Effects of topical nonsteroidal antiinflammatory drugs on the expression of matrix metalloproteinases in the cornea

PURPOSE: To assess the effects of nonsteroidal antiinflammatory drug (NSAID) eyedrops on the expression of matrix metalloproteinases in corneal tissue. SETTING: Ocular Microbiology and Immunology Laboratory, Refractive Surgery Research Laboratory, The Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, USA. METHODS: Seventy rats were divided into 2 groups: intact and debrided epithelium. Uniform central corneal epithelial defects were created in the right eye of the debrided corneal group. Each group was divided into 4 subgroups, each receiving 1 of the following eyedrops or artificial tears: The 3 NSAIDs were diclofenac sodium 0.1% (Falcon or Voltaren) and preservative-free ketorolac 0.5% (Acular PF). The artificial tears were carboxymethylcellulose sodium 0.5% (Refresh Plus PF). The eyedrops were administered 4 times a day for 1 week. The rats were killed on days 2 and 7. The corneas were excised and processed for immunohistochemical staining, Western blot assay, and zymography studies to determine the localization of the production of the following matrix metalloproteinases (MMPs): MMP-1, MMP-2, MMP-8, and MMP-9. RESULTS: Matrix metalloproteinase-1, MMP-8, and MMP-2 were detected in rat corneas at 48 hours in the debrided and intact epithelium groups treated with NSAID eyedrops. The MMP-1 and MMP-8 expression levels were higher in intact corneas in the diclofenac sodium groups than in the ketorolac and artificial tears groups. The expression was localized mostly in the epithelial cells and occasionally in keratocytes. CONCLUSION: This study provides preliminary evidence that topical application of some NSAIDs can induce the early expression of MMP-1, MMP-2, and MMP-8 in the cornea, suggesting that MMPs play a role in the corneal cytotoxicity of certain NSAIDs.     

Effect of topical fluoroquinolones on the expression of matrix metalloproteinases in the cornea

Background  

Matrix metalloproteinases play an important role in extracellular matrix deposition and degradation. Based on previous clinical observations of corneal perforations during topical fluoroquinolone treatment, we decided to evaluate the comparative effects of various fluoroquinolone eye drops on the expression of matrix metalloproteinases (MMPs) in cornea.     

Corneal complications associated with topical ophthalmic use of nonsteroidal antiinflammatory drugs

PURPOSE: To explore the potential association between adverse corneal events and the use of topical nonsteroidal antiinflammatory drugs (NSAIDs). SETTING: Practice-based reports. METHODS: A detailed case-reporting form and request for medical records were sent to all practices reporting cases of corneal or conjunctival pathology in association with the use of topical NSAIDs to the American Society of Cataract and Refractive Surgery. Cases were classified as "mild," "moderate," or "severe" according to predetermined clinical criteria. RESULTS: Records of 140 eyes (129 patients) were reviewed; 51 cases (36.4%) were mild, 55 (39.3%) moderate, and 34 (24.3%) severe. An association with a specific topical NSAID was confirmed in 117 cases (81.8%). Most confirmed cases (53.8%) involved generic diclofenac (Falcon). Cases associated with brand diclofenac (Voltaren, CIBA Vision) and ketorolac (Acular, Allergan) were more likely to have ocular comorbidity and to have received significantly higher total doses of NSAIDs. Neither "off-label" use nor use of any specific agent was associated with severe compared to mild or moderate disease. However, patients with more severe adverse events were more likely to have a history of diabetes, previous surgery in the affected eye, and surgery other than cataract. Cases not occurring in the perioperative period had significantly worse outcomes, had significantly more ocular comorbidities, and received nearly 3 times the dose of NSAIDs. CONCLUSIONS: While topical NSAIDs as a class may be associated with severe adverse events, such events appeared to require potentiation in the form of high total doses, ocular comorbidities, or both with Acular and Voltaren. Severe adverse events might have been more likely to occur at lower doses and in routine postoperative settings with generic diclofenac.     

