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Conjunctival Goblet Cell Density Following Cataract Surgery With Diclofenac Versus Diclofenac and Rebamipide: A Randomized Trial
Accepted Manuscript Conjunctival Goblet Cell Density Following Cataract Surgery with Diclofenac Versus Diclofenac and Rebamipide: A Randomized Trial Kumiko Kato, Kensaku Miyake, Nagako Kondo, Sayaka Asano, Junko Takeda, Akiko Takahashi, Yuko Takashima, Mineo Kondo PII:
S0002-9394(17)30261-1
DOI:
10.1016/j.ajo.2017.06.016
Reference:
AJOPHT 10174
To appear in:
American Journal of Ophthalmology
Received Date: 13 April 2017 Revised Date:
14 June 2017
Accepted Date: 19 June 2017
Please cite this article as: Kato K, Miyake K, Kondo N, Asano S, Takeda J, Takahashi A, Takashima Y, Kondo M, Conjunctival Goblet Cell Density Following Cataract Surgery with Diclofenac Versus Diclofenac and Rebamipide: A Randomized Trial, American Journal of Ophthalmology (2017), doi: 10.1016/j.ajo.2017.06.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Abstract Purpose: To determine the effects of topical diclofenac or betamethasone with concomitant application of topical rebamipide on the conjunctival goblet cell density in eyes after cataract
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surgery. Design: Randomized clinical trial.
Participants: Eighty patients who were scheduled for cataract surgery.
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Methods: Patients were randomized into four groups according to the postoperative topical drugs to be given; Group A, diclofenac alone; Group B, diclofenac and rebamipide; Group C,
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betamethasone alone; and Group D, betamethasone and rebamipide. Impression cytology was performed before and at one month after the surgery, and the mean density of goblet cells was determined.
Results: The mean (± SD) density of goblet cells before the surgery in Group A was 257.0 ±
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188.7 cells/mm2, and it decreased significantly to 86.5 ± 76.7 cells/mm2 at one month after the surgery (P = 0.002). In Group B, the goblet cell density was not statistically different between before (238.5 ± 116.6 cells/mm2) and at one month after the surgery (211.3 ± 184.4
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cells/mm2, P = 0.55). In groups C and D, the mean density of goblet cells was decreased at
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one month after the surgery, but the decreases were not significant (P = 0.11 and 0.52, respectively).
Conclusion: After cataract surgery with postoperative topical diclofenac, the conjunctival goblet cell density was significantly reduced, and this reduction was blocked by the concomitant use of topical rebamipide. These results suggest that the concomitant use of topical rebamipide with non-steroidal anti-inflammatory drugs is beneficial especially in cases with postoperative dry eyes.
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Conjunctival Goblet Cell Density Following Cataract Surgery with Diclofenac Versus Diclofenac and Rebamipide:
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A Randomized Trial
KUMIKO KATO, KENSAKU MIYAKE, NAGAKO KONDO, SAYAKA ASANO,
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JUNKO TAKEDA, AKIKO TAKAHASHI, YUKO TAKASHIMA, AND MINEO KONDO
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Short title: Goblet cell density after cataract surgery
Key Words: cataract surgery; goblet cell; impression cytology; non-steroidal anti-
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inflammatory drug (NSAID); diclofenac; rebamipide; steroid
From the Department of Ophthalmology, Mie University Graduate School of Medicine, Tsu,
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Japan (K.K., Y.T., M.K.); and the Shohzankai Medical Foundation Miyake Eye Hospital, Nagoya, Japan (K.M., N.K., S.A., J.T., A.T). Inquiries to Kumiko Kato, Department of Ophthalmology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan; phone number: +81-59-231-5027; Fax number: +81-59-231-3036; e-mail: [email protected]
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ACCEPTED MANUSCRIPT Introduction Cataract surgery with intraocular lens (IOL) implantation is generally accepted as a safe surgical procedure that provides very good postoperative outcomes. However, some challenges remain such as improving the postoperative comfort and restoring high quality
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vision. Factors related to these challenges are the optics of the IOL including the so-called premium IOLs,1 corneal configurations,2 and pathologies of the tear film and the ocular surface including the dry eye syndrome.3,4
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The dry eye syndrome or dry eye disease is a common condition which affects the tear film and the ocular surface. The 2007 International Dry Eye Workshop stated that, “dry
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eye is a multifactorial disease of the tears and ocular surface that results in symptoms such as ocular discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface”.3 Numerous factors have been designated as risk factors for postoperative dry eyes.5-14 Although the precise mechanism(s) for postoperative dry eye is not completely
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understood, past studies have suggested that eyes with this type of abnormality have a short tear film break-up time (BUT),15 a decrease in the density of goblet cells, and metaplasia of the corneal and conjunctival epithelial cells16-18 as seen in eyes with the conventional dry eye
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syndrome.19
The main medications used to treat the postoperative inflammation after cataract
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surgery are steroidal and non-steroidal anti-inflammatory drugs (NSAIDs). The NSAIDs were first used in Japan to prevent intraoperative miosis,20 postoperative breakdown of the bloodaqueous barrier, and cystoid macular edema.21,22 More than 60 peer reviewed studies have appeared worldwide that reported on the effects of topical NSAIDs. Recent reviews and meta-analysis have shown that NSAIDs are even more effective than steroids.23-26 However, NSAIDs can have side effects on the ocular surface and tear film.11,27-30 The NSAIDs have been recently suggested to be risk factors for postoperative dry eye31 although the mechanism for this has not been determined. 2
ACCEPTED MANUSCRIPT Recently, two anti-dry eye medications, diquafosol and rebamipide, have become commercially available in Japan.32,33 Rebamipide acts by promoting mucin secretions and improving the homeostasis of the ocular surface, and its topical application has led to a proliferation of goblet cells and the production of mucin. These changes stabilize the barrier
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function of the corneal epithelium and thereby enhance its anti-inflammatory effects.33,34
Experimental and clinical studies have reported that rebamipide eyedrops are effective in treating dry eyes without significant side effects even after long follow-up times.35-37
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Oral rebamipide was initially used to treat and prevent gastric ulcers, and it is currently widely used to prevent the oral NSAIDs-induced gastric mucosal damage.38,39
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Therefore, it is reasonable to evaluate the effects of topical rebamipide in preventing the side effects of topical NSAIDs which are widely used today as a postoperative anti-inflammatory agent.
Thus, the purpose of this study was to determine the effects of preservative-free
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steroids or NSAIDs eyedrops alone or together with rebamipide eyedrops on the conjunctival
Methods
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goblet cells density assessed by impression cytology.
Study design and approvals
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This was a randomized clinical trial conducted at the Mie University Hospital and Miyake Eye Hospital between June 2015 and February 2017. The procedures used were approved by the Medical Ethics Committee of Mie University Hospital (No. 2855) and Miyake Eye Hospital (No. 2016002). The procedures conformed to the tenets of the Declaration of Helsinki of the World Medical Association. All patients signed a written informed consent after they were provided with information on the procedures to be used. The study was also registered International Clinical Trial Registry Platform (UMIN Clinical Trials Registry, UMIN000026310, http://www.umin.ac.jp/ctr/index-j.htm). 3
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Subjects The eligibility criteria for the subjects were patients who required cataract surgery and were ≥50-years-of-age. The exclusion criteria included prior keratoconjunctival diseases, uveitis,
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glaucoma, diabetes mellitus, and eyes that had undergone any type of surgery of the ocular surface or intraocular surgery within 12 months of the beginning of this study. Before the surgery, we checked whether the subjects had any symptoms of dry eye using the Dry Eye
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Ocular Surface Disease Index (OSDI) Questionnaire.40 We excluded subjects who had
painful or sore eyes, and also subjects who had tear film BUT of less than 5 seconds or had
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corneal staining.
