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To investigate which cytokines, chemokines and growth factors are involved in the immunopathogenesis of idiopathic uveitis, and whether cytokine profiles are associated with. Serum and aqueous humor (AH) samples of 75 patients with idiopathic uveitis were analyzed by multiplex immunoassay. Infectious controls consisted of 16 patients with ocular toxoplasmosis all confirmed by intraocular fluid analyses. Noninfectious controls consisted of 7 patients with Behçet disease related uveitis and 15 patients with sarcoidosis related uveitis. The control group consisted of AH and serum samples from 47 noninflammatory control patients with age-related cataract. In each sample, 27 immune mediators ± IL-21 and IL-23 were measured. In idiopathic uveitis, 13 of the 29 mediators, including most proinflammatory and vascular mediators such as IL-6, IL-8, IL-12, G-CSF, GM-CSF, MCP-1, IP-10, TNF-α and VEGF, were significantly elevated in the aqueous humor when compared to all controls. Moreover, IL-17, IP-10, and IL-21, were significantly elevated in the serum when compared to all controls. We clustered 4 subgroups of idiopathic uveitis using a statistical analysis of hierarchical unsupervised classification, characterized by the order of magnitude of concentrations of intraocular cytokines. The pathogenesis of idiopathic uveitis is characterized by the presence of predominantly proinflammatory cytokines and chemokines and vascular endothelial growth factor with high expression levels as compared to other causes of uveitis. There are indications for obvious Th-1/ IL21-Th17 pathways but also IL9-Th9 and increased IFN-γ-inducing cytokine (IL12) and IFN-γ-inducible CXC chemokine (IP-10). The combined data suggest that immune mediator expression is different among idiopathic uveitis. This study suggests various clusters among the idiopathic uveitis group rather than one specific uveitis entity., Competing Interests: The authors have declared that no competing interests exist.

Background: Alzheimer's disease (AD) and age-related macular degeneration (AMD) present similarities, particularly with respect to oxidative stress, including production of 4-Hydroxy-2- nonenal (HNE). AMD has been named the AD in the eye. The Müller cells (MC) function as a principal glia of the retina and maintain water/potassium, glutamate homeostasis and redox status. Any MC dysfunction results in retinal neurodegeneration., Objectives: We investigated the effects of HNE in human MC., Results: HNE induced an increase of the reactive oxygen species associated with mitochondrial dysfunction and apoptosis. HNE induced endoplasmic reticulum (ER) stress (upregulation of GRP78/Bip, and the proapoptotic factor, CHOP). HNE also impaired expression of genes controlling potassium homeostasis (KCNJ10), glutamate detoxification (GS), and the visual cycle (RLBP1). MC adaptive response to HNE included upregulation of amyloid-β protein precursor (AβPP). To determine the role of AβPP, we overexpressed AβPP in MC. Overexpression of AβPP induced strong antioxidant and anti-ER stress (PERK downregulation and GADD34 upregulation) responses accompanied by activation of the prosurvival branch of the unfolded protein response. It was also associated with upregulation of major genes involved in MC-controlled retinal homeostasis (KCNJ10, GS, and RLBP1) and protection against HNE-induced apoptosis. Therefore, AβPP is an ER and oxidative stress responsive molecule, and is able to stimulate the transcription of major genes involved in MC functions impaired by HNE., Conclusion: Our study suggests that targeting oxidative and ER stress might be a potential therapeutic strategy against glia impairment in AMD and AD, in light of the common features between the two pathologies., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Purpose: Prospective evaluation of aqueous flare following intravitreal bevacizumab (Avastin, Genentech Inc., San Francisco, CA, USA) injections in eyes with choroidal neovascularization due to age-related macular degeneration., Patients and Methods: Sixteen eyes of eight patients were recruited. Aqueous humor flare was determined by laser flare meter every month after one intravitreal injection of 1.25mg of bevacizumab at baseline followed by a second injection at month3 (day 100±21days). Four patients received an injection at month6 (±10days), and one patient received an injection at month7., Results: Two months after the first intravitreal bevacizumab injection, flare values decreased from 10±5.57 (mean±standard deviation) to 5.2±1.69photon count/ms (P=0.0207) and from 8.3±3.59 to 5.4±0photon counts/ms, 2months after the second injection (P=0.02)., Conclusion: Significantly decreased aqueous humor flare levels were noted after repeated injections of bevacizumab., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

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., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Purpose: To investigate the interactions between conjunctival cells and peripheral blood lymphocytes (PBLs) in vitro and to analyze the role of benzalkonium chloride (BAC)-induced apoptosis in this model., Methods: Wong-Kilbourne derivative (WKD) cells were cocultured in cell-contact cultures or on cell inserts for 1 to 7 days with PBLs, activated or not with phorbol 12-myristate 13-acetate (PMA). Morphologic analyses of cell interactions were performed using membrane stainings (green PKH67 for WKD cells and red PKH26 for PBL), F-actin immunostaining, and scanning electron microscopy. Sub-G(1) peak, CD95/Fas, and HLA-DR expression were assessed by flow cytometry (FCM). Specific interactions through the E-cadherin-CD103 complex were studied with FCM and standard immunofluorescence. Five different concentrations of BAC were tested in microplate cytofluorometry assays, to evaluate cytotoxic effects on cell viability and apoptosis., Results: WKD/PBL coculture allowed obvious cell interactions, as shown through plasma membrane exchanges. Direct-contact coculture potentiated the BAC cytotoxic effects and increased HLA-DR and CD95/Fas expression on WKD cells. Trichostatin A-pretreated WKD/PBL coculture induced a slight increase in CD103 expression on PBLs. Moreover, the presence of PBLs during the recovery period after WKD cell BAC stimulation reduced WKD cell apoptosis., Conclusions: These results suggest that the in vitro interaction of PBLs with WKD cells participates in BAC-induced epithelial toxicity regulation, probably through cell membrane contacts.

