F. Brignole-Baudouin

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

Aim: To compare the conjunctival and corneal reactions of commercially available solution of latanoprost (Xalatan) and preservative-free (PF) tafluprost in rabbits. Methods: 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. Results: 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. Conclusion: The authors confirm that rabbit corneoconjunctival surfaces presented a better tolerance when treated with PF-tafluprost compared with commercially available latanoprost or BAK solution.

Multiple Endpoint Analysis of the 3D-Reconstituted Corneal Epithelium after Treatment with Benzalkonium Chloride: Early Detection of Toxic Damage

PURPOSE To investigate the effects of benzalkonium chloride (BAK) on the human reconstituted corneal epithelial model (HCE) and to optimize the operating potential of this model in the field of ophthalmic toxicology. METHODS The HCEs were treated with 0.001% to 0.5% BAK for 24 hours followed or not by a 24-hour postincubation period. To complete the histologic analysis, the authors designed a new MTT procedure to assess cellular viability. Frozen sections were analyzed by using fluorescence confocal microscopy for the presence of TUNEL, activated caspase-3, Ki67, ICAM-1, HLA-DR, E-cadherin, and occludin. Occludin gene expression was also investigated by using quantitative RT-PCR. RESULTS The MTT test revealed a dose-dependent response of BAK with significant toxic effects for concentrations as low as 0.005%. Increasing BAK concentrations induced an increased number of apoptotic cells, found from the superficial to the deeper layers, with the activation of caspase-3 at 0.01% and 0.02% concentrations. The number of Ki67- and ICAM-1-positive cells increased with 0.01% BAK and with 0.001% to 0.01% BAK, respectively. BAK induced the dose-dependent disappearance of occludin in the superficial layers while increasing its gene expression up to the 0.02% BAK concentration. CONCLUSIONS Fluorescence techniques conjugated with confocal microscopy on 3D-reconstructed corneal epithelia were well suited for the investigation of toxicological markers such as cell junction alteration, apoptosis, cell activation, and proliferation and gave relevant results compared with the known human data. They complement the new sensitive MTT test and improve the operating potential of this new, valuable 3D model in ophthalmic toxicology.

Méthodes d’évaluation de la surface oculaire dans les syndromes secs

Le syndrome d’œil sec est une entite clinico-pathologique complexe impliquant le film lacrymal, les glandes lacrymales, les paupieres, ainsi qu’un grand nombre des cellules de la surface oculaire incluant les cellules epitheliales, les cellules inflammatoires et immunitaires, et les cellules a mucus. Des methodes d’investigation variees ont ete developpees pour evaluer le film lacrymal tant sur le plan structurel que fonctionnel : mesure du menisque des larmes, fluorophotometrie, meibometrie, interferometrie, taux d’evaporation, osmolarite des larmes, ou encore thermographie. Le syndrome d’œil sec se traduit egalement au niveau de la surface oculaire par des irregularites corneennes, qui peuvent etre explorees par les techniques de videokeratographie ou de microscopie confocale in vivo , et des perturbations optiques confirmees par aberrometrie. Les empreintes conjonctivales restent une methode classique pour apprecier les alterations cellulaires de la surface oculaire. Cette technique a largement beneficie des progres en matiere de microscopie confocale, de biologie moleculaire, et de cytometrie en flux. L’etude biologique des proteines des larmes ou d’autres mediateurs est aussi tres utile, mais presente des limites importantes liees au recueil des larmes, et ce, particulierement chez les patients avec un syndrome sec, et au manque de standardisation de la majorite des mesures. L’osmolarite des larmes, l’electrophorese et le dosage des proteines normales des larmes comme le lysozyme ou la lactoferrine, restent les tests les plus utiles. Enfin, des explorations extra-oculaires comme le dosage des anticorps antinucleaires ou une biopsie des glandes salivaires accessoires peuvent etre necessaires pour confirmer le diagnostic de syndrome de Sjogren.

