Effect of Diquafosol on Hyperosmotic Stress-induced Tumor Necrosis Factor-α and Interleukin-6 Expression in Human Corneal Epithelial Cells.

PURPOSE: Diquafosol is a pharmaceutical drug used for dry eye treatment with a novel mechanism of action. It is a purinergic P2Y2 receptor agonist that promotes the secretion of tears and healing of corneal epithelial wounds. However, its inhibitory effect on hyperosmotic stress-induced inflammation in human corneal epithelial cells (HCECs) remains unclear. METHODS: A hyperosmotic stress model was established by transferring HCECs from isosmotic (312 mOsm/kg to hyperosmotic medium (500 mOsm/kg). HCECs were incubated with 500 mOsm/kg hyperosmotic medium for 30 minutes, and then treated with diquafosol (0.6-6 mg/mL) for 4 or 24 hours. Cells were then harvested and analyzed by western blot, immunocytochemistry, and real-time polymerase chain reaction to evaluate the expression of interleukin-6, tumor necrosis factor-alpha, and the phosphorylation status of nuclear factor-kappa B. RESULTS: Diquafosol significantly decreased the mRNA and protein expression of hyperosmotic stress-induced tumor necrosis factor-alpha and interleukin-6. These results were supported by immunofluorescence staining and quantitative real-time polymerase chain reaction analysis. Furthermore, diquafosol inhibits nuclear factor-kappa B activation by suppressing the phosphorylation and degradation of the inhibitor of кB. CONCLUSIONS: This study shows that diquafosol inhibits nuclear factor-kappa B signaling and inflammatory factors induced by hyperosmotic stress in HCECs. This suggests that using diquafosol for the improvement of dry eye syndrome could be effective in the treatment of inflammation-related corneal and conjunctival diseases.

Efficacy and Safety of a Large Conjunctival Autograft for Recurrent Pterygium.

PURPOSE: To evaluate the efficacy and safety of pterygium excision using a large conjunctival autograft for the treatment of recurrent pterygium. METHODS: The medical records of 120 patients (126 eyes) with recurrent pterygium were reviewed. For each affected eye, pterygium excision with a large conjunctival autograft was performed. The graft was harvested from the superior bulbar area and measured more than 8 × 10 mm in size. Only patients who completed at least six months of follow-up were included. Postoperative clinical outcomes, recurrence rate, and complications were analyzed. Patients with any evidence of recurrence after surgery received a subconjunctival bevacizumab injection. RESULTS: The average patient age was 56.5 ± 10.2 years, and 45 out of 120 patients were male. The mean study follow-up period was 17.7 ± 17.6 months. Most patients were satisfied with the cosmetic outcome. Postoperative visual acuity improved from 0.69 to 0.75 (p < 0.05). Postoperative refractive astigmatism and corneal astigmatism decreased by 0.55 and 2.73 diopters, respectively (p < 0.05). The postoperative recurrence rate was 4.0%, and the average recurrence period was 7.4 ± 0.6 weeks. A subconjunctival injection of 5 mg bevacizumab was performed in cases of recurrence; no progression of the pterygium was observed following the injection. Postoperative complications included 2 cases of conjunctival graft edema in 2 eyes, 5 donor site scars in 5 eyes, 13 pyogenic granulomas in 13 eyes, and a conjunctival epithelial inclusion cyst in 7 eyes. CONCLUSIONS: Pterygium excision with a large conjunctival autograft for the treatment of recurrent pterygium produced an excellent cosmetic outcome, a low recurrence rate, and minimal complications. A subconjunctival bevacizumab injection given in cases of recurrence following surgery might be effective in preventing progression of the pterygium.

Inhibition of Pterygium Fibroblast Migration and Outgrowth by Bevacizumab and Cyclosporine A Involves Down-Regulation of Matrix Metalloproteinases-3 and -13.