Cataract Surgery
Cataract surgery was performed by seven experienced surgeons in the Mie University
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Hospital and Miyake Eye Hospital. After topical or sub-Tenon’s capsule anesthesia by lidocaine hydrochloride, a standard microincision cataract surgery was performed including a 2.4-mm corneal or corneoscleral incision, continuous curvilinear capsulorhexis,
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phacoemulsification, and implantation of a foldable intraocular lens. There was no incision on
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the lower bulbar conjunctiva where the impression cytology was to be performed.
Randomization
Before the surgery, the 80 eyes were randomized into four groups using an allocation table made by a random numbers table (Fig. 1, YT). The allocation sequence was concealed until interventions were assigned. No information was provided to the cataract surgeons or rater of the results of impression cytology and tear film BUT about which group each eye belonged to. After the surgery, Groups A and B received 0.1% diclofenac without preservative 4
ACCEPTED MANUSCRIPT (Nitten Pharmaceutical Co., Ltd. Nagoya, Japan), and Groups C and D received 0.1% bethamethasone without preservative (Nitten Pharmaceutical Co., Ltd. Nagoya, Japan). These treatments were begun one day after the surgery and performed three times/day until 28 days after the surgery. In addition to these anti-inflammatory eyedrops, Groups B and D
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also received 2% topical rebamipide ocular solution (Otsuka Pharmaceutical Co., Ltd. Tokyo, Japan) four times/day from day 1 after the surgery and continued for 28 days. All eyes also received 0.5% moxifloxacin (Alcon Inc., Tokyo, Japan), a topical antibiotic, from day 1 after
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Impression cytology
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the surgery and continued for 28 days.
The primary endpoint of this study was the goblet cell density at one month after the surgery. Our methods of impression cytology have been described in detail.41 Impression cytology42 was performed before the surgery and one month after the surgery by five examiners (KK,
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NK, SA, JT, and AT). To perform impression cytology, the conjunctiva was topically anesthetized, and nitrocellulose membrane strips with 0.22 µm pore size (Merck Millipore Ltd, Darmstade, Germany) were placed on the inferior bulbar conjunctiva. We collected the
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specimens for impression cytology from only the inferior bulbar conjunctiva because we wanted to minimize the effects of the incision used for the cataract surgery on the results of
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impression cytology. The strips remained in contact with the conjunctiva for approximately 10 seconds and then peeled off with forceps. The strips were immediately placed into 95% alcohol for at least 2 hours, and then stained with Periodic Acid-Schiff (PAS). The strips were mounted with Entellan (Merck, Darmstadt, Germany) on glass microscopic slides and were examined and photographed under a light microscope at a magnification of 200x. The number of goblet cells was counted in three adjacent rectangular fields of 1.28 mm x 0.96 mm. The mean density of the goblet cells, cells/mm2, was determined for each specimen. No information was provided to the raters of the results of the impression cytology (KK) about 5
ACCEPTED MANUSCRIPT which group each specimen belonged to.
Measurements of tear film break-up time (BUT) and aqueous flare The tear film BUT was measured before the surgery and at one and two months after the
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surgery. No information was provided to the raters (KK, NK, SA, JT, and AT) of the tear film BUT about which group each patient belonged to. The aqueous flare value was assessed with a laser flare-cell photometer (Kowa FC-1000 LFCM, Kowa Co. Ltd. Tokyo) before the
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surgery and at one and two months after the surgery (Fig. 1).
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Sample size
The sample size was determined by the following estimation: level of significance, 5%; estimated value of standard deviation of goblet cell density, 150 cells/mm2; scientific significant difference of goblet cell density between the before and after the surgery, 150
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cells/mm2; and power, 90%. This estimation resulted in a required sample size of 13. However, in consideration of 10-20% dropout ratio and the lack of previous knowledge on the conjunctival goblet cell density before and after cataract surgery, we set the sample size 20
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for each group.
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Statistical analyses
A one-way layout analysis of variance (ANOVA) and Chi-square tests were used to examine the homogeneity of the background factors in the four groups. After confirming that the data was approximately normally distributed by the calculation of the skewness and kurtosis, paired t tests were used to determine if the density of goblet cells before the surgery was significantly different from that at one month after the surgery within the same group. Unpaired t tests were used to examine if the changes in the goblet cell density between two groups were significant. Dunnett's multiple comparison tests were used if the tear film BUTs 6
ACCEPTED MANUSCRIPT or aqueous flare values at one and two months after the surgery were different from those before the surgery. Results were considered statistically significant when P <0.05.
Results
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All 80 eyes had successful cataract surgery without any complications. After the surgery, one patient of Group A received an unplanned anti-dry eye medication because of severe dry eye symptoms. Two patients of Group B and D could not visit the hospital as scheduled. In
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addition, twelve eyes were excluded because the quality of the impression cytology was not sufficient to measure the goblet cell density. In the end, a total of 65 eyes were used for the
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final analyses (Fig. 1).
The demographic data of the four groups are shown in Table. There were no significant differences in the age, sex, preoperative tear film BUT, preoperative aqueous flare value, preoperative goblet cell density, type of anesthesia, incision, and operation time
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among the four groups.
Photographs of the impression cytology strips obtained from the eyes of representative patients from the four groups are shown in Figure 2. These images were
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obtained from specimens collected before the surgery and at one month after the surgery of the same subjects. At a glance, the density of PAS-positive goblet cells tended to decrease
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at one month after the surgery for all groups. The reduction of goblet cell density after the surgery appeared to be most marked in Group A (diclofenac without rebamipide, upper left panel of Fig. 2).
The mean (± SD) density of goblet cells before the surgery in Group A was 257.0 ± 188.7 cells/mm2, and it decreased significantly to 86.5 ± 76.7 cells/mm2 at one month after the surgery (P = 0.002, paired t test, left panel of Fig. 3). In Group B, the mean density of goblet cells decreased slightly from 238.5 ± 116.6 cells/mm2 to 211.3 ± 184.4 cells/mm2 at one month after the surgery but this difference was not significant (P = 0.55, middle panel of 7
ACCEPTED MANUSCRIPT Fig. 3). The decrease in the goblet cell density between the two groups was also compared at one month after the surgery. We found that the decrease in the mean goblet cell density was significantly greater in Group A (170.5 ± 195.1 cells/mm2) than in Group B (27.2 ± 172.4 cells/mm2, P = 0.03, unpaired t test, right panel of Fig. 3).
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Similar comparisons were made for Groups C and D (Fig. 4). The mean goblet cell density decreased slightly at one month after the surgery, but the decrease was not
significant for both Groups C and D (P = 0.11 and 0.52. respectively, left and middle panels
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of Fig. 4). The decrease in the mean goblet cell density was also not significantly different between Groups C and D (P = 0.46, right panel of Fig. 4).
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Next, we examined whether there were significant changes in the tear film BUT before and after the surgery for the four groups (Fig. 5). The BUT tended to decrease after the surgery for all four groups. We found that the reduction in the mean tear film BUT was significant at one and two months after the surgery in Group A (P <0.001 and P = 0.03,
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respectively, Dunnett's multiple comparison test, upper left panel of Fig. 5), and at one month after the surgery in Group B (P = 0.03, upper right panel of Fig. 5). In Groups C and D, the differences in the BUT before and after the surgery were not significant (lower panels of Fig.
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5).
Finally, we studied the changes in the aqueous flare value before and after the
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surgery for all four groups (Fig. 6). The flare values tended to increase at one month after the surgery, then it decreased slightly at two months after the surgery for all groups. A significant increase in the aqueous flare value was seen only at one month after the surgery in Group C (P = 0.008, Dunnett's multiple comparison test, lower left panel of Fig. 6). There were no serious ocular or systemic adverse events with relation to cataract surgery and examinations. One patient of Group A complained of severe dry eye symptoms after the cataract surgery and received an unplanned anti-dry eye medication (Fig. 1).