RGTA-based matrix therapy in severe experimental corneal lesions: Safety and efficacy studies

Corneal alteration potentially leading to ulceration remains a major health concern in ocular surface diseases. A treatment that would improve both the quality and speed of healing and control the inflammation would be of great interest. Regenerating agents (RGTAs) have been shown to stimulate wound healing and modulate undesired fibrosis in various in vivo systems. We investigated the effects of RGTA-OTR4120(®) in a rabbit corneal model in order to assess its potential use in ocular surface diseases. First, we assessed its safety for 7 and 28 days using the Draize test criteria in healthy rabbit eyes; then, we investigated the effect of a single dose (50μl, 5μg) in an alkali-burned cornea model. Daily follow-up of clinical signs of healing was scored, and histology was performed at D7. RGTA was well tolerated; no signs of ocular irritation were observed. In the corneal alkali-burn model, non-RGTA-treated eyes showed inflammatory clinical signs, and histology confirmed a loss of superficial corneal layers with epithelial disorganization, neovascularization and infiltration of inflammatory cells. When compared to NaCl control, RGTA treatment appeared effective in reducing clinical signs of inflammation, enhancing re-epithelialization, and improving histological patterns: edema, fibrosis, neovascularization and inflammation. Three to four layers of epithelial cells were already organized, stroma was virtually unvascularized and keratocytes well implanted in parallel collagen fibers with an overall reorganization similar to normal cornea. RGTA appears to be a promising agent for controlling ocular surface inflammation and promoting corneal healing and was well tolerated. This study offers preclinical information and supports the findings of other (compassionate or pilot) studies conducted in patients with various ocular surface diseases.

An in vivo confocal microscopy and impression cytology evaluation of pterygium activity.

Purpose: To correlate pterygium activity with in vivo confocal microscopy (IVCM) and ex vivo impression cytology (IC) analyses. Methods: Twenty-eight pterygia from 16 patients were analyzed in this study. A pterygium activity score was obtained by summing four scores of ocular discomfort, pterygium hyperemia, keratitis, and the presence of Fuchs patches. Patients underwent pterygium IVCM analysis and collection of IC specimens on the pterygium. IC specimens were analyzed quantitatively by immunohistochemistry for goblet cell and dendritiform inflammatory cell density. IVCM images and IC results were compared to search for a possible correlation with the pterygium activity score. Results: The presence of inflammatory cells, numerous blood vessels, and an irregular limit between the cornea and the pterygium with infiltration of hyperreflective cells in the adjacent corneal epithelium were the signs observed with IVCM associated with pterygium activity. Using IC, the density of goblet cells and dendritiform inflammatory cells was significantly correlated to the pterygium activity score (Spearman coefficient 0.765, P < 0.0001 and 0.799, P < 0.0001, respectively). In the same pterygium, dendritic inflammatory cell density and goblet cell density were also significantly correlated (Spearman coefficient 0.952; P < 0.0001). Conclusions: The correlation of pterygium cell changes with pterygium activity supports the role of inflammation and its relationship with goblet cells in the pathogenesis of pterygium.

Les conservateurs des collyres : vers une prise de conscience de leur toxicité

Preservatives are present in numerous multidose eyedrops and provide the sterility of the solution against bacteria and fungi. However, numerous studies have shown their toxicity for the ocular surface, particularly in long-term treatments. The most widely used preservative in eyedrops is benzalkonium chloride. This quaternary ammonium acts as a detergent, antiseptic, disinfectant, fungicide, bactericide, and spermicide. Its use on the ocular surface therefore has significant consequences. Indeed, the preservatives are pro-apoptotic, pro-inflammatory and they cause the dissolution of the lachrymal film. The prolonged administration of one or several eye drops containing preservatives induces changes in the superficial structures (conjunctiva, cornea) as well as in deeper structures (trabecula, lens). The least severe symptoms are irritation and discomfort, including sensation of a foreign body, itching, or burning sensations. However, more severe side effects have been described, such as chronic inflammation of variable intensity or the progressive development of fibrosis with higher risk of failure after glaucoma filtering surgery. Ideally, preservative-free eyedrops should be recommended, or at least a reduction of the number of instilled preserved eyedrops should be considered. All these strategies could increase patient comfort, quality of life, and compliance, with better outcome at the time of filtering surgery.

