[Glaucomatous trabeculum: An inflammatory trabeculopathy? [French translation]]. / Trabéculum glaucomateux : une trabéculopathie inflammatoire ?

Glaucoma is an optic neuropathy in which the primary risk factor is increased intraocular pressure (IOP), attributed to increased resistance to trabecular outflow of aqueous humor (AH). This resistance is believed to result from trabecular degeneration secondary to chronic oxidative stress and cellular senescence but may also involve inflammatory mechanisms whose roles are little known. In fact, inflammatory processes play a major role in the pathophysiology of glaucoma to varying degrees, affecting all structures of the eye, including the ocular surface, the anterior and posterior segments, and even the visual pathways of the brain. These processes are thought to result from dysfunction of a regulatory, protective para-inflammation, becoming chronic and harmful in glaucoma. While the mechanisms of the retinal inflammation which accelerates the degeneration of retinal ganglion cells (RGC) as well as the inflammation of the ocular surface aggravated by long-term use of preserved glaucoma eye drops have been described for several years, very little is known about the pathophysiology of trabecular inflammation in glaucoma. The objective of this literature review is to provide a synthesis of knowledge on the roles and mechanisms of inflammation in both the healthy and glaucomatous trabecular meshwork, as well as its role in the pathophysiology of glaucoma. Therefore, after a review of the mechanisms of cellular senescence and oxidative stress - sources of trabecular inflammation, we will approach the study of the expression and roles of the main inflammatory mediators within the trabecular meshwork. Finally, we will discuss current knowledge on the toxicity of glaucoma eye drops and their preservatives on the ocular surface and trabecular meshwork as well as their role in trabecular inflammation.

The trabecular meshwork in glaucoma: An inflammatory trabeculopathy?

Glaucoma is an optic neuropathy in which the primary risk factor is increased intraocular pressure (IOP), attributed to increased resistance to trabecular outflow of aqueous humor (AH). This resistance is believed to result from trabecular degeneration secondary to chronic oxidative stress and cellular senescence but may also involve inflammatory mechanisms whose roles are little known. In fact, inflammatory processes play a major role in the pathophysiology of glaucoma to varying degrees, affecting all structures of the eye, including the ocular surface, the anterior and posterior segments, and even the visual pathways of the brain. These processes are thought to result from dysfunction of a regulatory, protective para-inflammation, becoming chronic and harmful in glaucoma. While the mechanisms of the retinal inflammation which accelerates the degeneration of retinal ganglion cells (RGCs) as well as the inflammation of the ocular surface aggravated by long-term use of preserved glaucoma eye drops have been described for several years, very little is known about the pathophysiology of trabecular inflammation in glaucoma. The objective of this literature review is to provide a synthesis of knowledge on the roles and mechanisms of inflammation in both the healthy and glaucomatous trabecular meshwork, as well as its role in the pathophysiology of glaucoma. Therefore, after a review of the mechanisms of cellular senescence and oxidative stress - sources of trabecular inflammation, we will approach the study of the expression and roles of the main inflammatory mediators within the trabecular meshwork. Finally, we will discuss current knowledge on the toxicity of glaucoma eye drops and their preservatives on the ocular surface and trabecular meshwork as well as their role in trabecular inflammation.

[The trabecular meshwork: Structure, function and clinical implications. A review of the littérature (French translation of the article)]. / Le trabéculum : structure, fonction et implications cliniques. Une revue de la littérature.

Glaucoma is a blinding optic neuropathy, the main risk factor for which is increased intraocular pressure (IOP). The trabecular meshwork, located within the iridocorneal angle, is the main pathway for drainage of aqueous humor (AH) out of the eye, and its dysfunction is responsible for the IOP elevation. The trabecular meshwork is a complex, fenestrated, three-dimensional structure composed of trabecular meshwork cells (TMC) interdigitated into a multilayered organization within the extracellular matrix (ECM). The purpose of this literature review is to provide an overview of current understanding of the trabecular meshwork and its pathophysiology in glaucoma. Thus, we will present the main anatomical and cellular bases for the regulation of aqueous humor outflow resistance, the pathophysiological mechanisms involved in trabecular dysfunction in the various types of glaucoma, as well as current and future therapeutic strategies targeting the trabecular meshwork.

The trabecular meshwork: Structure, function and clinical implications. A review of the literature.