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Discussion The results showed that the conjunctival goblet cell density was significantly reduced after the cataract surgery with IOL implantation and application of topical diclofenac. However, this reduction was significantly attenuated by the concomitant use of topical rebamipide. The
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reduction of goblet cell density was also found in eyes treated with postoperative topical betamethasone although the decrease was not statistically significant. These results are significant because the studies were performed using preservative-free steroids or NSAIDs
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which minimized the negative effects of preservatives on the ocular surface.10 The reduction of goblet cell density was greater with diclofenac than with betamethasone, and rebamipide
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blocked this reduction.
The NSAIDs inhibit the biosynthesis of prostaglandin E2 (PGE2) by inhibiting the cyclooxygenase (COX) pathway.43 PGE2 can combine with four types of receptors and then have various biological activities.44 Homeostasis of the ocular surface is maintained by
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mutual interactions between the mucous epithelium, the immune system, and the neural elements. PGE2 enhances the metaplasia and proliferation of the mucous epithelium, accelerates or inhibits inflammation, and promotes hypersensitivity.45 Suppression of these
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biological actions of PGE2 is the main side effects of NSAIDs that leads to ocular surface epithelial disorders which then induce the main symptoms of dry eye.
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A reduction in the goblet cell density is the main morphological change seen in
eyes inflicted with dry eye,19 and this is manifested by a shortening of the tear film BUT, epithelial cell metaplasia, inflammation, and abnormal sensations.3,4 The reduction of the goblet cell density due to the use of NSAIDs is directly related to the reduction of mucin secretion and is closely related to metaplasia of the epithelial cells and epithelial barrier breakdown.19 Thus, the use of topical NSAIDs is considered to be a significant postoperative risk factor for dry eye.11,27-30 NSAIDs and preservatives of the topical solutions applied postoperatively, as well as several intraoperative factors, all contribute to the postoperative 9
ACCEPTED MANUSCRIPT dry eyes following cataract surgery.5-14 These findings agree with the results from a large epidemiological research study that reported that cataract surgery itself is a risk factor for dry eyes.46-48 Our findings showed that the concomitant use of topical rebamipide prevented the
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reduction in the goblet cell density induced by topical diclofenac. Rebamipide was originally used to treat or prevent the side effects of oral NSAIDs on the gastrointestinal mucosa.38,39 The mechanism for its effect can be explained by the biological actions on prostaglandin E2
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(PGE2).38,39,45 In the gastrointestinal mucosal membrane, rebamipide has been shown to promote metaplasia and proliferation of the mucous epithelial cells and to inhibit inflammation
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and pain.49 While the NSAIDs inhibit PGE2 biosynthesis,43 rebamipide promotes its synthesis,50 and PGE2 induces these biological effects mainly by combining with receptors such as EP2 and EP4. All of these actions contribute to maintaining the homeostasis of the gastrointestinal mucosal membranes.49 Also, receptors such as EP2, EP3, and EP4 are
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expressed in normal human cornea and conjunctival epithelium, however in severe dry eye such as those with Stevens-Johnson syndrome, their expressions are reduced. This suggests a close relationship between these receptors and dry eye.51-53 These findings
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explain how topical rebamipide maintains the homeostasis of the ocular surface and prevents the side effects of topical NSAIDs.
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When the tear film BUT of the diclofenac-treated groups were compared, the tear
film BUT was significantly reduced at one and two months after surgery compared to that before the surgery in the group without the concomitant use of rebamipide (Group A, upper left panel of Fig. 5). However, in the group with concomitant use of rebamipide (Group B), the tear film BUT remained unchanged at 2 months after surgery (upper right panel of Fig. 5). Furthermore, when the tear film BUT in the betamethasone-treated groups were compared, there was no significant change before and after surgery with or without the concomitant use of rebamipide (Group C & D, lower panels of Fig. 5). These results do not contradict the 10
ACCEPTED MANUSCRIPT impression cytology findings that the goblet cell density was reduced in cases treated with topical diclofenac and the concomitant use of topical rebamipide prevented this reduction. In cases with topical betamethasone, the reduction of the goblet cell density was not significant. The reason for the larger number of goblet cells but lower tear film BUTs at one month after
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the surgery in Group B might be due to the different timing of recovery of the morphology and function of the goblet cells after the surgery.
When the aqueous flare values of the diclofenac-treated groups were compared,
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there was no significant difference between the values before and at one and two months after the surgery regardless of the concomitant use of rebamipide (upper panels of Fig. 6).
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On the other hand, when the aqueous flare values of the betamethasone-treated groups were compared, there was a significant increase in the aqueous flare at one month after the surgery compared to that before the surgery in the subgroup without the concomitant use of rebamipide (lower left panel of Fig. 6). This agrees with the results from previous reviews and
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meta-analysis confirming that the NSAIDs are more effective than steroids in ameliorating postoperative inflammation.23-26
There are many risk factors associated with postoperative iatrogenic dry eye.5-14
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We have shown that the NSAIDs are also among the risk factors and have described the mechanism of developing dry eye. NSAIDs are reported to be effective in preventing
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postoperative inflammation,20-26 in treating postoperative pain and eye discomfort,54 and in treating dry eye.55 There are merits and demerits on the use of NSAIDs which reflect the complex biological activity of PGE2. There are two limitations in this study. First, we did not measure the conjunctival goblet cell densities at two months after the surgery because we wanted to minimize invasive examinations in these patients. However, if the goblet cell densities at two months were examined, then we could learn the degree of recovery of the goblet cell density after the termination of dicrofenac or betamethasone. 11
ACCEPTED MANUSCRIPT Second, we demonstrated that the goblet cell density was significantly reduced after the surgery in Group A (diclofenac only), but did not determine which of two possible factors, the use of diclofenac or the cataract surgery, was more strongly correlated with the reduction of goblet cell density. We could not do this because we did not have any control
Further studies are needed to clarify this point.
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groups, e.g., diclofenac without surgery or surgery without any anti-inflammatory drugs.
In conclusion, we found that topical diclofenac after cataract with IOL implantation
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surgery reduces the conjunctival goblet cell density but the concomitant use of topical
rebamipide prevents this reduction. Cataract with IOL implantation surgical techniques have
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improved to be less invasive and as a result, postoperative inflammation is reduced. However, even with the advances in cataract and IOL surgical techniques, the incidence of postoperative dry eye is higher than expected.15 One critical review reported that there is a lack of level I evidence that supports the long-term visual benefit of NSAID therapy when
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applied solely or in combination with corticosteroid therapy to prevent vision loss resulting from CME after cataract surgery.56 Another recent meta-analysis confirms the effects of NSAIDs on reducing the incidence of CME and/or the postoperative pain.57 This pain closely
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relates to the onset of pain as a sign of postoperative dry eye syndrome. After a comprehensive review of these conflicting information, it is important to consider what has
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been believed as the most adequate combination and duration of medications including the use of antibiotics, anti-inflammatory drugs and anti-dry eye drugs.
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ACCEPTED MANUSCRIPT a. FUNDING/SUPPORT: Grant-in-Aid for Scientific Research C (KK,16K11266; MK, 26462683) from Ministry of Education, Culture, Sports, Science and Technology of Japan. (http://www.jsps.go.jp/). b. Financial Disclosures: KK received honoraria from Alcon, Eizai, Otsuka, and Santen. KM
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received honoraria from Alcon, Hoya, Kowa, Otsuka, Santen, Seed and Senju. MK is a
consultant to Senjyu, and received financial research support from Alcon, Hoya, Nidek,
Novartis, Otsuka, Pfizer, Santen, and Senju, and honoraria from Alcon, Bayer, Hoya, Nidek,
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Novartis, Otsuka, Pfizer, Sanofi, Santen, Sanwa, and Senju. Other authors have no financial disclosures.