Revue généraleLes conservateurs des collyres : vers une prise de conscience de leur toxicitéPreservatives in eye drops: Toward awareness of their toxicity

Preservatives are present in numerous multidose eyedrops and provide the sterility of the solution against bacteria and fungi. However, numerous studies have shown their toxicity for the ocular surface, particularly in long-term treatments. The most widely used preservative in eyedrops is benzalkonium chloride. This quaternary ammonium acts as a detergent, antiseptic, disinfectant, fungicide, bactericide, and spermicide. Its use on the ocular surface therefore has significant consequences. Indeed, the preservatives are pro-apoptotic, pro-inflammatory and they cause the dissolution of the lachrymal film. The prolonged administration of one or several eye drops containing preservatives induces changes in the superficial structures (conjunctiva, cornea) as well as in deeper structures (trabecula, lens). The least severe symptoms are irritation and discomfort, including sensation of a foreign body, itching, or burning sensations. However, more severe side effects have been described, such as chronic inflammation of variable intensity or the progressive development of fibrosis with higher risk of failure after glaucoma filtering surgery. Ideally, preservative-free eyedrops should be recommended, or at least a reduction of the number of instilled preserved eyedrops should be considered. All these strategies could increase patient comfort, quality of life, and compliance, with better outcome at the time of filtering surgery.

Confocal biomicroscopy of corneal intraepithelial neoplasia regression following interferon alpha 2b treatment

Recombinant interferon alpha (IFN2B) is a proposed therapy for corneal intraepithelial neoplasia (CIN). Clinical response to this treatment is frequently challenging due to the delay between initiation of the IFN2B topical application and the beginning of CCIN regression. We present the case of a successful IFN2B treatment with prospective in vivo confocal microscopy imaging. A 50-year-old man was referred to our department for a recurrence of a conjunctival–corneal intraepithelial neoplasia (CCIN) in his right eye. A 2-month course of topical 0.02% mitomycin C (MMC) therapy was withdrawn a month earlier due to lid allergy. The lesion extended nasally from conjunctiva to peripheral cornea. Despite a surgical excision with cryotherapy, the intraepithelial neoplasia recurred 1 month later in the peripheral cornea (figs 1, 2). Since the large limbal resection and MMC treatment presented the risk of corneal epithelial stem cell deficiency, IFN2B was chosen as a subsequent therapeutic procedure.1 The follow-up included slit lamp and in vivo confocal microscopy examination on a monthly basis during 6 months. Under this regimen, healing was observed within …

Activation des voies TH1/TH2 détectée par l'expression de CCR4 et CCR5 sur la surface oculaire de patients glaucomateux traités au long cours