Glaucoma is a blinding optic neuropathy, the main risk factor for which is increased intraocular pressure (IOP). The trabecular meshwork, located within the iridocorneal angle, is the main pathway for drainage of aqueous humor (AH) out of the eye, and its dysfunction is responsible for the IOP elevation. The trabecular meshwork is a complex, fenestrated, three-dimensional structure composed of trabecular meshwork cells (TMC) interdigitated into a multilayered organization within the extracellular matrix (ECM). The purpose of this literature review is to provide an overview of current understanding of the trabecular meshwork and its pathophysiology in glaucoma. Thus, we will present the main anatomical and cellular bases for the regulation of aqueous humor outflow resistance, the pathophysiological mechanisms involved in trabecular dysfunction in the various types of glaucoma, as well as current and future therapeutic strategies targeting the trabecular meshwork.

IgE Ratio in Tears: A Predictive Tool of Ocular Allergic Inflammation.

PURPOSE: To demonstrate the tear IgE (measured/exuded) ratio (R) as a useful biological marker of ocular allergy in order to distinguish severe from less severe inflammatory status. METHODS: Tear samples and sera from 78 ocular allergy patients and 19 control subjects were analyzed. Total IgE and albumin were measured for calculating the tear IgE-R defining two subgroups (SG) of samples: R ≥ 4-SG and R < 4-SG. Eosinophil cationic protein, Th1 and Th2 cytokines (IFN-γ, IL-4, -5, -6, -8 and -10) and protein electrophoretic profiles were also investigated in tears. RESULTS: The R < 4-SG compared to the R ≥ 4-SG shows higher levels of tear albumin, eosinophil cationic protein, and Th1 and Th2 cytokines. Moreover, each subgroup presents a specific protein profile. CONCLUSION: This study showed that an IgE-R lower than four must be carefully interpreted as a warning sign of a severe inflammatory context and should be also associated with an exploration of immunological profile.

Correlation of clinical symptoms and signs with conjunctival gene expression in primary Sjögren syndrome dry eye patients.

PURPOSE: The aim of this study was to characterize the expression of inflammation-related genes on the ocular surface of Sjögren syndrome (SS) patients and to evaluate their correlations with clinical symptoms and signs. METHODS: The study enrolled 30 patients with SS dry eye and 15 healthy controls. Symptoms were evaluated using OSDI questionnaire. The clinical signs were investigated using corneal fluorescein staining (CFS), tear breakup time (TBUT), Schirmer test and tear osmolarity measurement. Conjunctival superficial cells were collected using conjunctival impression cytology and total RNAs were extracted for analysis using the NanoString® nCounter technology. The Mann-Whitney nonparametric statistical test and Spearman correlations were used to explore the correlations between the up/downregulated genes and the clinical signs and symptoms. RESULTS: Twenty-seven genes were upregulated and 13 were downregulated with statistically significant fold changes ranging from 1.5 to 16.7 and 0.3 to 0.8, respectively. OSDI and CFS were the most significantly correlated parameters with 21 and 19 inflammatory genes, respectively. Among all the upregulated genes, 14 were positively correlated with both OSDI and CFS. Two downregulated genes (GNGT1, HSPB2) were negatively correlated with OSDI and CFS. IL1RN was the only gene positively correlated with the Schirmer test. CONCLUSIONS: These results highlight the differentially expressed genes in primary Sjögren syndrome and their relationships between the inflammatory genes expressed and the patient symptom score and corneal damage. The inflammatory genes implicated in SS-associated dry eye could be important tools to determine the pathophysiological profiles of SS and potentially useable as specific signatures.

[Hyperosmolarity: Intracellular effects and implication in dry eye disease]. / Hyperosmolarité : effets intracellulaires et implication dans la sécheresse oculaire.

Dry eye disease is a multifactorial disease affecting the lacrimal functional unit and which has a significant impact on the quality of life of patients. This pathology works as a vicious circle at the ocular surface in which hyperosmolarity of the tear film plays a key role. This review intends to describe the different reported intracellular effects induced by hyperosmolarity in cells: alteration of cytoskeleton, cell cycle slowdown, adaptation mechanisms triggered as restoration of cell volume and accumulation of compatible osmolytes, the crucial role of the osmoprotectant factor Nuclear Factor of the Activated T cells-5 (NFAT5), apoptosis, as well as oxidative stress and inflammatory responses caused by this particular condition. Reported effects of hyperosmolarity in the experimental studies specific of dry eye disease concerning ocular surface cells will be described in parallel. Indeed, these data allow to understand a part of the pathophysiology of the disease, and specially the links between tear hyperosmolarity and inflammation of the ocular surface, the second key of the pathology phenomenon.