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c. Other Acknowledgements: We thank Kengo Ikesugi, Masahiko Sugimoto and Hisashi Matsubara of Mie University Hospital, and Ichiro Ota, Goichiro Miyake and Tetsu Asami of Miyake Eye Hospital for performing cataract surgery in our patients. We also thank Professor Duco I. Hamasaki of the Bascom Palmer Eye Institute of the University of Miami for critical
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discussion and final manuscript revisions.
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29. Khalifa YM, Mifflin MD. Keratitis and corneal melt with ketorolac tromethamine after conductive keratoplasty. Cornea 2011;30(4):477-478.
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30. Gokhale NS, Vemuganti GK. Diclofenac-induced acute corneal melt after collagen crosslinking for keratoconus. Cornea 2010;29(1):117-119. 31. Movahedan A, Djalilian AR. Cataract surgery in the face of ocular surface disease. Curr Opin Ophthalmol 2012;23(1):68-72. 32. Koh S, Ikeda C, Takai Y, Watanabe H, Maeda N, Nishida K. Long-term results of treatment with diquafosol ophthalmic solution for aqueous-deficient dry eye. Jpn J Ophthalmol 2013;57(5):440-446. 33. Ríos JD, Shatos M, Urashima H, Tran H, Dartt D. OPC-12759 increases proliferation of 16
ACCEPTED MANUSCRIPT cultured rat conjunctival goblet cells. Cornea 2006;25(5):573-581. 34. Ríos JD, Shatos MA, Urashima H,Dartt DA. Effect of OPC-12759 on EGF receptor activation, p44/p42 MAPK activity, and secretion in conjunctival goblet cells. Exp Eye Res 2008;86(4):629-636.
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35. Tanaka H, Fukuda K, Ishida W, Harada Y, Sumi T, Fukushima A. Rebamipide increases barrier function and attenuates TNFα-induced barrier disruption and cytokine expression in human corneal epithelial cells. Br J Ophthalmol 2013;97(7):912-916.
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36. Kinoshita S, Awamura S, Oshiden K, Nakamichi N, Suzuki H, Yokoi N. Rebamipide
(OPC-12759) in the treatment of dry eye: a randomized, double-masked, multicenter,
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placebo-controlled phase II study. Ophthalmology 119(12);2471-2478:2012. 37. Kinoshita S, Awamura S, Nakamichi N, Suzuki H, Oshiden K, Yokoi N. A multicenter, open-label, 52-week study of 2% rebamipide (OPC-12759) ophthalmic suspension in patients with dry eye. Am J Ophthalmol 2014;157(3):576-583.
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38. Arakawa T, Watanabe T, Fukuda T, Yamasaki K, Kobayashi K. Rebamipide, novel prostaglandin-inducer accelerates healing and reduces relapse of acetic acid-induced rat gastric ulcer. Comparison with cimetidine. Dig Dis Sci 1995;40(11):2469-2472.
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39. Zhang S, Qing Q, Bai Y, et al. Rebamipide helps defend against nonsteroidal antiinflammatory drugs induced gastroenteropathy: a systematic review and meta-analysis.
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Dig Dis Sci 2013;58(7):1991-2000. 40. Schiffman RM, Christianson MD, Jacobsen G, Hirsch JD, Reis BL. Reliability and validity of the Ocular Surface Disease Index. Arch Ophthalmol 2000;118(5):615-621. 41. Kato K, Takashima Y, Matsunaga K, et al. Effect of topical rebamipide on conjunctival goblet cell recovery after vitrectomy. Sci Rep 2016;6:19516. 42. Calonge M, Diebold Y, Saez V, et al. Impression cytology of the ocular surface: a review. Exp Eye Res 2004;78(3):457-472. 43. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like 17
ACCEPTED MANUSCRIPT drugs. Nature New Biol 1971;231(25):232-235. 44. Narumiya S, Furuyashiki T. Fever, inflammation, pain and beyond: prostanoid receptor research during these 25 years. FASEB J 2011;25(3):813-818. 45. Nakanishi M, Rosenberg DW. Multifaceted roles of PGE2 in inflammation and cancer.
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Semin Immunopathol 2013;35(2):123-137.
46. Moss SE, Klein R, Klein BEK. Prevalence of and risk factors for dry eye syndrome. Arch Ophthalmol 2000;118(9):1264-1268.
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47. Vehof J, Kozareva D, Hysi PG, Hammond CJ. Prevalence and risk factors of dry eye disease in a British female cohort. Br J Ophthalmol 2014;98(12):1712-1717.
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48. Ahn JM, Lee SH, Rim THT, et al. Prevalence of and risk factors associated with dry eye: the Korea National Health and Nutrition Examination Survey 2010-2011. Am J Ophthalmol 2014;158(6):1205-1214.
49. Takeuchi K, Kato S, Amagase K. Prostaglandin EP receptors involved in modulating
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gastrointestinal mucosal integrity. J Pharmacol Sci 2010;114(3):248-261. 50. Kleine A, Kluge S, Peskar BM. Stimulation of prostaglandin biosynthesis mediates gastroprotective effect of rebamipide in rats. Dig Dis Sci 1993;38(8):1441-1449.
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51. Ueta M, Matsuoka T, Yokoi N, Kinoshita S. Prostaglandin E2 suppresses polyinosine– polycytidylic acid (polyI:C)-stimulated cytokine production via prostaglandin E2 receptor
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(EP) 2 and 3 in human conjunctival epithelial cells. Br J Ophthalmol 2011;95(6):859-863. 52. Ueta M, Matsuoka T, Sotozono C, Kinoshita S. Prostaglandin E2 suppresses poly I: Cstimulated cytokine production via EP2 and EP3 in immortalized human corneal epithelial cells. Cornea 2012;31(2):1294-1298. 53. Ueta M, Sotozono C, Yokoi N, Inatomi T, Kinoshita S. Prostaglandin E receptor subtype EP3 expression in human conjunctival epithelium and its changes in various ocular surface disorders. PLoS One 2011;6(9):e25209. 54. Donnenfeld ED, Nichamin LD, Hardten DR, et al. Twice-daily, preservative-free ketorolac 18
ACCEPTED MANUSCRIPT 0.45% for treatment of inflammation and pain after cataract surgery. Am J Ophthalmol 2011;151(3):420-426. 55. Liu X, Wang S, Kao AA, Long Q. The effect of topical pranoprofen 0.1% on the clinical
1239.
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evaluation and conjunctival HLA-DR expression in dry eyes. Cornea 2012;31(11):1235-
56. Kim SJ, Schoenberger SD, Thorne JE, Ehlers JP, Yeh S, Bakri SJ. Topical nonsteroidal anti-inflammatory drugs and cataract surgery: a report by the American Academy of
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Ophthalmology. Ophthalmology 2015;122(11):2159-2168.
57. Duan P, Liu Y, Li J. The comparative efficacy and safety of topical non-steroidal anti-
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inflammatory drugs for the treatment of anterior chamber inflammation after cataract surgery: a systematic review and network meta-analysis. Graefes Arch Clin Exp
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Ophthalmol 2017;255(4):639-649.
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Figure Legends Figure 1: Study design, examinations, randomization, and dropouts. Before the surgery, 80 patients were randomized into four groups based on the postoperative topical drops to be used. After the surgery, one patient of Group A received an unplanned anti-dry eye
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medication because of severe dry eye symptoms. Two patients of Group B and D could not visit the hospital as scheduled. In addition, twelve eyes were excluded because the quality of
density. Thus, a total of 65 eyes were analyzed.
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the impression cytology specimens was not sufficient for the measurements of the goblet cell
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Figure 2: Photomicrographs of impression cytology with Periodic Acid-Schiff (PAS) staining of specimens obtained from the eyes of representative patients in the four groups (A–D). Impression cytology images before and one month after the cataract surgery in the same subjects are shown. Group A are the eyes that had received topical diclofenac without
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rebamipide. Group B are the eyes that received topical diclofenac with rebamipide. Group C are the eyes that received topical betamethasone without rebamipide. Group D are the eyes
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that received betamethasone with rebamipide. Scale bars, 50 µm.