We examined the connection between matrix metalloproteinase (MMP) expression/activity and pterygium fibroblast migration, and how these were affected by bevacizumab and/or cyclosporine A (CsA). Fibroblasts were obtained from 20 pterygia and 6 normal conjunctival specimens. Expression levels of MMP-3 and MMP-13 were examined after bevacizumab administration. Immunofluorescence staining was used to examine expression of both MMPs in fibroblasts migrating out from explanted pterygium tissues. Rates of cell migration from explant-cultured pterygia tissues and scratch-wounded confluent pterygium fibroblasts were examined in the presence of MMP-3 or MMP-13 inhibitors, as well as bevacizumab and/or CsA. A scratch wound healing migration assay was performed to determine the effects of bevacizumab and/or CsA. Protein expression of both MMPs in pterygium tissues and in cells migrating from organ-cultured pterygium tissues was greater than that observed in normal cells. Inhibition of the activities of both MMPs decreased their expression levels; these were also significantly reduced in bevacizumab-injected pterygium tissues. Bevacizumab significantly reduced the expression of both MMPs and cell migration. Pretreatment with CsA prior to bevacizumab exposure markedly inhibited cell migration and the expression of both MMPs. CsA enhanced the inhibitory effects of bevacizumab on pterygium fibroblast migration in vitro, possibly by inhibiting expression of both MMPs. These findings suggest that combined CsA and bevacizumab treatment may provide a potential therapeutic strategy for reducing the rate of pterygium recurrence.

Cyclosporine A inhibits TGF-ß2-induced myofibroblasts of primary cultured human pterygium fibroblasts.

Cyclosporine A (CsA), an immunomodulatory drug, and is increasingly used to treat moderate dry eye syndrome and ocular surface inflammation. However, any inhibitory effect on differentiation of fibroblasts to myofibroblasts remains unclear. Here, we show that the inhibitory effect of CsA on transforming growth factor-beta2 (TGF-ß2)-induced myofibroblasts in primary cultured human pterygium fibroblasts. CsA significantly decreased mRNA and protein expression of myofibroblast-related markers including α-SMA, laminin, and fibronectin. These findings were supported by the results from immunofluorescence staining. Taken together, these results indicate the therapeutic potential of CsA against pterygium progression. Further studies are necessary to elucidate the precise intracellular signal mechanism responsible for CsA-induced downregulation of myofibroblast markers in pterygium fibroblasts.

The neuroprotective effect of staurosporine on mouse retinal ganglion cells after optic nerve injury.

This study aimed to investigate the neuroprotective properity of staurosporine (STS) and identify the neuroprotective mechanism of staurosporine in mouse retina ganglion cell after optic nerve injured. Mice (C57BL/6) were anaesthetised with a mixture of 5 mg/kg xylazine hydrochloride and 40 mg/kg tiletamine/zolazepam (Zoletil®). Optic nerves of the mice were crushed (Templeton JP et al., 2012). With micro-forceps, the bulbar conjunctiva was grasped and retracted, rotating the globe nasally. The exposed optic nerve was grasped approximately 1-3 mm from the globe with Dumont #N7 cross-action forceps for 10 s. One day after crushing, intravitreal injections of STS (500 nM) were administered using a Narishige IM-300 air pressure regulator. For analysing the change in ganglion cell number, the mice were allowed to live for 30 days, after which they were killed and the ganglion cell survival was assessed. A significant and marked loss of fluorescent spots was found after 30 days, with fewer 4',6-diamidino-2-phenylindole (DAPI)-expressing retinal ganglion cells (RGCs) remaining in the injured and phosphate buffered saline (PBS)-injected group than those in non-injured PBS-injected controls. However, RGC cell numbers dramatically increased in the STS intravitreal injection group. Moreover, degradation of nerve fibre (NF) was markedly reduced in the STS injection group compared with the injured and PBS-injected group by inducing astrocyte expression of Bcl-2. Our data suggested that injection of STS into the vitreous may have a potential therapeutic effect in retinal diseases such as glaucoma.

Comparison of the Efficacy of Fluorometholone With and Without Benzalkonium Chloride in Ocular Surface Disease.