Changes in mechanical, chemical, and thermal sensitivity of the cornea after topical application of nonsteroidal anti-inflammatory drugs

PURPOSE: In addition to their well-known anti-inflammatory actions, some of the nonsteroidal anti-inflammatory drugs (NSAIDs) appear to have an analgesic effect. In human subjects, the changes in threshold and intensity of sensations evoked by mechanical, chemical, and thermal stimulation of the cornea induced by topical administration of two commercial NSAIDs, diclofenac sodium (Voltaren; Novartis, Basel, Switzerland) and flurbiprofen (Ocuflur; Allergan, Irvine, CA), were studied. METHODS: Corneal sensitivity was measured in 10 young, healthy subjects with a gas esthesiometer. Chemical (10%-70% CO2 in air), mechanical (0-264 mL/min), and thermal (corneal temperature changes between -4.5 degrees C and +3 degrees C around the normal value) stimuli were applied to the center of the cornea. The intensity and perceived magnitude of the psychophysical attributes of the evoked sensation were scored at the end of the pulse in a 10-cm, continuous visual analog scale (VAS). The threshold was expressed as the stimulus intensity that evoked a VAS score >0.5. Sensitivity was measured in both eyes of each subject on two separate days, one without treatment and the other 30 minutes after topical application of 0.03% flurbiprofen (seven subjects) or 0.1% diclofenac sodium (six subjects). RESULTS: Diclofenac attenuated significantly all the sensation parameters evoked by high-intensity mechanical, chemical, and thermal stimuli. Flurbiprofen produced a slight reduction of the sensations evoked by mechanical and chemical stimulation that became significant only for the irritation caused by chemical stimuli of maximum intensity (70% CO2). None of the drugs modified significantly the detection threshold of the different stimuli. CONCLUSIONS: Flurbiprofen had a very limited effect on sensations evoked by corneal stimulation, whereas diclofenac reduced the intensity of sensations evoked by stimuli of different modality, suggesting a mild local anesthetic effect of this drug on all types of corneal sensory fibers. Such anesthetic action could explain the analgesic effect that has been reported after topical application of diclofenac in inflamed human eyes.     

Infrared irradiation alters the expression of matrix metalloproteinases and glycosaminoglycans in the cornea and crystalline lens

Background

Prolonged exposure to infrared (IR) radiation is associated with different types of damage to cornea and lens. The aim of our study was to investigate the effect of acute and chronic exposure to IR radiation on the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 and on the expression of glycosaminoglycans (GAG) in the rabbit cornea and crystalline lens.

Methods

New Zealand rabbits were subjected to IR radiation for 4 months (chronic exposure to IR) or to normal light (control group). In experiments regarding acute exposure, animals were subjected to IR radiation or normal light for 12 h, in the presence of 0.1 % diclofenac sodium (eye drops instilled in the right eye of animals) or saline (instilled in the left eye of animals). The cornea and lens were dissected away and homogenized. The activity of MMP-2 and MMP-9 was assayed by gelatine zymography. Total GAG were isolated from tissue specimens after lipid extraction and extensive digestion with pronase and DNase and characterized by treatment with GAG-degrading enzymes, followed by electrophoresis on cellulose acetate membranes.

Results

Acute or chronic exposure to IR radiation induced the activity of MMP-2 in cornea and lens, whereas only acute IR radiation increased the content of heparan sulphate in crystalline lens. Local administration of diclofenac sodium did not prevent the above effects of acute IR radiation.

Conclusions

The detrimental effects of excessive or prolonged exposure of the eyes to IR radiation are associated with induced activity of MMP-2 in cornea and lens and alterations in the content of heparan sulphate in lens. Thus, MMP and GAG may offer alternative targets for pharmacological intervention to confront ocular damages associated with IR radiation.     

非甾体抗炎药与皮质类固醇抑制角膜上皮细胞增殖的实验研究

目的 探讨非甾体抗炎药对角膜上皮细胞体外增殖的抑制作用。方法 选用原代及传代兔角膜上皮细胞，实验组中加入含不同浓度双氯芬酸钠、安贺拉(酮咯酸氨丁三醇)、地塞米松及洁霉素的培养液，对照组加入等量空白培养液，采用四甲基偶氮唑蓝(MTT)比色法检测其对细胞增殖的抑制，计算出抑制率并进行比较。结果 双氯芬酸钠、安贺拉(酮咯酸氨丁三醇)、地塞米松及洁霉素对角膜上皮细胞增殖均有抑制作用。双氯芬酸钠与安贺拉3个对应浓度的比较中，24h时双氯芬酸钠抑制率分别为61．4％、42．1％及0，安贺拉组均为0；48h时双氯芬酸钠抑制率分别为81．0％、70．2％、47．6％，安贺拉抑制率分别为38．1％、31．0％和0；72h时双氯芬酸钠抑制率分别为95．0％、95．0％、54．0％，安贺拉抑制率分别为59．1％、47．2％和33．2％．在双氯芬酸钠与地塞米松及洁霉素的原液浓度比较中，24、48、72h时双氯芬酸钠抑制率分别为93．0％、94．0％、96．0％，洁霉素为24．6％、51．2％、79．1％，地塞米松为45．6％、69．0％、80．0％．在双氯芬酸钠与地塞米松及洁霉素的二分之一原液浓度比较中，24、48、72h时双氯芬酸钠抑制率分别为68．4％、91．7％、95．0％，洁霉素为12．3％、26．2％、49．0％，地塞米松为12．3％、54．6％、55．0％．结论 非甾体抗炎药双氯芬酸钠与安贺拉对角膜上皮细胞增殖均有抑制作用，以双氯芬酸钠抑制作用最强，强于传统抗增殖药地塞米松，在抑制LASIK术后上皮植入方面有广泛的应用前景。     