Figure 3: Means and standard error of the means (SEMs) of goblet cell densities before and
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at one month after the cataract surgery in Groups A and B. (Left and middle) Goblet cell density before the cataract surgery and at one month after the surgery in Groups A (diclofenac without rebamipide) and B (diclofenac with rebamipide). The decrease of goblet cell density at one month after the surgery was statistically significant in Group A but not significant in Group B. (Right) The decrease of goblet cell density at one month after the surgery in Groups A and B. The decrease of goblet cell density at one month after the surgery was significantly greater in Group A than in Group B. 20
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Figure 4: Means and standard error of the means (SEMs) of goblet cell densities before and at one month after the cataract surgery in Group C (betamethasone without rebamipide) and D (betamethasone with rebamipide).
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(Left and middle) The mean goblet cell density was slightly lower at one month after the surgery in both Groups C and D but these differences were not statistically significant.
(Right) The decrease in the mean goblet cell density was not significantly different between C
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and D.
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Figure 5: Means and standard error of the means (SEMs) of the tear film break-up time (BUT) before and at one and two months after the cataract surgery for the four groups. (Top left) Eyes that received postoperative topical diclofenac without rebamipide. (Top right) Eyes that received postoperative topical diclofenac with rebamipide. (Bottom left) Eyes that
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received postoperative topical betamethasone without rebamipide. (Bottom right) Eyes that received postoperative topical betamethasone with rebamipide. The tear film BUT was decreased after the surgery for all four groups. The reduction of the mean tear film BUT was
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significant at one and two months after the surgery in Group A, and at one month after the
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surgery in Group B.
Figure 6: Means and standard error of the means (SEMs) of the aqueous flare value before and at one and two months after the cataract surgery for the four groups. (Top left) Eyes that received postoperative topical diclofenac without rebamipide. (Top right) Eyes that received postoperative topical diclofenac with rebamipide. (Bottom left) Eyes that received postoperative topical betamethasone without rebamipide. (Bottom right) Eyes that received postoperative topical betamethasone with rebamipide. The flare values tended to increase at one month after the surgery, then decreased slightly at two month after the surgery for all 21
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month in Group C.
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Table: Demographic data of our four groups Diclofenac
Betamethasone
Rebamipide (+)
Rebamipide (-)
Rebamipide (+)
Group A
Group B
Group C
Group D
18
15
17
15
71.8 ± 7.7
70.4 ± 5.9
70.5 ± 5.3
70.1 ± 7.1
0.88
6/12
4/11
10/7
8/7
0.19
Tear film break-up time (sec)
7.4 ± 2.7
6.9 ± 2.2
7.3 ± 2.3
6.8 ± 2.1
0.83
Aqueous flare value (photon/ms)
11.1 ± 8.0
14.0 ± 15.8
11.1 ± 7.7
11.6 ± 8.6
0.84
Goblet cell density (cells/mm2)
239 ±117
257 ± 189
249 ± 189
295 ± 172
0.80
Anesthesia (topical/sub-Tenon)
7/11
7/8
10/7
10/5
0.39
Incision (corneal/sclerocorneal)
18/0
14/1
16/1
15/0
0.54
14.7 ± 9.9
13.9 ± 14.6
0.85
Sex (male/female)
Operation time (min)
17.5 ± 10.6
15.5 ± 12.4
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Data are shown as the mean ± standard deviation.
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Age (yrs)
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No. of eyes
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Rebamipide (-)
p-value
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S0002-9394(17)30261-1
DOI:
10.1016/j.ajo.2017.06.016
Reference:
AJOPHT 10174
To appear in:
American Journal of Ophthalmology
Received Date: 13 April 2017 Revised Date:
14 June 2017
Accepted Date: 19 June 2017
Please cite this article as: Kato K, Miyake K, Kondo N, Asano S, Takeda J, Takahashi A, Takashima Y, Kondo M, Conjunctival Goblet Cell Density Following Cataract Surgery with Diclofenac Versus Diclofenac and Rebamipide: A Randomized Trial, American Journal of Ophthalmology (2017), doi: 10.1016/j.ajo.2017.06.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Abstract Purpose: To determine the effects of topical diclofenac or betamethasone with concomitant application of topical rebamipide on the conjunctival goblet cell density in eyes after cataract
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surgery. Design: Randomized clinical trial.
Participants: Eighty patients who were scheduled for cataract surgery.
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Methods: Patients were randomized into four groups according to the postoperative topical drugs to be given; Group A, diclofenac alone; Group B, diclofenac and rebamipide; Group C,
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betamethasone alone; and Group D, betamethasone and rebamipide. Impression cytology was performed before and at one month after the surgery, and the mean density of goblet cells was determined.
Results: The mean (± SD) density of goblet cells before the surgery in Group A was 257.0 ±
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188.7 cells/mm2, and it decreased significantly to 86.5 ± 76.7 cells/mm2 at one month after the surgery (P = 0.002). In Group B, the goblet cell density was not statistically different between before (238.5 ± 116.6 cells/mm2) and at one month after the surgery (211.3 ± 184.4
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cells/mm2, P = 0.55). In groups C and D, the mean density of goblet cells was decreased at
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one month after the surgery, but the decreases were not significant (P = 0.11 and 0.52, respectively).
Conclusion: After cataract surgery with postoperative topical diclofenac, the conjunctival goblet cell density was significantly reduced, and this reduction was blocked by the concomitant use of topical rebamipide. These results suggest that the concomitant use of topical rebamipide with non-steroidal anti-inflammatory drugs is beneficial especially in cases with postoperative dry eyes.
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Conjunctival Goblet Cell Density Following Cataract Surgery with Diclofenac Versus Diclofenac and Rebamipide:
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A Randomized Trial
KUMIKO KATO, KENSAKU MIYAKE, NAGAKO KONDO, SAYAKA ASANO,
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JUNKO TAKEDA, AKIKO TAKAHASHI, YUKO TAKASHIMA, AND MINEO KONDO
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Short title: Goblet cell density after cataract surgery
Key Words: cataract surgery; goblet cell; impression cytology; non-steroidal anti-
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inflammatory drug (NSAID); diclofenac; rebamipide; steroid
From the Department of Ophthalmology, Mie University Graduate School of Medicine, Tsu,
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Japan (K.K., Y.T., M.K.); and the Shohzankai Medical Foundation Miyake Eye Hospital, Nagoya, Japan (K.M., N.K., S.A., J.T., A.T). Inquiries to Kumiko Kato, Department of Ophthalmology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan; phone number: +81-59-231-5027; Fax number: +81-59-231-3036; e-mail: [email protected]
1
ACCEPTED MANUSCRIPT Introduction Cataract surgery with intraocular lens (IOL) implantation is generally accepted as a safe surgical procedure that provides very good postoperative outcomes. However, some challenges remain such as improving the postoperative comfort and restoring high quality
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vision. Factors related to these challenges are the optics of the IOL including the so-called premium IOLs,1 corneal configurations,2 and pathologies of the tear film and the ocular surface including the dry eye syndrome.3,4
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The dry eye syndrome or dry eye disease is a common condition which affects the tear film and the ocular surface. The 2007 International Dry Eye Workshop stated that, “dry
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eye is a multifactorial disease of the tears and ocular surface that results in symptoms such as ocular discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface”.3 Numerous factors have been designated as risk factors for postoperative dry eyes.5-14 Although the precise mechanism(s) for postoperative dry eye is not completely
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understood, past studies have suggested that eyes with this type of abnormality have a short tear film break-up time (BUT),15 a decrease in the density of goblet cells, and metaplasia of the corneal and conjunctival epithelial cells16-18 as seen in eyes with the conventional dry eye
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syndrome.19
The main medications used to treat the postoperative inflammation after cataract
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surgery are steroidal and non-steroidal anti-inflammatory drugs (NSAIDs). The NSAIDs were first used in Japan to prevent intraoperative miosis,20 postoperative breakdown of the bloodaqueous barrier, and cystoid macular edema.21,22 More than 60 peer reviewed studies have appeared worldwide that reported on the effects of topical NSAIDs. Recent reviews and meta-analysis have shown that NSAIDs are even more effective than steroids.23-26 However, NSAIDs can have side effects on the ocular surface and tear film.11,27-30 The NSAIDs have been recently suggested to be risk factors for postoperative dry eye31 although the mechanism for this has not been determined. 2
ACCEPTED MANUSCRIPT Recently, two anti-dry eye medications, diquafosol and rebamipide, have become commercially available in Japan.32,33 Rebamipide acts by promoting mucin secretions and improving the homeostasis of the ocular surface, and its topical application has led to a proliferation of goblet cells and the production of mucin. These changes stabilize the barrier
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function of the corneal epithelium and thereby enhance its anti-inflammatory effects.33,34
Experimental and clinical studies have reported that rebamipide eyedrops are effective in treating dry eyes without significant side effects even after long follow-up times.35-37
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Oral rebamipide was initially used to treat and prevent gastric ulcers, and it is currently widely used to prevent the oral NSAIDs-induced gastric mucosal damage.38,39
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Therefore, it is reasonable to evaluate the effects of topical rebamipide in preventing the side effects of topical NSAIDs which are widely used today as a postoperative anti-inflammatory agent.