PURPOSE: The purpose of this study was to compare the cytotoxicity and antiinflammatory effect of preserved and unpreserved 0.1% fluorometholone (FML). METHODS: Drug-induced morphological changes and cytotoxicity were examined in human corneal epithelial cells. Dry eye was induced in mice by treatment with 0.2% benzalkonium chloride (BAC) for the first 2 weeks, and then, the eyes (4 groups; Normal saline, BAC, preserved FML, and unpreserved FML) were treated thrice daily with each formulation for the next 2 weeks. Corneal tissues were embedded in paraffin and stained with hematoxylin and eosin for histopathological examination. Immunofluorescence staining was performed for tumor necrosis factor-α, interleukin-6, and human leukocyte antigen-DR. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay was performed to evaluate drug-induced cytotoxicity. RESULTS: BAC and preserved FML caused cell shrinkage and detachment from the plate in a dose-dependent manner, and cell viability decreased significantly. However, cytotoxicity was reduced on treatment with unpreserved FML. Hematoxylin-eosin staining revealed surface desquamation, irregular surface, loss of cell borders, and stromal shrinkage in the group treated with BAC. On BAC exposure, tumor necrosis factor-α, interleukin-6, and human leukocyte antigen-DR were strongly detected, and cytotoxicity was markedly increased, as evidenced by a positive result in the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Ocular surface damage and inflammation were slightly reduced on treatment with preserved FML. In comparison, unpreserved FML did not induce morphological changes; moreover, decreased cell cytotoxicity and ocular surface inflammation were observed. CONCLUSIONS: The cytotoxicity of antiinflammatory eye drops evaluated in this study was induced by the preservative BAC. Accordingly, unpreserved FML is more effective than preserved eye drops in decreasing ocular inflammation.

An Overview of Ophthalmologic Survey Methodology in the 2008-2015 Korean National Health and Nutrition Examination Surveys.

The Korea National Health and Nutrition Examination Survey (KNHANES) is a national program designed to assess the health and nutritional status of the noninstitutionalized population of South Korea. The KNHANES was initiated in 1998 and has been conducted annually since 2007. Starting in the latter half of 2008, ophthalmologic examinations were included in the survey in order to investigate the prevalence and risk factors of common eye diseases such as visual impairment, refractive errors, strabismus, blepharoptosis, cataract, pterygium, diabetic retinopathy, age-related macular degeneration, glaucoma, dry eye disease, and color vision deficiency. The measurements included in the ophthalmic questionnaire and examination methods were modified in the KNHANES IV, V, and VI. In this article, we provide detailed information about the methodology of the ophthalmic examinations in KNHANES in order to aid in further investigations related to major eye diseases in South Korea.

In Vivo Effects of Preservative-free and Preserved Prostaglandin Analogs: Mouse Ocular Surface Study.

PURPOSE: Chronic use of topical hypotensive agents induces several side effects caused by preservatives. The purpose of this study was to evaluate the effects of prostaglandin analogs with varying concentrations of benzalkonium chloride (BAC), preservative-free (PF), and alternative preservatives on mouse corneal tissue. METHODS: Thirty-five, 8- to 10-week-old female C57BL/6 mice (five mice for each group) were used for this study. To the control group, we applied normal saline, and to each drug-treated group we applied 0.02% BAC, bimatoprost 0.01% (with BAC 0.02%), latanoprost 0.005% (with BAC 0.02%), travoprost 0.004% (with 0.001% polyquad) or tafluprost 0.0015% with/without 0.001% BAC, once a day (9 p.m.) for 4 weeks. Corneal fluorescein staining was evaluated in all groups. After harvest, the corneal tissues were embedded in paraffin and then Hematoxylin-Eosin stain was performed for histopathological examination. Immunofluorescence staining was done against TNF-α, IL-6, HLA DR, pJNK, and pAkt. RESULTS: In corneal fluorescein staining, severe punctate epithelial keratitis was seen in the groups of 0.02% BAC, 0.02% BAC containing bimatoprost 0.01% and latanoprost 0.005%. The surface desquamation, irregular surface, loss of cell borders, anisocytosis and stromal shrinkage were observed in the groups of BAC-containing eye drops. Moreover, the groups treated with BAC-containing eye drops have high inflammatory markers, significantly decreased cell viability-related signal, pAkt, and higher apoptosis-inducing signal, pJNK, than the control group. On the other hand, travoprost 0.004% and PF tafluprost 0.0015% have less cellular morphologic changes, lower inflammation, and higher cellular viability than BAC-containing formulations. CONCLUSIONS: Corneal damage, increased inflammation and apoptosis and low cell viability were observed in BAC-containing groups. PF or alternatively preserved glaucoma medications seem to be a reasonable and viable alternative to those preserved with BAC.

Cyclosporine A Downregulates MMP-3 and MMP-13 Expression in Cultured Pterygium Fibroblasts.