Background: hyaluronan biopolymer is used in dermatology but the underlying mechanism and the impact of its molecular weight have not yet been investigated in skin wound healing. The aim of our work was to study the role of HA molecular weight in the proliferative phase of wound healing and to understand how this physiological biopolymer acts to promote wound healing on a human keratinocyte in vitro model., Methodology and Findings: wound healing closure was evaluated using scratch test assay, cell proliferation by counting cell with haemocytometer, expression of CD44 and ZO-1 (protein present in tight junctions specific of epithelia) using flow cytometry, and P2X7 receptor activation on living using a cytoflurometric method. Our study showed that medium hyaluronan fragment (MMW-HA, between 100 and 300 kDa) induced a significant increase in wound closure, increased ZO-1 protein expression and induced a slight activation of P2X7 receptor, contrary to high (between 1000 and 1400 kDa) and low (between 5 and 20 kDa) molecular hyaluronan fragments that had no healing effects. Basal activation of P2X7 receptor is already known to stimulate cell proliferation and this activation in our model plays a pivotal role in MMW-HA-induced wound healing. Indeed, we showed that use of BBG, a specific inhibitor of P2X7 receptor, blocked completely the beneficial effects of MMW-HA on wound healing., Conclusion: taken together, our results showed for the first time the relationship between P2X7 receptor and hyaluronan in wound healing, and that topical use of MMW-HA (fragment between 100 and 300 kDa) could represent a new therapeutic strategy to promote healing.

Introduction: The various forms of ophthalmic pharmaceutical presentation of steroids is proliferating on the market: solutions, gels, and suspensions. Suspensions are characterized by particles in solution and require agitation before instillation. This trial studied the impact of agitation on the corticoid concentration of eye drop solutions, gels, and suspensions., Methods: Corticosteroid levels in a drop of a dexamethasone solution or suspension or betamethasone suspension or gel were compared using liquid chromatography. These levels were measured after shaking for 5, 10, 30s, and 1 min using a vortex or without shaking., Results: The results of this study show that, whatever shaking time was used, the suspension form seems less suited to instillation of corticosteroids. The suspension did not deliver consistent levels of corticosteroids (mean between 23 and 99%) compared to solutions and gels, which released about 100% of the corticosteroid content in each drop., Conclusion: Physicians, ophthalmologists, and pharmacists should remind the patient of the proper use of these suspensions before instillation. In cases of treatment failure, it is necessary to check the instillation method before questioning patient compliance., (Copyright © 2011. Published by Elsevier Masson SAS.)

Objectives: Pralidoxime is an organic cation used as an antidote in addition to atropine to treat organophosphate poisoning. Pralidoxime is rapidly eliminated by the renal route and thus has limited action. The objectives of this work were as follows. 1) Study the role of organic cation transporters in the renal secretion of pralidoxime using organic cation transporter substrates (tetraethylammonium) and knockout mice (Oct1/2⁻/⁻; Oct3⁻/⁻). 2) Assess whether sustained high plasma concentrations increase pralidoxime antidotal activity toward paraoxon-induced respiratory toxicity., Setting: INSERM U705, Faculté de Pharmacie, Université Paris Descartes, 4 Avenue de l'Observatoire, 75006 Paris, France., Subjects: Rodents: Knockout mice (Oct1/2⁻/⁻; Oct3⁻/⁻) and Sprague-Dawley rats., Interventions: None., Measurements and Main Results: In rats, the renal clearance of pralidoxime was 3.6-fold higher than the creatinine clearance. Pretreatment with tetraethylammonium (75 mg/kg) in rats or deficiencies in organic cation transporters 1 and 2 in mice (Oct1/2⁻/⁻) resulted in a significant increase in plasma pralidoxime concentrations. Lack of Oct3 did not alter plasma pralidoxime concentrations. The antidotal activity of pralidoxime (50 mg/kg intramuscularly) was longer and with greater effect, resulting in a return to normal values when administered to rats pretreated with tetraethylammonium., Conclusions: Pralidoxime is secreted in rats and mice by renal Oct1 and/or Oct2 but not by Oct3. Modulation of organic cation transporter activity increased the plasma pralidoxime concentrations and the antidotal effect of pralidoxime with sustained return within the normal range of respiratory variables in paraoxon-poisoned rats. These results suggest a promising approach in an animal model toward the increase in efficiency of pralidoxime. However, further studies are needed before these results are extended to human poisoning.