Thus, the purpose of this study was to determine the effects of preservative-free
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steroids or NSAIDs eyedrops alone or together with rebamipide eyedrops on the conjunctival
Methods
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goblet cells density assessed by impression cytology.
Study design and approvals
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This was a randomized clinical trial conducted at the Mie University Hospital and Miyake Eye Hospital between June 2015 and February 2017. The procedures used were approved by the Medical Ethics Committee of Mie University Hospital (No. 2855) and Miyake Eye Hospital (No. 2016002). The procedures conformed to the tenets of the Declaration of Helsinki of the World Medical Association. All patients signed a written informed consent after they were provided with information on the procedures to be used. The study was also registered International Clinical Trial Registry Platform (UMIN Clinical Trials Registry, UMIN000026310, http://www.umin.ac.jp/ctr/index-j.htm). 3
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Subjects The eligibility criteria for the subjects were patients who required cataract surgery and were ≥50-years-of-age. The exclusion criteria included prior keratoconjunctival diseases, uveitis,
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glaucoma, diabetes mellitus, and eyes that had undergone any type of surgery of the ocular surface or intraocular surgery within 12 months of the beginning of this study. Before the surgery, we checked whether the subjects had any symptoms of dry eye using the Dry Eye
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Ocular Surface Disease Index (OSDI) Questionnaire.40 We excluded subjects who had
painful or sore eyes, and also subjects who had tear film BUT of less than 5 seconds or had
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corneal staining.
Cataract Surgery
Cataract surgery was performed by seven experienced surgeons in the Mie University
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Hospital and Miyake Eye Hospital. After topical or sub-Tenon’s capsule anesthesia by lidocaine hydrochloride, a standard microincision cataract surgery was performed including a 2.4-mm corneal or corneoscleral incision, continuous curvilinear capsulorhexis,
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phacoemulsification, and implantation of a foldable intraocular lens. There was no incision on
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the lower bulbar conjunctiva where the impression cytology was to be performed.
Randomization
Before the surgery, the 80 eyes were randomized into four groups using an allocation table made by a random numbers table (Fig. 1, YT). The allocation sequence was concealed until interventions were assigned. No information was provided to the cataract surgeons or rater of the results of impression cytology and tear film BUT about which group each eye belonged to. After the surgery, Groups A and B received 0.1% diclofenac without preservative 4
ACCEPTED MANUSCRIPT (Nitten Pharmaceutical Co., Ltd. Nagoya, Japan), and Groups C and D received 0.1% bethamethasone without preservative (Nitten Pharmaceutical Co., Ltd. Nagoya, Japan). These treatments were begun one day after the surgery and performed three times/day until 28 days after the surgery. In addition to these anti-inflammatory eyedrops, Groups B and D
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also received 2% topical rebamipide ocular solution (Otsuka Pharmaceutical Co., Ltd. Tokyo, Japan) four times/day from day 1 after the surgery and continued for 28 days. All eyes also received 0.5% moxifloxacin (Alcon Inc., Tokyo, Japan), a topical antibiotic, from day 1 after
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Impression cytology
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the surgery and continued for 28 days.
The primary endpoint of this study was the goblet cell density at one month after the surgery. Our methods of impression cytology have been described in detail.41 Impression cytology42 was performed before the surgery and one month after the surgery by five examiners (KK,
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NK, SA, JT, and AT). To perform impression cytology, the conjunctiva was topically anesthetized, and nitrocellulose membrane strips with 0.22 µm pore size (Merck Millipore Ltd, Darmstade, Germany) were placed on the inferior bulbar conjunctiva. We collected the
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specimens for impression cytology from only the inferior bulbar conjunctiva because we wanted to minimize the effects of the incision used for the cataract surgery on the results of
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impression cytology. The strips remained in contact with the conjunctiva for approximately 10 seconds and then peeled off with forceps. The strips were immediately placed into 95% alcohol for at least 2 hours, and then stained with Periodic Acid-Schiff (PAS). The strips were mounted with Entellan (Merck, Darmstadt, Germany) on glass microscopic slides and were examined and photographed under a light microscope at a magnification of 200x. The number of goblet cells was counted in three adjacent rectangular fields of 1.28 mm x 0.96 mm. The mean density of the goblet cells, cells/mm2, was determined for each specimen. No information was provided to the raters of the results of the impression cytology (KK) about 5
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Measurements of tear film break-up time (BUT) and aqueous flare The tear film BUT was measured before the surgery and at one and two months after the
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surgery. No information was provided to the raters (KK, NK, SA, JT, and AT) of the tear film BUT about which group each patient belonged to. The aqueous flare value was assessed with a laser flare-cell photometer (Kowa FC-1000 LFCM, Kowa Co. Ltd. Tokyo) before the
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surgery and at one and two months after the surgery (Fig. 1).
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Sample size
The sample size was determined by the following estimation: level of significance, 5%; estimated value of standard deviation of goblet cell density, 150 cells/mm2; scientific significant difference of goblet cell density between the before and after the surgery, 150
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cells/mm2; and power, 90%. This estimation resulted in a required sample size of 13. However, in consideration of 10-20% dropout ratio and the lack of previous knowledge on the conjunctival goblet cell density before and after cataract surgery, we set the sample size 20
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for each group.
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Statistical analyses
A one-way layout analysis of variance (ANOVA) and Chi-square tests were used to examine the homogeneity of the background factors in the four groups. After confirming that the data was approximately normally distributed by the calculation of the skewness and kurtosis, paired t tests were used to determine if the density of goblet cells before the surgery was significantly different from that at one month after the surgery within the same group. Unpaired t tests were used to examine if the changes in the goblet cell density between two groups were significant. Dunnett's multiple comparison tests were used if the tear film BUTs 6
ACCEPTED MANUSCRIPT or aqueous flare values at one and two months after the surgery were different from those before the surgery. Results were considered statistically significant when P <0.05.
Results
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All 80 eyes had successful cataract surgery without any complications. After the surgery, one patient of Group A received an unplanned anti-dry eye medication because of severe dry eye symptoms. Two patients of Group B and D could not visit the hospital as scheduled. In
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addition, twelve eyes were excluded because the quality of the impression cytology was not sufficient to measure the goblet cell density. In the end, a total of 65 eyes were used for the
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final analyses (Fig. 1).
The demographic data of the four groups are shown in Table. There were no significant differences in the age, sex, preoperative tear film BUT, preoperative aqueous flare value, preoperative goblet cell density, type of anesthesia, incision, and operation time
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among the four groups.