[Mesenchymal stem cell therapy, a new hope for eye disease]. / Thérapie cellulaire par cellules souches mésenchymateuses, un nouvel espoir pour les pathologies oculaires.

Mesenchymal stem cells (MSC) are adult stem cells, first identified in skeletal tissues and then found in the entire body. MSC are able to not only differentiate into specialized cells within skeletal tissue - chondrocytes, osteocytes, adipocytes and fibroblasts - but also secrete a large range of soluble mediators defining their secretome and allowing their interaction with a number of cell protagonists. Thus, in a general sense, MSC are involved in tissue homeostasis through their secretome and are specifically responsible for cell turn-over in skeletal tissues. For a decade and a half, safety and efficiency of MSC has led to the development of many clinical trials in various fields. However, results were often disappointing, probably because of difficulties in methods and evaluation. At a time when the first clinical trials using MSC are emerging in ophthalmology, the goal of this literature review is to gather and put into perspective preclinical and clinical results in order to better predict the future of this innovative therapeutic pathway.

Femtosecond and excimer laser-assisted endothelial keratoplasty (FELEK): a new technique of endothelial transplantation.

PURPOSE: To describe a new technique of endothelial keratoplasty (EK) that improves the quality of lamellar dissection of donor cornea. METHODS: We compared four techniques of donor cornea preparation for lamellar dissection on 8 donor corneas: mechanical dissection with a microkeratome, a single femtosecond laser lamellar cut, a double femtosecond laser lamellar cut and combined femtosecond laser lamellar dissection with excimer laser surface photoablation. The quality of the donor cornea interface was assessed and compared using scanning electron microscopy (SEM), and the most satisfactory technique was employed for EK on three patients. The postoperative anatomic results were analyzed with anterior segment spectral-domain optical coherence tomography (SD-OCT) and in vivo confocal microscopy (IVCM). RESULTS: The smoothest stromal interface was observed on SEM with the combined use of femtosecond laser dissection and excimer photoablation. The surgical procedures performed with donor cornea prepared by a combination of femtosecond and excimer lasers resulted in clear corneas after 1 month. SD-OCT showed good attachment of the endothelial graft and a hyperreflective interface. On IVCM, subepithelial haze, honeycomb-like activated keratocytes and needle-shaped particles were visible in the recipient corneal stroma as well as numerous hyperreflective particles on the donor-recipient interface. CONCLUSION: A new technique, femtosecond and excimer laser-assisted endothelial keratoplasty (FELEK), which refines the current limitations observed in Descemet-stripping automated endothelial keratoplasty (DSAEK), is described. Femtosecond laser dissection provides a thin and reproducible endothelial graft cut with a high level of safety and accuracy, while excimer photoablation yields a smooth, high-quality interface.

Comparison of preservative-free ketotifen fumarate and preserved olopatadine hydrochloride eye drops in the treatment of moderate to severe seasonal allergic conjunctivitis.

PURPOSE: To compare preservative-free ketotifen 0.025% ophthalmic solution to olopatadine 0.1% ophthalmic solution in with the treatment of seasonal allergic conjunctivitis (SAC) in clinical practice. METHODS: This was a comparative, randomised, investigator-masked, pilot clinical study in adult patients with documented history of SAC and presenting with moderate to severe itching and conjunctival hyperemia. Eligible patients initiated either ketotifen or olopatadine treatment at a dose of one drop twice daily for 28days. The resolution of ocular signs and symptoms was assessed on day 7 and day 28. Itching was also assessed within 15minutes following the first instillation (day 0). Conjunctival impression cytology was performed at each visit to assess the evolution of ICAM-1 expression (day 0, 7 and 28). RESULTS: Seventy-five patients were randomised (ketotifen: 38 patients; olopatadine: 37 patients). At day 28, the composite score for primary criteria (itching, tearing, and conjunctival hyperemia) improved from 6.8±1.2 to 0.9±1.0 in the Ketotifen group, without statistically significant difference between treatment groups (P=0.67). There was no relevant difference between treatment groups in other efficacy parameters, except a trend for a more rapid resolution of conjunctival hyperemia in the Ketotifen group. Both drugs were well tolerated, with a trend for a better tolerability reported by patients on ketotifen compared to those on olopatadine at day 7 (P=0.054). CONCLUSIONS: A rapid and comparable improvement in SAC was achieved after 28days of treatment with both preservative-free ketotifen and preserved olopatadine ophthalmic solutions, with a slightly better ocular tolerance with unpreserved ketotifen 0.025% eye drops.