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., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Purpose: We evaluated (1) 4 multipurpose lens care solutions and 3 contact lenses (soft and rigid) for cytotoxicity according to ISO 10993-5 standard (medical device biocompatibility) and (2) the protective effects of a marine cationic solution and hyaluronic acid., Methods: Low water soft lens, high water soft lens, and rigid lens were laid on a conjunctival cell line after being soaked in multipurpose solution (Optifree Express, Renu, Solocare Aqua, or Menicare Plus). Cell morphology was microscopically observed, and cell viability was evaluated using the neutral red test. Apoptosis was assessed after direct contact of multipurpose solutions (MPS) with conjunctival cells using fluorescence microscopy and flow cytometry. The ability of a controlled ionization marine solution and hyaluronic acid to prevent multipurpose solution's cytotoxicity was finally evaluated., Results: Contact lenses soaked in the MPS induced cell morphology alterations and loss of cell viability. Rinsing the lens with the marine solution improved cell viability and preincubating cells with hyaluronic acid inhibited apoptosis., Conclusions: MPS can be damaging for the ocular surface cells. We proposed to rinse the lens with a marine solution before insertion of the lens on the cornea to wash away the multipurpose solution and to use hyaluronic acid to protect the ocular surface cells against apoptosis induced by MPS.

Background: Olive oil and fish oils are known to possess beneficial properties for human health. We investigated whether different oils and fatty acids alone were able to decrease oxidative stress induced on corneal cells., Methods: In our in vivo study, rats were fed with marine oils rich in polyunsaturated fatty acids (PUFA) or refined olive oil during 28 days. At the end of the protocol, corneas were analysed for their fatty acids composition to study the incorporation of fatty acids in cell membranes. In our in vitro study, a human corneal cell line was incubated with marine oils or refined olive oil and subjected to oxidative stress (tBHP 50 muM, 1 hour). Effects on reactive oxygen species generation, mitochondria and caveolin-1 expression were studied using microcytofluorometry, flow cytometry and confocal microscopy., Results: Our results indicate that dietary oils changed the fatty acids composition of corneal cell membranes. According to our results, PUFA-rich oils and refined olive oil (free of antioxidants) blocked reactive oxygen species production. Oleic acid, the major fatty acid of olive oil, also decreased oxidative stress. Moreover, oleic acid modified caveolin-1 expression. Antioxidant properties of oleic acid could be due to disruption of membrane microdomains such as caveolae., Conclusion: Oleic acid, a potential potent modulator of oxidative stress, could be added to PUFA-rich oils to prevent oxidative stress-linked corneal pathology.

Purpose: The aim of this study was to investigate high-molecular-weight hyaluronan (HA-HMW) corneal protection against sodium lauryl sulfate (SLS)-induced toxic effects with in vitro and in vivo experimental approaches., Methods: In vitro experiments consisted of a human corneal epithelial cell line incubated with HA-HMW, rinsed, and incubated with SLS. Cell viability, oxidative stress, chromatin condensation, caspase-3, -8, -9, and P2X7 cell death receptor activation, interleukin-6, and interleukin-8 production were investigated. In vivo experiments consisted of 36 New Zealand white rabbits treated for 3 days, 3 times per day, with HA-HMW or phosphate-buffered salt solution. At day 4, eyes were treated with SLS. Clinical observation and in vivo confocal microscopy using the Rostock Cornea Module of the Heidelberg Retina Tomograph-II were performed to evaluate and to compare SLS-induced toxicity between eyes treated with HA-HMW and eyes treated with phosphate-buffered salt solution., Results: In vitro data indicate that exposure of human corneal epithelial cells to HA-HMW significantly decreased SLS-induced oxidative stress, apoptosis, and inflammation cytokine production. In vivo data indicate that SLS cornea injuries, characterized by damaged corneal epithelium, damaged anterior stroma, and inflammatory infiltrations, were attenuated with HA-HMW treatment., Conclusions: A good correlation was seen between in vitro and in vivo findings showing that HA-HMW decreases SLS-induced toxic effects and protects cornea.

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.

Purpose: To determine whether multipurpose solutions, widely used for contact lens disinfections, could be at the origin of ocular pathologies (contact lens intolerance and ocular infections)., Methods: An observational cohort study (questionnaire analysis) was carried out to estimate the number of contact lens wearers, type of infection, and type of lens care regimen used by patients. Besides, multipurpose solutions cytotoxicity (necrosis and apoptosis) was evaluated on a conjunctival cell line using cytofluorometry., Results: In the general population, 59% of contact lens wearers use multipurpose solutions whereas 35% use oxidative products. Of the questioned contact lens wearers with ocular infections, 80% used multipurpose solutions. Multipurpose solutions are therefore not efficient enough against microorganisms, and cannot be considered as disinfectant solutions but only as preservatives. However, preservatives are known to be toxic to ocular surface, so apoptosis induced by multipurpose solutions could lead to ocular surface diseases. Our cytofluorometry study allowed us to demonstrate that contact lens multipurpose solutions containing preservatives are cytotoxic through caspase 3 induction, chromatin condensation and P2X7 cell-death receptor activation, in contrast with unpreserved sterile saline solutions that were found inert., Conclusions: Multipurpose solutions seem to be preservative but not disinfecting solutions. They are not adapted to the final rinse of contact lenses because of apoptosis induction. It could explain part of lens intolerance.