Effect of prostaglandin analogues on tear proteomics and expression of cytokines and matrix metalloproteinases in the conjunctiva and cornea

The purpose of this work was to identify potential tear-film-based proteins and their effect on changes in the conjunctiva and cornea in eyes using prostaglandin (PG) analogues. Recruited subjects were individuals who had used PG for at least 1 year and comparison with eyes of normal controls and timolol using patients were done. Approximately 3-5?μL of tears were sampled from both eyes of each subject using glass microcapillaries. Proteomic analysis was done to compare the pooled tear samples from each group by Bradford assay and cytokine arrays. Impression cytology was used to gather mRNA from conjunctival epithelial cells, and target protein mRNA was quantified by PCR. Rabbits treated with PG were scarified, and changes in the corneal stroma were evaluated by immunohistochemical staining and western blot analysis. There were increased levels of IL-1β, IL-6, MMP-1, MMP-3, and MMP-9 and decreased levels of TIMP-1 and TIMP-2 in the tears of PG-treated patients. The mRNA of IL-1β, MMP-1, MMP-3, and MMP-9 was elevated and mRNA of TIMP-1 and TIMP-2 was decreased in the conjunctival epithelial cells. Rabbits treated with PG showed corneal thinning with decreased collagen type I expression. The protein of MMP-1 and MMP-9 was elevated and protein of TIMP-1 was decreased in the rabbit cornea by western blot analysis. Immunohistochemical staining showed elevated expression of MMP-1 and MMP-9 and the decreased expression of TIMP-1 in the corneal stroma. The topical use of PG analogues results in an altered balance between MMPs and TIMPs, which may be triggered by inflammatory cytokines. This results in an increase of matrix degradation and decrease of stromal collagens in the cornea by PG treatments.     

Effects of topical corticosteroids and nonsteroidal anti-inflammatory drugs on prostaglandin e2-induced aqueous flare elevation in pigmented rabbits

We evaluated the effects of anti-inflammatory potency of corticosteroids and nonsteroidal anti-inflammatory drugs on prostaglandin E2 (PGE2)-induced aqueous flare elevation in pigmented rabbits. Transcorneal diffusion of PGE2, 25 microg/ml (7.09 x 10(-2) mmol/l), with the use of a glass cylinder was achieved to produce aqueous flare elevation. Anti-inflammatory drugs were topically administered once before PGE2 application. Aqueous flare was measured with a laser flare-cell meter. Topical single instillation of dexamethasone sodium metasulfobenzoate 0.1%, dexamethasone sodium phosphate 0.1%, and fluorometholone 0.1% 6 h before PGE2 application inhibited 56, 59, and 43% of flare elevation, respectively. Topical single instillation of bromfenac sodium 0.1% and pranoprofen 0.1% 1 h before PGE2 application inhibited 33 and 15% of flare elevation, respectively. Indomethacin 0.5% did not inhibit flare elevation. Corticosteroid eyedrops needed several hours from topical instillation to exhibit inhibition of flare elevation. Most nonsteroidal anti-inflammatory drug eyedrops inhibited aqueous flare elevation when instilled 1 h before PGE2 application.     

Transplantation of amniotic membrane in murine herpes stromal keratitis modulates matrix metalloproteinases in the cornea