Photographs of the impression cytology strips obtained from the eyes of representative patients from the four groups are shown in Figure 2. These images were
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obtained from specimens collected before the surgery and at one month after the surgery of the same subjects. At a glance, the density of PAS-positive goblet cells tended to decrease
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at one month after the surgery for all groups. The reduction of goblet cell density after the surgery appeared to be most marked in Group A (diclofenac without rebamipide, upper left panel of Fig. 2).
The mean (± SD) density of goblet cells before the surgery in Group A was 257.0 ± 188.7 cells/mm2, and it decreased significantly to 86.5 ± 76.7 cells/mm2 at one month after the surgery (P = 0.002, paired t test, left panel of Fig. 3). In Group B, the mean density of goblet cells decreased slightly from 238.5 ± 116.6 cells/mm2 to 211.3 ± 184.4 cells/mm2 at one month after the surgery but this difference was not significant (P = 0.55, middle panel of 7
ACCEPTED MANUSCRIPT Fig. 3). The decrease in the goblet cell density between the two groups was also compared at one month after the surgery. We found that the decrease in the mean goblet cell density was significantly greater in Group A (170.5 ± 195.1 cells/mm2) than in Group B (27.2 ± 172.4 cells/mm2, P = 0.03, unpaired t test, right panel of Fig. 3).
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Similar comparisons were made for Groups C and D (Fig. 4). The mean goblet cell density decreased slightly at one month after the surgery, but the decrease was not
significant for both Groups C and D (P = 0.11 and 0.52. respectively, left and middle panels
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of Fig. 4). The decrease in the mean goblet cell density was also not significantly different between Groups C and D (P = 0.46, right panel of Fig. 4).
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Next, we examined whether there were significant changes in the tear film BUT before and after the surgery for the four groups (Fig. 5). The BUT tended to decrease after the surgery for all four groups. We found that the reduction in the mean tear film BUT was significant at one and two months after the surgery in Group A (P <0.001 and P = 0.03,
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respectively, Dunnett's multiple comparison test, upper left panel of Fig. 5), and at one month after the surgery in Group B (P = 0.03, upper right panel of Fig. 5). In Groups C and D, the differences in the BUT before and after the surgery were not significant (lower panels of Fig.
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Finally, we studied the changes in the aqueous flare value before and after the
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surgery for all four groups (Fig. 6). The flare values tended to increase at one month after the surgery, then it decreased slightly at two months after the surgery for all groups. A significant increase in the aqueous flare value was seen only at one month after the surgery in Group C (P = 0.008, Dunnett's multiple comparison test, lower left panel of Fig. 6). There were no serious ocular or systemic adverse events with relation to cataract surgery and examinations. One patient of Group A complained of severe dry eye symptoms after the cataract surgery and received an unplanned anti-dry eye medication (Fig. 1).
8
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Discussion The results showed that the conjunctival goblet cell density was significantly reduced after the cataract surgery with IOL implantation and application of topical diclofenac. However, this reduction was significantly attenuated by the concomitant use of topical rebamipide. The
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reduction of goblet cell density was also found in eyes treated with postoperative topical betamethasone although the decrease was not statistically significant. These results are significant because the studies were performed using preservative-free steroids or NSAIDs
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which minimized the negative effects of preservatives on the ocular surface.10 The reduction of goblet cell density was greater with diclofenac than with betamethasone, and rebamipide
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blocked this reduction.
The NSAIDs inhibit the biosynthesis of prostaglandin E2 (PGE2) by inhibiting the cyclooxygenase (COX) pathway.43 PGE2 can combine with four types of receptors and then have various biological activities.44 Homeostasis of the ocular surface is maintained by
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mutual interactions between the mucous epithelium, the immune system, and the neural elements. PGE2 enhances the metaplasia and proliferation of the mucous epithelium, accelerates or inhibits inflammation, and promotes hypersensitivity.45 Suppression of these
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biological actions of PGE2 is the main side effects of NSAIDs that leads to ocular surface epithelial disorders which then induce the main symptoms of dry eye.
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A reduction in the goblet cell density is the main morphological change seen in
eyes inflicted with dry eye,19 and this is manifested by a shortening of the tear film BUT, epithelial cell metaplasia, inflammation, and abnormal sensations.3,4 The reduction of the goblet cell density due to the use of NSAIDs is directly related to the reduction of mucin secretion and is closely related to metaplasia of the epithelial cells and epithelial barrier breakdown.19 Thus, the use of topical NSAIDs is considered to be a significant postoperative risk factor for dry eye.11,27-30 NSAIDs and preservatives of the topical solutions applied postoperatively, as well as several intraoperative factors, all contribute to the postoperative 9
ACCEPTED MANUSCRIPT dry eyes following cataract surgery.5-14 These findings agree with the results from a large epidemiological research study that reported that cataract surgery itself is a risk factor for dry eyes.46-48 Our findings showed that the concomitant use of topical rebamipide prevented the
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reduction in the goblet cell density induced by topical diclofenac. Rebamipide was originally used to treat or prevent the side effects of oral NSAIDs on the gastrointestinal mucosa.38,39 The mechanism for its effect can be explained by the biological actions on prostaglandin E2
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(PGE2).38,39,45 In the gastrointestinal mucosal membrane, rebamipide has been shown to promote metaplasia and proliferation of the mucous epithelial cells and to inhibit inflammation
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and pain.49 While the NSAIDs inhibit PGE2 biosynthesis,43 rebamipide promotes its synthesis,50 and PGE2 induces these biological effects mainly by combining with receptors such as EP2 and EP4. All of these actions contribute to maintaining the homeostasis of the gastrointestinal mucosal membranes.49 Also, receptors such as EP2, EP3, and EP4 are
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expressed in normal human cornea and conjunctival epithelium, however in severe dry eye such as those with Stevens-Johnson syndrome, their expressions are reduced. This suggests a close relationship between these receptors and dry eye.51-53 These findings
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explain how topical rebamipide maintains the homeostasis of the ocular surface and prevents the side effects of topical NSAIDs.
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When the tear film BUT of the diclofenac-treated groups were compared, the tear
film BUT was significantly reduced at one and two months after surgery compared to that before the surgery in the group without the concomitant use of rebamipide (Group A, upper left panel of Fig. 5). However, in the group with concomitant use of rebamipide (Group B), the tear film BUT remained unchanged at 2 months after surgery (upper right panel of Fig. 5). Furthermore, when the tear film BUT in the betamethasone-treated groups were compared, there was no significant change before and after surgery with or without the concomitant use of rebamipide (Group C & D, lower panels of Fig. 5). These results do not contradict the 10
ACCEPTED MANUSCRIPT impression cytology findings that the goblet cell density was reduced in cases treated with topical diclofenac and the concomitant use of topical rebamipide prevented this reduction. In cases with topical betamethasone, the reduction of the goblet cell density was not significant. The reason for the larger number of goblet cells but lower tear film BUTs at one month after
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the surgery in Group B might be due to the different timing of recovery of the morphology and function of the goblet cells after the surgery.
When the aqueous flare values of the diclofenac-treated groups were compared,
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there was no significant difference between the values before and at one and two months after the surgery regardless of the concomitant use of rebamipide (upper panels of Fig. 6).
