface in response to hyperosmolar and desiccating stress in dry eye. Homing and infiltrating T cells onto the ocular surface are predominantly CD4 + T cells in dry eye disease.38,39 Th-1-related chemokine receptors such as CCR5 and CXCR3, and their ligands have an important role in the trafficking of activated CD4 +
FIG. 8. Representative histograms showing percentage of CD44-positive cells in the conjunctiva of the EDE (A), 0.5% CMC-treated (B), isotonic 0.1% SH-treated (C), and hypotonic 0.18% SHtreated groups (D).
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T cells.20,23,40 CD40 appeared to be co-expressed with human leukocyte antigen-DR in patients with dry eye.41 It is involved in the regulation of immune response and cell apoptosis.42 In the present study, the conjunctiva of hypotonic 0.18% SH-treated mice showed decreased expression of IL-1b, IL-6, TNF-a, MIG, and IP-10 and decreased numbers of CD4 + CXCR3 + and CD40 + compared with the other isotonic solution groups. Our results indicate that hypotonic 0.18% SH has an advantage compared with other isotonic artificial tears in that it can effectively decrease inflammatory parameters on the ocular surface. It is known that conjunctival goblet cells can be damaged by inflammation of the lacrimal functional unit. The degree of CD4 + T-cell infiltration has an inverse correlation with the conjunctival goblet cell density in desiccating stressinduced dry eye.43 IL-6 and interferon-g can promote the loss of goblet cells on the ocular surface in desiccating stress.44,45 In the present study, the use of hypotonic 0.18% SH achieved higher goblet cell density compared with other isotonic eyedrops. Decreased inflammation on the ocular surface by hypotonic eyedrops might lead to an improvement of goblet cell density. CD44, known as a hyaluronate receptor, is a widely expressed cell surface glycoprotein that serves as an adhesion molecule in cell–substrate and cell–cell interactions, including lymphocyte homing, homeopoiesis, and migration.46 In mature lymphocytes, CD44 is up-regulated in response to antigenic stimuli and participates in the effector stage of immunological response.47 A previous study demonstrated that CD44 binding with hyaluronic acid was influenced by the type of macrophages and consequent cytokines.48 CD44 is also expressed in corneal and conjunctival cells.49 The level of in vitro and in vivo expression of CD44 on lymphocytes from patients with primary Sjo¨gren’s syndrome significantly increased compared with lymphocytes from normal subjects.50 Zhu et al.51 surmised that the regulation of CD44 was closely related to corneal inflammatory reactions. According to a recent study, the use of hypotonic SH eyedrops in patients with moderate dry eye was beneficial in improving symptoms and signs of dry eye and decreasing the number of CD44 + cells on the conjunctiva.37 They demonstrated that, after hyaluron bound with CD44 on the cell surface, the CD44-hyaluron complex was internalized and degraded within lysosomes, which was evidenced by increased CD63.37 We also found that the hypotonic 0.18% SH group had the lowest number of CD44 + cells among the groups. However, it is unclear how hypotonic SH could decrease the number of CD44 + cells. Since we think that CD4 + T cells play a major role in the pathogenesis of dry eye, the nature of CD44 + cells was not determined in the present study. We suppose that the decrease of CD44 by SH might be due to not only decreased migration of various inflammatory cells but also increased lysosomal degradation after binding. Interestingly, 0.1% SH showed lower levels of TNF-a, IL-6, CD40, and CD44 compared with 0.5% CMC. However, corneal staining was tolerable between 0.1% SH and 0.5% CMC. Lower levels of CD40 and CD44 indicated that SH itself may play a role in controlling the localized inflammation, as previously reported.12,52 Despite the precise mechanism of how hypotonic SH solutions reduced inflammation, our experimental results can support the current expectation that hypotonic artificial
OH ET AL.
tears can improve dry eye condition, where increased tear film osmolarity and consequently inflammation play a part in the pathogenesis of the ocular surface damage. Our study had limitations. Since we evaluated commercially available SH eyedrops from the same company, the concentration was not controlled. To confirm the role of hypotonic artificial tears, a comparison of isotonic and hypotonic SH eyedrops with the same concentration will be needed. Besides CD4 + T cells, antigen-presenting cells, macrophages, and B cells can also participate in the pathogenesis. Although we evaluated the efficacy of artificial tear eyedrops using a murine EDE model, clinical responses to the agents for dry eye might be variable in real situations. Further clinical studies are needed to confirm the correlation between inflammatory parameters and clinical signs and symptoms of dry eye after using hypotonic SH eyedrops. In conclusion, the use of hypotonic 0.18% SH eyedrops is more effective in improving clinical parameters and number of goblet cells and decreasing inflammatory cytokines, chemokines, and cells on the ocular surface compared with isotonic 0.5% CMC or 0.1% SH eyedrops in the treatment of EDE.
Acknowledgments This study was supported by Forest Science and Technology Projects (Project No. S121313L050100) provided by Korea Forest Service, and the Alcon Laboratory, Seoul, Korea.
Author Disclosure Statement The authors declare that there are no conflicts of interest.
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Received: March 7, 2013 Accepted: March 21, 2014 Address correspondence to: Dr. Kyung Chul Yoon Department of Ophthalmology Chonnam National University Medical School and Hospital 8 Hakdong, Donggu Gwangju 501-757 Korea E-mail: [email protected]