PURPOSE: To study matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in the corneas from mice with ulcerative herpes stromal keratitis (HSK) treated with amniotic membrane transplantation (AMT). METHODS: The corneas from BALB/c mice were infected with HSV-1. Mice with ulcerative HSK on postinfection (PI) day 14 were used for the experiments. In one group of mice, the corneas were treated with transplantation of amniotic membrane (AMT) that was secured with a tarsorrhaphy, and a control group underwent tarsorrhaphy alone. After 2 days, the appearance of corneal ulcers and stromal inflammation was judged clinically. Corneal sections were studied by immunohistochemistry for the expression of MMP-2, -8, and -9 and TIMP-1 and -2. MMP activity in the corneas was investigated by zymography, and the expression of the enzymes was measured by the Western blot technique. RESULTS: At day 14 PI, the ulcers stained intensely positive for MMP-2, -8, and -9 and TIMP-1 and -2. Ulceration (P < 0.001), stromal inflammation (P < 0.01) and inflammatory cell infiltration (P < 0.001) markedly improved by day 2 after AMT. This was associated with reduced expression (P < 0.01) and activity of MMP-8, and -9 and increased localization of TIMP-1 (P < 0.01), whereas TIMP-2 was not affected. In contrast, high levels of expression of MMP-8 and -9 remained in the cornea after tarsorrhaphy, and the TIMP-1 expression was only slightly upregulated. CONCLUSIONS: Rapid improvement of HSV-1-induced ulcerative keratitis is noted after amniotic membrane transplantation. This may be caused by reduced expression and activity of MMP-8 and -9, increased expression of TIMP-1, and sustained expression of TIMP-2.     

Effects of mechanical stretching on trabecular matrix metalloproteinases

PURPOSE: The homeostatic mechanisms responsible for intraocular pressure (IOP) regulation are not understood. Studies were conducted to evaluate the hypothesis that trabecular meshwork (TM) cells sense increases in IOP as stretching or distortion of their extracellular matrix (ECM) and respond by increasing ECM turnover enzymes. METHODS: Flow rates were increased in perfused human anterior segment organ cultures and the matrix metalloproteinase (MMP) levels and IOP were evaluated. Human TMs in stationary anterior segment organ culture were mechanically stretched, and MMP levels were analyzed. TM cells were grown on membranes, which were then stretched, and MMP levels were evaluated. Western immunoblots, zymography, and confocal immunohistochemistry were used to evaluate changes in MMPs and their tissue inhibitors, the TIMPS: RESULTS: Doubling the flow rate in perfused human organ cultures increased gelatinase A levels in the perfusate by 30% to 50% without affecting gelatinase B or stromelysin levels. Immediately after doubling the flow rate, the measured IOP doubled. However, over the next few days the IOP gradually returned to the initial level, although the flow rate was maintained at double the initial value. Stretching stationary organ cultures or stretching TM cells grown on membranes resulted in similar increases in gelatinase A without changes in gelatinase B or stromelysin levels. The gelatinase A increases occurred between 24 and 72 hours and were approximately proportional to the degree of stretching. Although coating the membranes with different ECM molecule affected the gelatinase A response, the optimum response occurred when the cells had been grown long enough to produce their own ECM. By Western immunoblot and confocal immunohistochemistry, the stretch-induced increases in gelatinase A were accompanied by strong decreases in TIMP-2 levels and moderate increases in one membrane type MMP, MT1-MMP. After mechanical stretching of the membrane, gelatinase A, MT1-MMP and TIMP-2 all exhibited a similar punctate immunostaining pattern over the TM cell surface. CONCLUSIONS: These results are compatible with the hypothesis that elevations in IOP are sensed by TM cells as ECM stretch/distortion. TM cells respond by increasing gelatinase A and MT1-MMP, while decreasing TIMP-2 levels. This will increase ECM turnover rates, reduce the trabecular resistance to aqueous humor outflow, and restore normal IOP levels. This hypothesis provides a regulatory feedback mechanism for IOP homeostasis.     

Roles of adherence and matrix metalloproteinases in growth patterns of fungal pathogens in cornea

PURPOSE: To investigate the roles of adherence and matrix metalloproteinases (MMPs) in growth patterns of major fungal pathogens in cornea. METHODS: Ninety-six eyes in 96 rabbits were equally divided into four groups receiving inoculation of fungal conidia of Aspergillus fumigatus, Candida albicans, Fusarium solani, and Penicillium citreo-viride, respectively, to induce fungal keratitis. Corneas in each group were obtained at 2, 8, 16 hr, and 1, 2, 3, 5, and 8 days after inoculation and were subjected to scanning electron microscopy, histopathological examination, and gelatin zymography. Eight saline-inoculated eyes in another eight rabbits served as controls. RESULTS: All eyes in the fungus-inoculated groups developed fungal keratitis. The binding of conidia to corneal epithelial basement membrane was initiated earlier in the A. fumigatus and C. albicans groups than in the F. solani and P. citreo-viride groups. Destruction of basement membrane began at 1 to 3 days. Histopathologically, infiltration of inflammatory cells was more evident in the A. fumigatus and C. albicans groups than the F. solani and P. citreo-viride groups at 3 days. The hyphae of A. fumigatus and C. albicans traversed the cornea in a plane perpendicular to the stromal lamellae, whereas the hyphae of F. solani and P. citreo-viride lay parallel to the corneal lamellae. MMP-9 and MMP-2 were found in all infected corneas. At 3 days, proteolysis was most active; the level of MMP-9 was higher in the A. fumigatus and C. albicans groups than in the F. solani and P. citreo-viride groups. There were positive correlations among the number of binding conidia, degree of inflammation, and level of MMP-9 (p < 0.05). CONCLUSIONS: The adherence ability, chemotaxis to neutrophils, and MMP-9 expression level differ in eyes with different fungal pathogens, which may contribute to the different growth patterns of fungi in cornea.     