Purpose: We investigated the effects of various rinsing and healing protocols on corneal wound repair and inflammation following alkali burn in rabbits., Methods: We conducted in vitro, in vivo and ex vivo studies. First, different rinse solutions were tested in vitro after incubation of ocular cells with methanol or NaOH. Cell viability was then assessed using the neutral red test (cytofluorometry). Second, NaOH was applied to rabbit corneas and associations of rinse solutions (NaCl 0.9% or controlled ionization marine solutions) with N-acetylcysteine or vegetable oils (from Calophyllum inophyllum and Aleurites moluccana) were tested in vivo. The regeneration of the corneal epithelium and the infiltration of inflammatory cells were evaluated using in vivo confocal microscopy and ex vivo histological cuts., Results: The association of a controlled ionization marine solution with 10% C. inophyllum oil and 90% A. moluccana oil induced regeneration of the corneal epithelium and a decrease in inflammatory cells., Conclusions: Irrigation with marine solution followed by treatment with a mixture of C. inophyllum and A. moluccana oils is a promising treatment for ocular burns., (Copyright 2008 S. Karger AG, Basel.)

Purpose: The aim of this study was to investigate high molecular weight hyaluronan (HMW-HA) protection on human corneal epithelial (HCE) cells against ultraviolet B (UVB) radiation-induced toxic effects., Methods: The HCE cell line was incubated with HMW-HA or phosphate-buffered salt solution (PBS), rinsed, and exposed to UVB radiation. Cell viability, reactive oxygen species (ROS) and glutathione (GSH) levels, 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) release, p53 phosphorylation, caspase-3, -8, -9 activation, and interleukin (IL)-6 and -8 production were assessed to evaluate and to compare UVB-induced toxicity between cells treated with HMW-HA and cells treated with PBS., Results: Data indicate that HMW-HA had significant protective effects against UVB radiation. HMW-HA increased HCE cell viability, decreased IL-6 and -8 production, and decreased caspase-3 and -8 activation. However, HMW-HA had no significant effect on ROS and GSH levels, 8-oxo-dG release, and p53 phosphorylation., Conclusions: To our knowledge, we report for the first time the ability of HMW-HA to protect cells against UV irradiation. According to our results, HMW-HA provides anti-inflammatory and anti-apoptotic signals to cells exposed to UVB.

Introduction: The retina is located in a highly oxygenated environment and is therefore particularly susceptible to oxidative damage. Blueberries have a high antioxidant potential since they are rich in anthocyans. The aim of this study was to investigate the cytoprotective role of blueberries on human retinal cells., Materials and Methods: Blueberry extract was incubated at 0.05% and 0.1% for 15 minutes or 24 hours on a human retinal cell line. Then oxidative stress was induced by 150 microM tert-butylhydroperoxide (tBHP) for 1 hour. Intracellular metabolism, reactive oxygen species, superoxide anion, and mitochondrial apoptosis were evaluated using Alamar blue, DCFDA, dihydroethidium, and nonylacridine orange dyes, respectively. Tests were performed using cytofluorometry adapted to microplates., Results: Blueberry protected cells against tBHP-induced cytotoxicity. It increased cell viability, decreased oxidative stress and mitochondrial apoptosis. After a 24-hour preincubation time, blueberry totally inhibited tBHP-induced cytotoxicity., Conclusion: Blueberry seems to be a potent antioxidant and could be easily added to food complements to prevent or limit ocular pathologies induced by oxidative stress.

Purpose: To propose an in vivo confocal microscopic scoring system for evaluation of irritant-induced corneal changes., Materials and Methods: Rat corneas were instilled with 0.01-0.5% benzalkonium chloride (BAC) and examined using high-resolution in vivo confocal microscopy (HRT-II) to measure corneal thickness and characterize corneal damage patterns. Severity scores were given for each predefined evaluation parameter and then totaled., Results: The scoring system revealed a dose-dependent effect of BAC and discriminated between high- and low-dose treatments., Conclusions: This HRT-II scoring standardizes damage evaluation at the cellular level, even when assessing irritating compounds at low concentrations.

Objective: Benzalkonium chloride (BAK) is one of the most often used preservative in pharmaceutical products and it is known to induce toxic effects. Hyaluronan (HA), a linear biopolymer, is involved in several biological processes. The aim of this work is to in vitro investigate if HA is able to decrease BAK toxicity., Methods: Two human epithelial cell lines were treated with different incubation time protocol with BAK and three different molecular weights HA (HA 20k Da, HA 100 kDa and HA 1000 kDa, 0.2%, w/v). Flow cytometry, fluorescence microscopy, microplate cytofluorometry and confocal microscopy were performed to evaluate expression of CD44 receptor, cell viability, oxidative stress, mitochondrial mass, chromatin condensation, plasma-membrane permeability, DNA fragmentation and cytoskeleton morphology., Results: The three HAs studied induce neither oxidative stress nor apoptosis. HA 1000 kDa significantly decreases oxidative stress, apoptosis and necrosis induced by BAK. Experiments with HA 20 kDa or HA 100 kDa did not show the same effects. For instance, the more molecular weight decreases, the more protection decreases. Moreover, we suggest that HA interacts with cell plasma-membrane and inhibits cell death receptors., Conclusion: High molecular weight HA (1000 kDa, 0.2%) is an effective protective agent against BAK.