PURPOSE: To investigate the regulation of matrix metalloproteinase (MMP)-3 and MMP-13 expression over time and in the presence of cyclosporine A (CsA) in primary cultured human pterygium fibroblasts. We also examined the effects of CsA on cultured human pterygium fibroblasts. METHODS: Primary cultured human pterygium fibroblasts subjected to scratch assays were exposed to 1 and 100 µg/mL of CsA for 3 or 10 minutes. Cells were washed with Dulbecco phosphate-buffered saline, and then incubated with serum-depleted Dulbecco modified Eagle medium/F-12 medium for 48 hours. Expression levels of MMP-3 and MMP-13 proteins and the corresponding mRNA transcripts were determined by western blotting and reverse transcription polymerase chain reaction assays, respectively. RESULTS: Migration of cultured pterygium fibroblast cells was suppressed by pretreatment with CsA compared with controls in a time-dependent and dose-dependent manner (3 minutes, 50.6% ± 1.1 in 1 µg/mL, 60.0% ± 1.2 in 100 µg/mL; 10 minutes, 59.8% ± 5.7 in 1 µg/mL, 60.5 ± 2.4 in 100 µg/mL, respectively, P < 0.01). Pretreatment with CsA also reduced the mRNA (P < 0.05) and protein expression levels (P < 0.05). CONCLUSIONS: CsA was actively involved in the migration of pterygium fibroblasts. Cell migration is inhibited in response to CsA through the inhibition of MMP-3 and MMP-13 expression. These findings reveal the therapeutic potential of CsA on pterygium progression. Further studies will be necessary to elucidate the precise intracellular signal mechanism responsible for CsA-induced downregulation of MMPs in pterygium fibroblasts.

In vitro effects of preservative-free and preserved prostaglandin analogs on primary cultured human conjunctival fibroblast cells.

PURPOSE: Long-term use of topical medication is needed for glaucoma treatment. One of the most commonly prescribed classes of hypotensive agents are prostaglandin analogs (PGs) used as both first-line monotherapy; as well as in combination therapy with other hypotensive agents. Several side effects of eye drops can be caused by preservatives. The purpose of this study was to evaluate the effects of PGs with varying concentrations of benzalkonium chloride (BAC), alternative preservatives, or no preservatives on human conjunctival fibroblast cells. METHODS: Primary human conjunctival fibroblast cells were used in these experiments. Cells were exposed to the following drugs: BAC at different concentrations, bimatoprost 0.01% (with BAC 0.02%), latanoprost 0.005% (with BAC 0.02%), tafluprost 0.0015% with/without 0.001% BAC and travoprost 0.004% (with 0.001% Polyquad) for 15 and 30 minutes. Cell cytotoxicity was evaluated by phase-contrast microscopy to monitor morphological changes of cells, Counting Kit-8 (CCK-8) assay to cell viability, and fluorescent activated cell sorting (FACS) analysis to measure apoptosis. RESULTS: BAC caused cell shrinkage and detachment from the plate in a dose-dependent manner. Morphological changes were observed in cells treated with bimatoprost 0.01% and latanoprost 0.005%. However, mild cell shrinkage was noted in cells treated with tafluprost 0.0015%, while a non-toxic effect was noted with travoprost 0.004% and preservative-free tafluprost 0.0015%. CCK-8 assay and FACS analysis showed all groups had a significantly decreased cell viability and higher apoptosis rate compared with the control group. However, travoprost 0.004% and preservative-free tafluprost 0.0015% showed lower cytotoxicity and apoptosis rate than other drugs. CONCLUSIONS: This in vitro study revealed that BAC-induced cytotoxicity is dose-dependent, although it is important to emphasize that the clinical significance of toxicity differences observed among the different PGs formulations has not yet been firmly established. Alternatively preserved or preservative-free glaucoma medications seem to be a reasonable and viable alternative to those preserved with BAC.

ADAM10 mediates N-cadherin ectodomain shedding during retinal ganglion cell differentiation in primary cultured retinal cells from the developing chick retina.