Buts L’objet de ce travail a ete d’etudier, sur des empreintes conjonctivales de patients glaucomateux traites au long cours, l’expression en cytofluorimetrie en flux, de deux recepteurs de chimiokines CCR4 et CCR5 lies respectivement aux systemes TH2 et TH1, et de les correler a l’expression de HLA-DR. Patients et methodes Des empreintes conjonctivales ont ete prises chez une serie de 35 patients glaucomateux traites depuis plus d’un an avec des collyres betabloquants (n = 7), a base de prostaglandines (n = 10), en multitherapie (n = 18), et chez 20 sujets normaux. Une fois dissociees, les cellules ont ete incubees avec des anticorps monoclonaux specifiques de CCR4, CCR5 et HLA-DR. Les prelevements ont ete analyses de maniere masquee en utilisant la cytofluorimetrie en flux pour evaluer le pourcentage de cellules positives pour chaque marqueur. Resultats Par rapport aux sujets normaux, l’antigene HLA-DR s’est avere significativement plus eleve chez les patients glaucomateux, avec une tendance a des niveaux plus eleves en cas de multitherapie et plus bas avec un traitement a base de prostaglandines. CCR4 et CCR5 etaient tous deux tres significativement plus exprimes chez les patients glaucomateux sous multitherapie ou sous monotherapie, quelle qu’elle soit, que chez les sujets normaux qui exprimaient ces recepteurs a des niveaux tres faibles, compatibles avec une expression sur les seules cellules inflammatoires locales. Conclusion Cette etude confirme la surexpression des recepteurs pour ces deux chimiokines par l’epithelium conjonctival chez les patients traites au long cours. Les resultats suggerent que l’utilisation chronique des traitements topiques puisse stimuler a la fois les systemes TH1 et TH2, comme en temoigne l’expression concomitante de CCR4 et CCR5. Ils evoquent des mecanismes inflammatoires combinant allergie et toxicite, et confirment la complexite des processus inflammatoires survenant au niveau de la surface oculaire des patients glaucomateux.

321 L’endothélium cornéen dans un modèle d’uvéite induite par endotoxine : corrélation entre microscopie confocale in vivo et immunohistochimie

Introduction L’objectif de l’etude etait d’analyser la participation de l’endothelium corneen dans l’uveite afin de mieux comprendre les mecanismes de formation et la composition des precipites retro-corneens. Les images de microscopie confocale in vivo ont ete correlees a l’immunohistochimie ex vivo de l’endothelium corneen dans un modele d’uveite induite par endotoxine. Materiels et Methodes L’uveite a ete induite chez des rats Lewis par une injection de lipopolysaccharide. Un examen a la lampe a fente et un examen en microscopie confocale in vivo ont ete realises a 6, 24, 48, 72 et 96 heures apres l’injection de lipopolysaccharide ; un groupe de rats a ete sacrifie a 24 heures et un autre a 96 heures. Une immunohistochimie de l’endothelium corneen, avec les anticorps diriges contre ICAM-1, CD68 (anti-macrophage), MA967 (anti-neutrophile), alpha beta-TCR (anti-lymphocyte), Zonula Occludens-1, occludine et le marqueur phalloidine (marqueur du cytosquelette), a ete realisee sur des cornees montees a plat et analysee par un microscope confocal laser a 3 dimensions. Resultats La microscopie confocale in vivo a montre de nombreuses particules arrondies hyper reflectives sur la surface de l’endothelium corneen, dans le stroma anterieur et dans la chambre anterieure, correspondant aux cellules inflammatoires jusqu’a 96 heures, avec un maximum a 24 heures apres l’injection. En immunomarquage, les cellules endotheliales montraient une surexpression d’ICAM-1 dans les rats avec uveite par rapport aux temoins. Zonula Occludens-1 et occludine avaient une expression endotheliale diminuee et une distribution plus heterogene dans les rats avec uveite par rapport aux temoins, montrant une rupture des jonctions cellulaires endotheliales. Comparee aux controles, l’expression de CD 68, MA967 et alpha beta-TCR etait augmentee dans les cornees des rats avec uveite. Les deux techniques ont montre un reseau de vaisseaux corneens peripheriques (derivant d’une structure vasculaire circonferentielle ressemblant au cercle arteriel de l’iris), siege d’une diapedese des cellules inflammatoires infiltrant le stroma anterieur. Discussion La correlation entre microscopie confocale in vivo et immunomarquage ex vivo de l’endothelium corneen dans l’uveite a permis d’ameliorer la connaissance et l’interpretation des images de microscopie confocale in vivo. Conclusion Ces deux techniques ont ete contributives et complementaires dans l’etude du role et des modifications de l’endothelium corneen dans l’uveite. A partir de ces resultats, l’analyse des precipites retro corneens et de l’endothelium en microscopie confocale in vivo pourrait fournir des informations sur l’etiologie ou la severite des uveites en pratique clinique.