Purpose: The purpose of this study was to investigate responses to toxic cellular stresses in different human ocular epithelia., Methods: Reactivity with a specific anti-P2X7 antibody was studied using confocal fluorescence microscopy on conjunctival, corneal, lens, and retinal cell lines as well as using impression cytology on human ocular cells. Activation of the P2X7 receptor by selective agonists (ATP and benzoylbenzoyl-ATP) and inhibition by antagonists (oATP, KN-62, and PPADS) were evaluated using the quinolinium,4-[(3-methyl-2-(3H)-benzoxazolylidene) methyl]-1-[3-(triethylammonio)propyl]di-iodide (YO-PRO-1) test in cytofluorometry. Different specific stresses were then induced by a chemical toxin (benzalkonium chloride) and a chemical oxidant (tert-butyl hydroperoxide) to assess the role of the P2X7 receptor. Modulation of P2X7 receptor activation was performed with several ionic solutions., Results: Our data show that four cell lines express the P2X7 cell death purinergic receptor as judged by reactivity with a specific anti-P2X7 antibody, activation by the selective P2X7 agonist benzoylbenzoyl-ATP and to a lesser extent by ATP (YO-PRO-1 dye uptake), and inhibition by three antagonists (oATP, KN-62, and PPADS). Benzalkonium chloride, a widely used preservative, induced dramatic membrane permeabilization through P2X7 pore opening on conjunctival and corneal epithelia. Reactive oxygen species, induced by tert-butyl hydroperoxide, lead to P2X7 receptor activation on retinal pigment epithelium. Modulation of P2X7 receptor activation was obtained with extracellular Ca(2+) and Mg(2+) and with a controlled ionization marine solution rich in different divalent cations. This marine solution could be proposed as a new ophthalmic solution., Conclusions: Our observations reveal a novel pathway for epithelial cells apoptosis/cytolysis by inducing different toxic stresses and their modulation by using ionic solutions.

Aims: This study's objective was to evaluate the tolerance and safety of a new ophthalmic solution based on ReGeneraTing agent (RGTA) technology in a pilot noncontrolled exploration on compassion use for corneal ulcers and severe chronic dystrophies resistant to the usual treatments., Rationale: RGTAs are large biopolymers engineered to replace heparan sulfates specifically bound to matrix proteins and growth factors destroyed after a lesion has occurred. The RGTA-bound proteins are protected from proteolysis and this allows the extracellular matrix microenvironment to restore its original proper organization. The initial endogenous signals needed for tissues to regenerate are back on the restored matrix. They are expected to trigger the natural onset of events, signaling cells to migrate and multiply with the cascades and equilibrium found in tissue homeostasis. RGTA-induced matrix therapy is a possible alternative to cell or gene therapy in regenerative medicine. In a rabbit preclinical model of alkali-induced severe corneal ulcers, a single instillation of RGTA ophthalmic solution was found sufficient to enhance speed and quality of healing, restoring an almost normal corneal histology after only 1 week. These data prompted us to initiate this study., Patients and Methods: Eleven eyes from ten patients were included in this study. All patients had severe dystrophic cornea or painful corneal ulcers rated over 50 on the VAS pain scale ranging from 0 to 100 and had undergone unsuccessful treatments. The RGTA ophthalmic solution was administered by the investigator during each weekly consultation as a single drop over 1 month. Tolerance and efficacy were judged on subjective criteria based on pain evaluation and functional inconvenience as well as on objective clinical criteria through a complete ophthalmic examination at days 3, 7, 14, 21, 28 and after 2 and 3 months from the beginning of the treatment., Results: The study was conducted to completion for all patients included at the beginning. Tolerance was excellent both locally and generally: no uneasiness during instillation, no worsening of the initial pathology, no occurrence of ocular inflammation or increase in ocular pressure, and no general side effects were observed. In addition, we observed a noticeable analgesic effect, increasing with time and instillations, but pain reappeared in the majority of cases as treatment ended. The mean visual analog scale pain score was 72.73 +/- 7.86, it decreased significantly with the first drops of treatment. After 1 month, the mean visual analog scale pain score was 32+/-15.49, then it increased after the end of the treatment, confirming the link between the effects observed and the treatment. Efficacy on keratitis was moderate but with an overall tendency toward improvement. The initial Oxford Score was 3.37 +/- 1.06. After 1 month, it decreased significantly to 1.57 +/- 0.97 and then it rose again after the end of the treatment. As for corneal ulcers, of the five cases included, four healed during the protocol. Two reversed when the treatment stopped, two healed without reversion at the last follow-up visit. The last case was characterized by stem cell deficiency and no improvement was noted. It is important to keep in mind that these ulcers were all resistant to usual therapies., Conclusion: This RGTA ophthalmic solution is the first matrix therapy product in ophthalmology. The RGTA OTR4120 was used in treating chronic and severe corneal dystrophies as well as corneal ulcers resistant to usual treatments. It was very well tolerated with no side effects. It significantly reduced pain and favored corneal healing in almost all corneal ulcers. Weekly instillation of a single drop seems insufficient and these very promising data need to be confirmed on a larger population in a controlled trial with more adapted dosages. Based on these preliminary data, a RGTA-based matrix therapy product may be a very innovative solution to unresolved pain and corneal surface healing problems.