Here, we examined the role of ADAM10 during retinal cell differentiation in retinal sections and in vitro cultures of developing chick retinal cells from embryonic day 6 (ED6). Immunohistochemistry showed that ADAM10 is abundantly expressed in the inner zone of neuroblastic layer at ED5, and it becomes more highly expressed in the ganglion cell layer at ED7 and ED9. Western blotting confirmed that ADAM10 was expressed as an inactive pro-form that was processed to a shorter, active form in control cultured cells, but in cultures treated with an ADAM10 inhibitor (GI254023X) and ADAM10-specific siRNA, the level of mature ADAM10 decreased. Phase-contrast microscopy showed that long neurite extensions were present in untreated cultures 24 h after plating, whereas cultures treated with GI254023X showed significant decreases in neurite extension. Immunofluorescence staining revealed that there were far fewer differentiated ganglion cells in ADAM10 siRNA and GI254023X-treated cultures compared to controls, whereas the photoreceptor cells were unaltered. The Pax6 protein was more strongly detected in the differentiated ganglion cells of control cultures compared to ADAM10 siRNA and GI254023X-treated cultures. N-cadherin ectodomain shedding was apparent in control cultures after 24 h, when ganglion cell differentiation was observed, but ADAM10 siRNA and GI254023X treatment inhibited these processes. In contrast, N-cadherin staining was strongly detected in photoreceptor cells regardless of ADAM10 siRNA and GI254023X treatment. Taken together, these data indicate that the inhibition of ADAM10 can inhibit Pax6 expression and N-cadherin ectodomain shedding in retinal cells, possibly affecting neurite outgrowth and ganglion cell differentiation.

Prevalence of eye diseases in South Korea: data from the Korea National Health and Nutrition Examination Survey 2008-2009.

PURPOSE: The aim of this study is to report on preliminary data regarding the prevalence of major eye diseases in Korea. METHODS: We obtained data from the Korea National Health and Nutrition Examination Survey, a nation-wide cross-sectional survey and examinations of the non-institutionalized civilian population in South Korea (n = 14,606), conducted from July 2008 to December 2009. Field survey teams included an ophthalmologist, nurses, and interviewers, traveled with a mobile examination unit and performed interviews and ophthalmologic examinations. RESULTS: The prevalence of visual impairment, myopia, hyperopia and astigmatism in participants over 5 years of age was 0.4 ± 0.1%, 53.7 ± 0.6%, 10.7 ± 0.4%, and 58.0 ± 0.6%, respectively. The prevalence of strabismus and blepharoptosis in participants over 3 years of age was 1.5 ± 0.1% and 11.0 ± 0.8%, respectively. In participants over 40 years of age, the prevalence of cataract, pterygium, early and late age-related macular degeneration, diabetic retinopathy and glaucoma was 40.2 ± 1.3%, 8.9 ± 0.5%, 5.1 ± 0.3%, 0.5 ± 0.1%, 13.4 ± 1.5%, and 2.1 ± 0.2%, respectively. CONCLUSIONS: This is the first nation-wide epidemiologic study conducted in South Korea for assessment of the prevalence of eye diseases by both the Korean Ophthalmologic Society and the Korea Center for Disease Control and Prevention. This study will provide preliminary information for use in further investigation, prevention, and management of eye diseases in Korea.

Conjunctivolimbal autograft using a fibrin adhesive in pterygium surgery.

PURPOSE: To evaluate the efficacy and safety of fibrin bioadhesive in conjunctivolimbal autograft surgery for primary pterygium. METHODS: Thirty-six eyes in 34 patients were reviewed with nasal primary pterygium who were treated with pterygium excision with superior conjunctivolimbal transplantation with fibrin bioadhesive. Surgical durations were recorded and the patients were followed up on the first day after surgery and then at 1, 2, 4, 8 and 12 weeks postoperatively. The graft-recipient site attachments were examined and subjective symptoms of patients were recorded at every follow-up examinations. RESULTS: The mean patient age was 57.9+/-10.1 (ranging from 33 to 83) years. The mean follow-up period was 22.05+/-5.78 weeks. The mean surgery time was 18.04+/-5.65 minutes. The subjective symptoms (pain, foreign body sensation, tearing and discomfort) disappeared in 23 of 36 eyes (64%) in one week after surgery, and all discomforts subsided within two weeks after surgery in all patients. The conjunctivolimbal autograft was correctly positioned and fixed in 34 of 36 eyes (94.4%) throughout the follow-up period. Graft dehiscence was seen in two eyes (5.6%), one eye was treated with remedial sutures, and the other eye showed a spontaneous healing without remedial sutures. Transient graft edema occurred in four eyes (11.2%) but subsided spontaneously within a month. There were no cases of pterygium regrowth or complications due to the fibrin bioadhesive. CONCLUSIONS: The use of fibrin bioadhesive in conjunctivolimbal autograft surgery in primary pterygium simplifies surgical techniques, shorten surgical duration, and produce less postoperative subjective symptoms . Therefore, the fibrin bioadhesive is a safe and effective tool to attach conjunctivolimbal autograft in primary pterygium surgery.