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On the other hand, when the aqueous flare values of the betamethasone-treated groups were compared, there was a significant increase in the aqueous flare at one month after the surgery compared to that before the surgery in the subgroup without the concomitant use of rebamipide (lower left panel of Fig. 6). This agrees with the results from previous reviews and
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meta-analysis confirming that the NSAIDs are more effective than steroids in ameliorating postoperative inflammation.23-26
There are many risk factors associated with postoperative iatrogenic dry eye.5-14
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We have shown that the NSAIDs are also among the risk factors and have described the mechanism of developing dry eye. NSAIDs are reported to be effective in preventing
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postoperative inflammation,20-26 in treating postoperative pain and eye discomfort,54 and in treating dry eye.55 There are merits and demerits on the use of NSAIDs which reflect the complex biological activity of PGE2. There are two limitations in this study. First, we did not measure the conjunctival goblet cell densities at two months after the surgery because we wanted to minimize invasive examinations in these patients. However, if the goblet cell densities at two months were examined, then we could learn the degree of recovery of the goblet cell density after the termination of dicrofenac or betamethasone. 11
ACCEPTED MANUSCRIPT Second, we demonstrated that the goblet cell density was significantly reduced after the surgery in Group A (diclofenac only), but did not determine which of two possible factors, the use of diclofenac or the cataract surgery, was more strongly correlated with the reduction of goblet cell density. We could not do this because we did not have any control
Further studies are needed to clarify this point.
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groups, e.g., diclofenac without surgery or surgery without any anti-inflammatory drugs.
In conclusion, we found that topical diclofenac after cataract with IOL implantation
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surgery reduces the conjunctival goblet cell density but the concomitant use of topical
rebamipide prevents this reduction. Cataract with IOL implantation surgical techniques have
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improved to be less invasive and as a result, postoperative inflammation is reduced. However, even with the advances in cataract and IOL surgical techniques, the incidence of postoperative dry eye is higher than expected.15 One critical review reported that there is a lack of level I evidence that supports the long-term visual benefit of NSAID therapy when
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applied solely or in combination with corticosteroid therapy to prevent vision loss resulting from CME after cataract surgery.56 Another recent meta-analysis confirms the effects of NSAIDs on reducing the incidence of CME and/or the postoperative pain.57 This pain closely
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relates to the onset of pain as a sign of postoperative dry eye syndrome. After a comprehensive review of these conflicting information, it is important to consider what has
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been believed as the most adequate combination and duration of medications including the use of antibiotics, anti-inflammatory drugs and anti-dry eye drugs.
12
ACCEPTED MANUSCRIPT a. FUNDING/SUPPORT: Grant-in-Aid for Scientific Research C (KK,16K11266; MK, 26462683) from Ministry of Education, Culture, Sports, Science and Technology of Japan. (http://www.jsps.go.jp/). b. Financial Disclosures: KK received honoraria from Alcon, Eizai, Otsuka, and Santen. KM
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received honoraria from Alcon, Hoya, Kowa, Otsuka, Santen, Seed and Senju. MK is a
consultant to Senjyu, and received financial research support from Alcon, Hoya, Nidek,
Novartis, Otsuka, Pfizer, Santen, and Senju, and honoraria from Alcon, Bayer, Hoya, Nidek,
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Novartis, Otsuka, Pfizer, Sanofi, Santen, Sanwa, and Senju. Other authors have no financial disclosures.
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c. Other Acknowledgements: We thank Kengo Ikesugi, Masahiko Sugimoto and Hisashi Matsubara of Mie University Hospital, and Ichiro Ota, Goichiro Miyake and Tetsu Asami of Miyake Eye Hospital for performing cataract surgery in our patients. We also thank Professor Duco I. Hamasaki of the Bascom Palmer Eye Institute of the University of Miami for critical
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discussion and final manuscript revisions.
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57. Duan P, Liu Y, Li J. The comparative efficacy and safety of topical non-steroidal anti-
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inflammatory drugs for the treatment of anterior chamber inflammation after cataract surgery: a systematic review and network meta-analysis. Graefes Arch Clin Exp
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Figure Legends Figure 1: Study design, examinations, randomization, and dropouts. Before the surgery, 80 patients were randomized into four groups based on the postoperative topical drops to be used. After the surgery, one patient of Group A received an unplanned anti-dry eye
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medication because of severe dry eye symptoms. Two patients of Group B and D could not visit the hospital as scheduled. In addition, twelve eyes were excluded because the quality of
density. Thus, a total of 65 eyes were analyzed.
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the impression cytology specimens was not sufficient for the measurements of the goblet cell
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Figure 2: Photomicrographs of impression cytology with Periodic Acid-Schiff (PAS) staining of specimens obtained from the eyes of representative patients in the four groups (A–D). Impression cytology images before and one month after the cataract surgery in the same subjects are shown. Group A are the eyes that had received topical diclofenac without
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rebamipide. Group B are the eyes that received topical diclofenac with rebamipide. Group C are the eyes that received topical betamethasone without rebamipide. Group D are the eyes
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that received betamethasone with rebamipide. Scale bars, 50 µm.
Figure 3: Means and standard error of the means (SEMs) of goblet cell densities before and
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at one month after the cataract surgery in Groups A and B. (Left and middle) Goblet cell density before the cataract surgery and at one month after the surgery in Groups A (diclofenac without rebamipide) and B (diclofenac with rebamipide). The decrease of goblet cell density at one month after the surgery was statistically significant in Group A but not significant in Group B. (Right) The decrease of goblet cell density at one month after the surgery in Groups A and B. The decrease of goblet cell density at one month after the surgery was significantly greater in Group A than in Group B. 20
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Figure 4: Means and standard error of the means (SEMs) of goblet cell densities before and at one month after the cataract surgery in Group C (betamethasone without rebamipide) and D (betamethasone with rebamipide).
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(Left and middle) The mean goblet cell density was slightly lower at one month after the surgery in both Groups C and D but these differences were not statistically significant.
(Right) The decrease in the mean goblet cell density was not significantly different between C
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and D.
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Figure 5: Means and standard error of the means (SEMs) of the tear film break-up time (BUT) before and at one and two months after the cataract surgery for the four groups. (Top left) Eyes that received postoperative topical diclofenac without rebamipide. (Top right) Eyes that received postoperative topical diclofenac with rebamipide. (Bottom left) Eyes that
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received postoperative topical betamethasone without rebamipide. (Bottom right) Eyes that received postoperative topical betamethasone with rebamipide. The tear film BUT was decreased after the surgery for all four groups. The reduction of the mean tear film BUT was
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significant at one and two months after the surgery in Group A, and at one month after the
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surgery in Group B.
Figure 6: Means and standard error of the means (SEMs) of the aqueous flare value before and at one and two months after the cataract surgery for the four groups. (Top left) Eyes that received postoperative topical diclofenac without rebamipide. (Top right) Eyes that received postoperative topical diclofenac with rebamipide. (Bottom left) Eyes that received postoperative topical betamethasone without rebamipide. (Bottom right) Eyes that received postoperative topical betamethasone with rebamipide. The flare values tended to increase at one month after the surgery, then decreased slightly at two month after the surgery for all 21
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Table: Demographic data of our four groups Diclofenac
Betamethasone
Rebamipide (+)
Rebamipide (-)
Rebamipide (+)
Group A
Group B
Group C
Group D
18
15
17
15
71.8 ± 7.7
70.4 ± 5.9
70.5 ± 5.3
70.1 ± 7.1
0.88
6/12
4/11
10/7
8/7
0.19
Tear film break-up time (sec)
7.4 ± 2.7
6.9 ± 2.2
7.3 ± 2.3
6.8 ± 2.1
0.83
Aqueous flare value (photon/ms)
11.1 ± 8.0
14.0 ± 15.8
11.1 ± 7.7
11.6 ± 8.6
0.84
Goblet cell density (cells/mm2)
239 ±117
257 ± 189
249 ± 189
295 ± 172
0.80
Anesthesia (topical/sub-Tenon)
7/11
7/8
10/7
10/5
0.39
Incision (corneal/sclerocorneal)
18/0
14/1
16/1
15/0
0.54
14.7 ± 9.9
13.9 ± 14.6
0.85
Sex (male/female)
Operation time (min)
17.5 ± 10.6
15.5 ± 12.4
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Data are shown as the mean ± standard deviation.
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Age (yrs)
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No. of eyes
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Rebamipide (-)
p-value
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