Effects of antiinflammatory drugs on migration of the rabbit corneal epithelium

PURPOSE: To evaluate and compare the effects of diclofenac sodium 0.1% and betamethasone phosphate 0.1% on corneal wound healing. SETTING: Department of Ophthalmology, Wakayama Medical College, Wakayama, Japan. METHODS: Using the method described by Nishida et al., corneal epithelial spreading was measured in vitro in a rabbit corneal block in the presence or absence of 2 antiinflammatory agents at various doses. RESULTS: At clinical doses (1 microg/mL and 10 microg/mL), the drugs did not suppress migration of the corneal epithelium. At high doses (20 microg/mL, 50 microg/mL, and 100 microg/mL), they did inhibit the migration. There was no between-group difference in corneal epithelial migration at clinical doses. At high doses, corneal epithelial migration was inhibited in the diclofenac sodium group compared with the betamethasone group. CONCLUSIONS: At clinical doses, diclofenac sodium and betamethasone did not inhibit corneal epithelial migration. However, these drugs should be prescribed cautiously in patient with high-risk diseases such as diabetes mellitus and glaucoma.     

Keratitis, ulceration, and perforation associated with topical nonsteroidal anti-inflammatory drugs

PURPOSE: To report corneal complications associated with topical nonsteroidal anti-inflammatory drugs (NSAIDs). DESIGN: Retrospective, noncomparative interventional case series. PARTICIPANTS: Eighteen eyes of 16 patients with adverse corneal events associated with NSAID use. METHODS: Evaluation of 16 patients referred for management of corneal complications during use of topical NSAIDs (ketorolac tromethamine [Acular], diclofenac sodium [Voltaren], diclofenac sodium [Falcon DSOS]). MAIN OUTCOME MEASURES: Type and severity of corneal complications. RESULTS: Of the 16 patients, two experienced severe keratopathy, three experienced ulceration, six experienced corneal or scleral melts, and five experienced perforations. Eleven patients had recent cataract surgery; nine of these were on concurrent topical steroids and antibiotics. Another patient who did not have recent surgery was using concurrent topical steroids without antibiotics for sarcoid uveitis. Systemic associations included two patients with rheumatoid arthritis, one patient with asymptomatic Sjogren's syndrome, and two with rosacea. CONCLUSIONS: Topical NSAIDs were associated with corneal complications in 18 eyes of 16 patients. Potential risk factors include conditions that predispose the patient to corneal melting, concurrent topical steroids, and epithelial keratopathy in the early postoperative period.     

Time-dependent matrix metalloproteinases and tissue inhibitor of metalloproteinases expression change in fusarium solani keratitis

AIM: To investigate matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) expression during the progress of fusarium solani (F.solani) keratitis in a rat model. METHODS: A rat model of F.solani keratitis was produced using corneal scarification and a hand-made contact lens. MMPs and TIMPs expressiond were explored in this rat model of F.solani keratitis using real-time polymerase chain reaction (PCR) and DIF. GM6001 (400 μmol/mL) was used to treat infected corneas. The keratitis duration, amount and area of corneal neovascularization (CNV) were evaluated. RESULTS: MMP-3 expression was 66.3 times higher in infected corneas compared to normal corneas. MMP-8, -9, and -13 expressions were significantly upregulated in the mid-period of the infection, with infected-to-normal ratios of 4.03, 39.86, and 5.94, respectively. MMP-2 and -7 expressions increased in the late period, with the infected-to-normal ratios of 5.94 and 16.22, respectively. TIMP-1 expression was upregulated in the early period, and it was 43.17 times higher in infected compared to normal corneas, but TIMP-2, -3, and -4 expressions were mildly downregulated or unchanged. The results of DIF were consistent with the result of real-time PCR. GM6001, a MMPs inhibitor, decreased the duration of F.solani infection and the amount and area of CNV. CONCLUSION: MMPs and TIMPs contributed into the progress of F.solani keratitis.