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.

[Efficacy assessment of Nutrilarm®, a per os omega-3 and omega-6 polyunsaturated essential fatty acid dietary formulation versus placebo in patients with bilateral treated moderate dry eye syndrome]. / Évaluation de l'efficacité du Nutrilarm®, complément nutritionnel à base d'acides gras essentiels polyinsaturés oméga 3 et oméga 6, versus placebo chez des patients atteints de sécheresse oculaire bilatérale modérée et traitée.

INTRODUCTION: Inflammation is one of the main mechanisms common to all forms of dry eye. Since polyunsaturated acids are known to show biological anti-inflammatory properties, the aim of this study was to evaluate the efficacy of dietary n-6 and n-3 fatty acids in patients suffering from ocular dryness. PATIENTS AND METHODS: One hundred and eighty-one patients diagnosed with bilateral moderate dry eye who were already treated with lachrymal substitutes were randomized in a double-blind international study to receive placebo or Nutrilarm(®) capsules (combination of omega-3 and omega-6), twice a day for 6 months. In all subjects, dryness feeling, overall subjective comfort, and ocular symptoms (burning, stinging, sandy and/or gritty sensation, light sensitivity, reflex tearing, and ocular fatigue) were evaluated at each visit. Furthermore, fluorescein tests (break-uptime and Oxford scheme) and lissamine green test were performed at each visit. The Schirmer test was performed at inclusion and after 6 months of treatment. RESULTS: After 6 months of supplementation with Nutrilarm(®), both the BUT scores and ocular fatigue were significantly improved when compared with placebo (P=0.036 and P=0.044, respectively). There was a trend in favor of Nutrilarm(®) in terms of the efficacy evaluated by the investigator (P=0.061). Fewer patients experienced a feeling of severe dryness with Nutrilarm(®) compared with placebo after 6 months of treatment (2.5 and 9.3%, respectively), but the difference was not statistically significant. CONCLUSION: Oral administration of a double supplementation dietary n-6 and n-3 fatty acids present an additional therapeutic advantage in patients suffering from ocular dryness who were already treated with lachrymal substitutes.

Multiple endpoint analysis of BAC-preserved and unpreserved antiallergic eye drops on a 3D-reconstituted corneal epithelial model.

PURPOSE: To compare the effects of benzalkonium chloride (BAC)-preserved and unpreserved antiallergic eye drops on the human 3D-reconstituted corneal epithelial model (3D-HCE). METHODS: 3D-HCE were treated for 24 h followed or not by a 24 h post-incubation recovery period (24 h+24 h) with phosphate-buffered saline (PBS), 0.01% BAC, unpreserved formulations of ketotifen, N Acetyl-Aspartyl Glutamic Acid (NAAGA), cromoglycate, or BAC-preserved commercial formulations of ketotifen, olopatadine, epinastine, and levocabastine. The 3D-HCE viability was evaluated using the 3-(4,5-Dimethylthiazol-2-yl) -2,5-Diphenyltetrazolium Bromide (MTT) test at 24 h and 24 h+24 h. At 24 h, the numbers of Cluster of Differentiation 54 (CD54)- and Ki67-immunopositive cells as well as the number of apoptotic deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were evaluated on 3D-HCE frozen sections. The expression of the tight junction-associated protein occludin was also assessed using fluorescence confocal microscopy on flat-mounted 3D-HCE epithelia. RESULTS: The MTT and the TUNEL tests revealed a significant decrease of cell viability and an increased apoptosis in the superficial layers of the 3D-HCE only when treated with BAC-containing formulations and in a BAC concentration-dependent manner. The expression of CD54 and Ki67 in the basal layers was also increased in this group. A concentration-dependent disorganization of occludin distribution in the epithelium treated with BAC-containing solutions was also observed. The unpreserved formulations induced effects comparable to the control. CONCLUSIONS: BAC-preserved solutions decreased cell viability and induced apoptosis in a concentration-dependent manner. Moreover, they induced CD54 expression, proliferation in the basal layers, and changes in the distribution of occludin, which is consistent with a disorganization of the tight-junctions and suggests the loss of the epithelial barrier function. On the contrary, the unpreserved solutions did not impair cell structures and viability, suggesting a better tolerance for the ocular surface. As allergic patients often exhibit impaired and inflammatory ocular surface, BAC-free compounds should be the first choice when treating allergic conjunctivitis.