Purpose: This study compared the toxicity profiles of three antiglaucoma prostaglandin F2alpha analogs, latanoprost, travoprost, and bimatoprost which contain benzalkonium chloride (BAK), with tafluprost, a new preservative-free prostaglandin analog., Methods: IOBA-NHC cells were exposed to BAK-containing prostanoid solutions, their respective BAK concentrations, and preservative-free tafluprost solution for 30 min. Membrane integrity, apoptosis, oxidative stress, and cells morphology were evaluated., Results: Preservative-free tafluprost resulted in significantly higher membrane integrity and lower pro-apoptotic and pro-oxidative effects than preservative-containing prostaglandin analog preparations., Conclusions: These results suggest that tafluprost, a new preservative-free prostaglandin analog, has very low or no pro-apoptotic, pro-necrotic, or pro-oxidative effects in vitro compared to preservative-containing formulations.

Purpose: The Wong-Kilbourne derivative of Chang conjunctiva-derived cell line has been widely used for toxicological and functional in vitro studies on the ocular surface. The common reserve to this cell line is the reported contamination with HeLa cells. Thus, the IOBA-NHC spontaneously immortalized conjunctival epithelial cell line has been recently developed and did not show other cell type contamination. Our purpose was to determine whether both cell lines would be equally suitable for in vitro toxicological studies. Therefore, we compared in these two cell types the toxic effects of the preservative, benzalkonium chloride (BAC); its toxicity has been often reported on conjunctival in vivo and in vitro models., Methods: The necrotic, apoptotic, and oxidative effects of BAC were evaluated on Chang and IOBA-NHC cell lines using microplate cytofluorometry tests (neutral red, 2,7- dichlorofluorescein diacetate dye [H(2)DCF-DA], hydroethidine, and Yopro-1), flow cytometry (Annexin V/7-AAD and DNA content tests), and standard immunofluorescence stainings. Cells were exposed to five concentrations of BAC (10(-2)%, 5.10(-3)%, 10(-3)%, 10(-4)%, and 10(-5)%) for two incubation times: 15 min of treatment and 15 min of treatment followed by 24 h of cell recovery in complete medium., Results: All parameters of toxicity increased in a BAC dose-dependent manner on both cell lines., Conclusions: The comparison of BAC toxicity on both cell lines supported the use of IOBA-NHC and Chang cells for toxicological in vitro studies. Drawbacks of both cell lines have to be known and considered in studies performed on these cell lines.

Galantamine, an acetylcholinesterase inhibitor used to enhance memory in AD patients by acetylcholinesterase inhibition, has been tested for its protective properties on an in vitro model of H(2)O(2)-induced oxidative stress. SK-N-SH cells treated with H(2)O(2) for 2h showed an increase in ROS production (54%) and in NO production (52%) together with a marked reduction of the mitochondrial membrane potential (19%). These features, typical of the oxidative injury that accompanies AD, were partly recovered by galantamine. Galantamine reduced the release of reactive oxygen species (up to 50%) and prevented loss in mitochondrial activity. When SK-N-SH cells were treated with H(2)O(2) for 24h, nitrite generation was increased by twice compared with 2h. Galantamine treatment resulted in a significant inhibition of H(2)O(2)-induced nitrite generation whatever the concentration tested with a return to control values. Galantamine also concentration-dependently inhibited AChE activity (28-88%) in H(2)O(2)-SK-N-SH cells after 24h. This drug, which facilitates cholinergic neurotransmission, is also neuroprotective by lowering oxidative injury. Our study provides a better understanding of the mechanisms of protection of this acetylcholinesterase inhibitor which also has antioxidative properties.

Objective: To investigate the cytotoxicity of multipurpose solutions used for contact lens disinfection., Methods: Four multipurpose solutions (Optifree containing quaternary ammonium as preservative, Renu, Solocare and Complete containing polyhexamethylene biguanide as preservative) were incubated on a conjunctival cell line to evaluate their capacity to induce necrosis (neutral red test), intracellular redox status alteration (Alamar Blue test), reactive oxygen species overproduction (DCFH-DA and dihydroethidium tests), mitochondrial alterations (NonylAcridine Orange and JC-1 tests) and to activate P2X7 cell death receptor (YO-PRO-1 test). Tests were performed using cytofluorometry and inverted fluorescence microscopy., Results: Our results showed that multipurpose solutions induced necrosis, in addition to oxidative stress. Optifree induced oxidative stress with increased mitochondrial mass, Renu, Solocare and Complete induced whether a decrease in reactive oxygen species production with mitochondrial alterations, or an increase in reactive oxygen species production, but each solution stimulated P2X7 cell death receptor activation. Early stimulation of P2X7 cell death receptor, prior to any superoxide production, demonstrated the possible involvement of this receptor in the oxidative stress process., Conclusion: These new findings suggest that most of multipurpose solutions are toxic with oxidative stress and apoptosis induction.

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.

