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      Conjunctival and corneal reactions in rabbits following short- and repeated exposure to preservative-free tafluprost, commercially available latanoprost and 0.02% benzalkonium chloride

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          To compare the conjunctival and corneal reactions of commercially available solution of latanoprost (Xalatan) and preservative-free (PF) tafluprost in rabbits.


          The rabbits received 50 μl of phosphate-buffered saline (PBS), PF-tafluprost 0.0015%, latanoprost 0.005% or benzalkonium chloride (BAK) 0.02%; all solutions were applied at 5 min intervals for a total of 15 times. The ocular surface toxicity was investigated using slit-lamp biomicroscopy examination, flow cytometry (FCM) and on imprints for CD45 and tumour necrosis factor-receptor 1 (TNFR1) conjunctival impression cytology (CIC) and corneal in vivo confocal microscopy (IVCM). Standard immunohistology also assessed inflammatory/apoptotic cells.


          Clinical observation and IVCM images showed the highest ocular surface toxicity with latanoprost and BAK, while PF-tafluprost and PBS eyes presented almost normal corneoconjunctival aspects. FCM showed a higher expression of CD45+ and TNFR1+ in latanoprost- or BAK-instilled groups, compared with PF-tafluprost and PBS groups. Latanoprost induced fewer positive cells for inflammatory marker expressions in CIC specimens compared with BAK-alone, both of which were higher than with PF-tafluprost or PBS. Immunohistology showed the same tendency of toxic ranking.


          The authors confirm that rabbit corneoconjunctival surfaces presented a better tolerance when treated with PF-tafluprost compared with commercially available latanoprost or BAK solution.

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          Most cited references 27

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          Effects of benzalkonium chloride on growth and survival of Chang conjunctival cells.

          The aim of this study was to investigate the action of benzalkonium chloride (BAC), used as a preservative in most ophthalmic topical solutions, on epithelial conjunctival cells in vitro. A continuous human conjunctival cell line (Wong-Kilbourne derivative of Chang conjunctiva) was exposed to BAC solutions at various concentrations (0.1%-0.0001%) during a period of 10 minutes. Cells were examined before treatment and 3, 24, 48, and 72 hours later, after reexposure to normal cell culture conditions. Cell number and viability were assessed with crystal violet and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide colorimetric assays. The expression of the apoptotic marker Apo 2.7, nuclear antigen p53, membrane proteins Fas and Fas ligand, and DNA content was studied by flow cytometry. Morphologic aspects of cell nuclei were analyzed on slides with a nucleic acid-specific dye, 4',6'-diamidino-2-phenylindole dihydrochloride. Cytoskeleton was labeled with a monoclonal anti-pancytokeratin antibody. In addition, apoptosis was measured by DNA electrophoresis assays in agarose gel. Cell exposure to 0.1% and 0.05% BAC induced cell lysis immediately after treatment. All cells (100%) treated with 0.01% BAC died in a delayed manner within 24 hours, with most of the characteristics of apoptosis (chromatin condensation and DNA fragmentation, reduction in cell volume, expression of the apoptotic marker Apo 2.7, and apoptotic changes in DNA content). Aliquots of 0.005%, 0.001%, 0.0005%, and 0.0001% BAC induced growth arrest and apoptotic cell death in a dose-dependent manner between 24 and 72 hours after treatment. The expressions of Fas and p53 did not vary after BAC treatment. Fas ligand was always negative. These results suggest that BAC induces cell growth arrest and death at a concentration as low as 0.0001%. The mode of BAC-induced cell death is dose-dependent. Cells die by necrosis after BAC treatment at high concentrations and by apoptosis if low concentrations of BAC are applied. This new aspect of in vitro toxicity of BAC could in part explain some ocular surface disorders observed in patients undergoing long-term topical treatments with preservative-containing drugs.
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            Ocular surface inflammatory changes induced by topical antiglaucoma drugs: human and animal studies.