[Preservatives in eye drops: toward awareness of their toxicity]. / 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.

[Evaluation of NAAGA efficacy in dry eye syndrome]. / Evaluation de l'efficacité du NAAGA dans la sécheresse oculaire.

OBJECTIVES: The aim of this study was to assess the efficacy of Naabak((R)) eyedrops in reducing inflammation in dry eye syndrome. PATIENTS AND METHODS: This pilot, multicenter, randomized, double-blind, parallel study was carried out in adult patients suffering from moderate dry eye syndrome. Patients were treated for three months with preservative-free NAAGA (Naabak((R))) or with sodium chloride 0.9% without preservative (Larmabak(R)). They received the treatment four to six times a day during the 1(st) month and three to four times a day during the 2(nd) and 3(rd) months. At each visit (D28 and D84), clinical tests were performed as well as a biological evaluation of HLA-DR and MUC5AC expression on conjunctival imprints using flow cytometry. RESULTS: After three months of treatment, the ocular surface symptoms and overall discomfort were improved in patients treated with Naabak(R) and in those treated with Larmabak(R) with no significant difference between the groups. Cytological impression showed a significant decrease in the expression of inflammatory markers, notably antigen HLA-DR, in the Naabak(R) group. CONCLUSION: This study confirms the anti-inflammatory property of preservative-free NAAGA (Naabak(R)) in the context of dry eye syndrome with a similar clinical efficacy compared to sodium chloride solution (Larmabak(R)). Naabak(R) could present an additional advantage compared to artificial tears and could be indicated in the treatment of moderate inflammatory dry eye syndrome.

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 microl 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.

Reduction of quaternary ammonium-induced ocular surface toxicity by emulsions: an in vivo study in rabbits.

PURPOSE: To evaluate and compare the toxicological profiles of two quaternary ammonium compounds (QAC), benzalkonium chloride (BAK), and cetalkonium chloride (CKC), in standard solution or cationic emulsion formulations in rabbit eyes using newly developed in vivo and ex vivo experimental approaches. METHODS: Seventy eyes of 35 adult male New Zealand albino rabbits were used in this study. They were randomly divided into five groups: 50 microl of phosphate-buffered saline (PBS), PBS containing 0.02% BAK or 0.002% CKC (BAK Sol and CKC Sol, respectively), and emulsion containing 0.02% BAK or 0.002% CKC (BAK Em and CKC Em, respectively) were applied to rabbit eyes 15 times at 5-min intervals. The ocular surface changes induced by these eye drops were investigated using slit-lamp examination, flow cytometry (FCM), impression cytology (IC) on conjunctiva, and corneal in vivo confocal microscopy (IVCM). Standard immunohistology in cryosections was also examined for cluster of differentiation (CD) 45+ infiltrating and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL)+ apoptotic cells. RESULTS: Clinical observations and IVCM showed that the highest toxicity was induced by BAK Sol, characterized by damaged corneal epithelium and a high level of inflammatory infiltration. BAK Em and CKC Sol presented moderate effects, and CKC Em showed the lowest toxicity with results similar to those of PBS. Conjunctival imprints analyzed by FCM showed a higher expression of RLA-DR and TNFR1 markers in BAK Sol-instilled eyes than in all other groups, especially at 4 h. Immunohistology was correlated with in vivo and ex vivo findings and confirmed this toxicity profile. A high level of infiltration of CD45+ inflammatory cells and TUNEL+ apoptotic cells was observed in limbus and conjunctiva, especially in QAC solution-receiving eyes compared to QAC emulsion-instilled eyes. CONCLUSIONS: The acute administration of 15 instillations at 5 min intervals was a rapid and efficient model to assess quaternary ammonium toxicity profiles. This model showed the highest toxicity, induced by the BAK solution, and the lowest level of toxicity, induced by the CKC emulsion. These in vivo and ex vivo experimental approaches demonstrated that ocular surface toxicity was reduced by using an emulsion instead of a traditional solution and that a CKC emulsion was safe for future ocular administration.