We report the first case of endophthalmitis caused by Phoma glomerata. A 32-year-old man who underwent retinal detachment surgery consecutive to a penetrating globe injury presented with endophthalmitis 7 days after surgery. Anterior chamber tap and intravitreal injection of antibiotics (ceftazidime and vancomycin) were performed systematically. Fungus was observed at microscopic examination of the aqueous humor and treatment with intravitreal injection of amphotericin B was decided. The patient failed to improve with intravitreal amphotericin B but responded clinically to intravitreal voriconazole. The fungus was identified after culture as Phoma glomerata. The MIC for amphotericin B was 1microg/ml, for caspofungin was 2microg/ml, and for itraconazole was 8microg/ml or more. The MIC for voriconazole was up to 8microg/ml. The clinical response after intravitreal injection may be related to the high concentrations reached in the vitreous. Because of severity and ominous prognosis of intraocular fungal infections and posttraumatic Phoma ocular infections, aggressive management is required by intravitreal voriconazole administration.

Purpose: 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., Design: Case-control study., Participants: 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., Methods: 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., Main Outcome Measures: HLA-DR and chemokine receptors (CCR4 and CCR5) in ICSs., Results: 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., Conclusions: 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.

Purpose: To assess the usefulness of noninvasive combined technologies used to observe ocular surface changes in toxicology studies., Methods: Benzalkonium chloride (BAC) at 0.01%, 0.1%, 0.25%, and 0.5% was applied to rat corneas for 11 days. The eye was evaluated macroscopically from day (D)0 to D52. The cornea was examined with the slit lamp, a fluorescein test was performed, and a confocal microscope was used in vivo to calculate corneal thickness, score corneal epithelial and endothelial defects, and quantify corneal stromal inflammation and neovascularization. Conjunctival impression and brush cytology specimens were taken for labeling with MUC-5AC antibodies and sub-G1 peak analysis by flow cytometry, respectively. Histologic analyses were performed on D11., Results: Although macroscopic and slit lamp examinations revealed signs of ocular irritation in the 0.25% and 0.5% BAC-treated eyes only, in vivo confocal microscopy revealed epithelial defects in the 0.01% and 0.1% BAC-treated corneas, and sub-G1 peak analyses showed increased apoptosis for all the BAC concentrations on D8 and D11. BAC at 0.25% and 0.5% induced increased corneal thickness, loss of goblet cells, reversible corneal inflammation, and persistent neovascularization., Conclusions: Sub-G1 peak analysis of conjunctival brushings, in conjunction with in vivo confocal microscopy of the cornea and immunolabeling of conjunctival imprints, constitutes a noninvasive, reliable, and sensitive tool to evaluate toxic drug-induced ocular surface damage in rats, in addition to standard clinical assessments and at a wide range of concentrations, including the lowest ones. This study is consistent with the international strategy aimed at reducing the use of animals and refining animal toxicologic models.

Purpose: Ocular side effects in patients using eye drops may be due to intolerance to the vector used in eye drops. Castor oil is the commonly used lipophilic vector but has been shown to be cytotoxic. Effects on cells of four oils (olive, camelina, Aleurites moluccana, maize) were compared with those of castor oil in human conjunctival cells., Methods: Human conjunctival cells were incubated with the oils for 15 minutes. After a 24-hour recovery period, cells were tested for viability, proliferation, apoptosis (P2X7 cell death receptor and caspase 3 activation), intracellular redox potential, and reactive oxygen species production. Fatty acid incorporation in cell membranes was also analyzed. In vivo ocular irritation was assessed using the Draize test., Results: Compared to the four other oils, castor oil was shown to induce significant necrosis and P2X7 cell death receptor and caspase 3 activation and to enhance intracellular reactive oxygen species production. Aleurites moluccana and camelina oils were not cytotoxic and increased cell membrane omega-3 fatty acid content. None of the five tested oils showed any in vivo ocular irritation., Conclusions: The results demonstrated that castor oil exerts cytotoxic effects on conjunctival cells. This cytotoxicity could explain the side effects observed in some patients using eye drops containing castor oil as a vehicle. The lack of cytotoxic effects observed with the four other oils, Aleurites, camelina, maize, and olive, suggest that they could be chosen to replace castor oil in ophthalmic formulations.

The aim of this study was to investigate how the brain is affected during systemic inflammation. For this purpose, Swiss mice were challenged with a single intraperitoneal dose of lipopolysaccharide (LPS; 250microg/mouse) to mimic aspects of systemic infection. Spatial learning in Y-maze test demonstrated a differential learning profile during the training test between control and LPS-treated mice, with an alteration in the latter group. We show that systemic LPS-induced inflammation and oxidative injury as assessed by reactive oxygen species (ROS) and nitrites/nitrates (NOx) production associated with reduced glutathione (GSH) depletion, cyclooxygenase-2 (COX-2) expression, and lipid peroxidation. LPS also induced a loss in mitochondrial integrity as shown by a significant decrease in membrane potential and impairment in mitochondrial redox activity. Thus, peripheral inflammation by producing brain inflammation and oxidative injury causes mnesic deficits. It remains to determine whether such events can induce neuronal dysfunction/degeneration and, with time, lead to cholinergic deficiency, amyloid deposits and cognitive impairments as they occur in Alzheimer's disease.