            To investigate conjunctival and trabecular specimens from patients with glaucoma according to the duration and number of drugs received before filtration surgery, and to confirm, in a complementary experimental model, the role of preservative by comparing the effects of preserved and nonpreserved timolol. Experimental animal and human tissue study. Paired specimens of conjunctiva and trabeculum were taken from 61 patients undergoing trabeculectomy. Twenty-six patients were treated with 2 or more drugs for at least 1 year; 30 had received a beta-blocker for more than 1 year and 5 underwent primary surgery. A second study was performed in 25 rats receiving topical solutions in both eyes for 1 month. Immunohistochemistry was performed in all biopsy specimens using 12 different monoclonal antibodies. Ocular structures from rats treated for 1 month with preserved 0.5% timolol, nonpreserved 0.5% timolol, or 0.01% benzalkonium chloride were similarly investigated in an experimental study. Inflammatory cell infiltrates and fibroblasts were evaluated in biopsies, as well as in animal specimens, together with histologic changes induced by the drugs applied. Twenty-four of 26 conjunctivae and 21 of 24 trabecular pieces from multitreated patients were found to be abnormally infiltrated by cells expressing inflammatory or fibroblastic markers or both. Nineteen of 30 conjunctivae and 9 of 22 trabeculums in the monotherapy group and only 1 of 5 specimens from the primary surgery group were abnormal. In rats, preserved timolol and benzalkonium similarly showed infiltrates together with toxic histopathologic changes as compared to the nonpreserved timolol and control groups. These two combined studies confirmed histopathologic effects of antiglaucomatous drugs on the conjunctiva and showed similar effects in the trabecular meshwork. The experimental study showed that benzalkonium chloride is at least, to a large part, responsible for these toxic or immunoinflammatory effects or both on the ocular structures.
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              The ocular surface of glaucoma patients treated over the long term expresses inflammatory markers related to both T-helper 1 and T-helper 2 pathways.

              To investigate the expression of CCR5 and CCR4, two chemokine receptors, as markers of the T helper (Th) 1 and Th2 pathways, respectively, and class II antigen HLA-DR as a hallmark of inflammation on conjunctival cells obtained from patients receiving long-term glaucoma treatment. Case-control study. A total of 18 normal subjects and 70 glaucoma patients treated with topical antiglaucoma drugs for more than 1 year: 14 receiving a beta-blocker as monotherapy, 38 treated with a prostaglandin analog alone (19 with latanoprost, 6 with travoprost, 13 with bimatoprost), and 18 receiving multiple treatments. Impression cytologic specimens (ICSs) were obtained from 1 eye of the patients and processed for flow cytometry. Conjunctival cells were extracted and incubated with monoclonal antibodies against CCR4, CCR5, HLA-DR, or their specific controls to measure, in a masked manner, the percentages of conjunctival cells positive for the 3 markers. HLA-DR and chemokine receptors (CCR4 and CCR5) in ICSs. Compared with all other groups, HLA-DR expression was raised significantly in the multitreatment group, whereas all monotherapies showed slight and nonsignificant increases. Both CCR4 and CCR5 were increased significantly in all 5 glaucoma groups compared with normal subjects, with no between-group differences. This study demonstrates the overexpression of 2 chemokine receptors in the conjunctival epithelium of glaucoma patients treated over the long term. These results show the simultaneous overexpression of CCR4 and CCR5, suggesting that the chronic use of topical treatments may stimulate both the Th1 and Th2 systems simultaneously. These results also suggest that inflammatory mechanisms combining allergy with toxicity are at work and illustrate the complexity of inflammatory reactions occurring in the ocular surface of glaucoma patients.

                Author and article information

                Br J Ophthalmol
                The British Journal of Ophthalmology
                BMJ Publishing Group (BMA House, Tavistock Square, London, WC1H 9JR )
                September 2008
                22 August 2008
                22 August 2008
                : 92
                : 9
                : 1275-1282
                [1 ]Department of Toxicology, Faculty of Biological and Pharmacological Sciences, University Paris Descartes, Paris, France
                [2 ]INSERM UMR S 592, Institute of Vision, University of Paris 6, Paris, France
                [3 ]Department of Ophthalmology III, Quinze-Vingts National Ophthalmology Hospital, Paris, France
                [4 ]Ambroise Paré Hospital, APHP, University of Versailles, Paris, France
                Author notes
                Correspondence to: Dr F Brignole-Baudouin, INSERM UMR S 592, Institute of Vision, University of Paris 6, Paris, France, 17, rue Moreau, 75012, Paris, France; frbaudouin@ 123456aol.com
                © Liang et al 2008

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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                Laboratory Science

                Ophthalmology & Optometry


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