Purpose: With use of the Wong-Kilbourne derivative Chang conjunctival cell line, this study compared in vitro the ocular toxicity of three topical intraocular pressure (IOP)-lowering agents: travoprost 0.004% containing 0.015% benzalkonium chloride (BAK), travoprost Z 0.004%, a new formulation without BAK, and latanoprost 0.005% containing 0.02% BAK., Methods: Neutral red, Alamar blue, YOPRO-1, and annexin V/7-AAD assays were used to evaluate the effects of the IOP-lowering agents and BAK on cellular viability, membrane integrity, and apoptosis in the conjunctival cell line using microtitration fluorometric analysis and flow cytometry. All assessments were performed in a masked manner., Results: Assessment of cell viability and membrane integrity revealed a significant effect by latanoprost with BAK or BAK alone but no effect by travoprost Z without BAK or buffer alone (P < 0.0001). Latanoprost with BAK, travoprost with BAK, and BAK alone were cytotoxic in Chang conjunctival cells, whereas no cytotoxicity was observed in cells exposed to travoprost Z without BAK or in cells treated with buffer (P < 0.0001). No increase in apoptosis or necrosis was observed in cells treated with control or travoprost Z without BAK compared with BAK, travoprost with BAK, and latanoprost with BAK (P < 0.0001)., Conclusions: Latanoprost with BAK, travoprost with BAK, and BAK alone have significant cytotoxic effects on human conjunctiva-derived cells and are associated with apoptosis. These effects likely result from BAK used as a preservative. IOP-lowering agents with alternative preservatives instead of BAK will most likely have fewer ocular surface adverse effects than agents containing BAK.

Modified melanoma cells (B16-F0.MOD) characterized by inhibited IGF-I, CD9 low but not their wild-type counterparts (B16-F0.WT), IGF-I positive, CD9 high, were shown to be immunogenic for syngeneic hosts. C57BL/6 syngeneic recipients vaccinated with B16-F0.MOD cells developed immune effectors that were observed at the humoral as well as cellular levels. These immune effectors were shown to be capable of controlling in vitro tumour growth and in vivo tumour progression.

Purpose: To evaluate and compare the proinflammatory and apoptotic effects of lipopolysaccharide (LPS) in three rabbit corneal injury models using a new in vivo confocal microscope (IVCM) and immunohistological techniques., Methods: Adult male New Zealand albino rabbits were used in this study. Three corneal models were tested: corneal incision, corneal epithelium scraping, and corneal suture. Ten rabbits were used in each model and these three groups were subdivided into two subgroups: with or without LPS instillation (with saline used as control) for eight days. Rabbit corneas were analyzed in vivo by using the Rostock Cornea Module (RCM) of the Heidelberg Retina Tomograph (HRT)-II. Immunohistology was used to evaluate inflammatory, proliferating, and apoptotic cells in the different injury models following saline or LPS instillations., Results: Clinically, LPS induced earlier and higher levels of inflammation and corneal neovascularization in eyes subjected to scraping and suturing compared to saline. The RCM/HRT successfully presented high-quality images allowing analysis of all pathological corneal layers. Compared to groups receiving saline, LPS caused earlier and greater surface and stromal inflammatory infiltration as well as neovascularization. Immunohistology was correlated with in vivo findings and confirmed these results by showing greater infiltration of KI 67+ proliferating cells, TUNEL+ apoptotic cells, and TNF-alpha+, TNFR1+, TLR4/MD2+, ICAM-1+, RLA-DR+, CD11b+, and CD11c+ inflammatory cells, in eyes receiving LPS compared to those receiving saline., Conclusions: These results indicate that in various models of corneal injury, LPS is a potent proinflammatory stimulus and its exposure has major effects on determinants of inflammation, angiogenesis, and apoptosis.

Background: The purpose of the study was to compare toxic effects and responses to histamine and IFN gamma associated with the use of some widely used anti-allergic eye drops commercially available today., Methods: For dynamic studies, the Wong-Kilbourne cell line was stimulated for 24 h with histamine or IFN gamma in the presence or absence of anti-allergic eye drops. Supernatants of histamine-stimulated cells were evaluated for the production of IL-6 and IL-8 by ELISA, while the expression of ICAM-1 was evaluated by flow cytometry on IFN gamma-stimulated cells. Toxicological assays were performed using cold light cytofluorometry: viability and apoptosis as well as reactive oxygen species (ROS) and O2(.)- production were assessed using neutral red, Hoechst/propidium iodide, H(2)-DCFDA and hydroethidine tests, respectively., Results: Antihistamines reduced IL-6 release and presented dose-dependent inhibitory effects on IL-8 production. None of the eye drops decreased the basal or IFN gamma-stimulated expression of ICAM-1. Conversely, eye drops preserved with benzalkonium chloride (BAC) induced even higher ICAM-1 expression levels on IFN gamma-stimulated cells than did IFN gamma alone, whereas unpreserved drugs had no effect. Toxicological assays confirmed the pivotal role of BAC in proportionally reducing cell viability while increasing apoptosis and oxidative stress., Conclusions: The ability of topical ocular anti-H(1) drugs to significantly reduce the production of IL-6 and IL-8 argues that they may help treat the inflammatory processes occurring in allergic ocular surface disorders. Nevertheless, preserved ophthalmic formulations may enhance epithelial conjunctival expression of ICAM-1 in the presence of a low inflammatory stimulus, such as IFN gamma, and displayed toxic as well as pro-oxidative effects on these cells. Therefore, BAC used as preservative might in part interfere with the potential anti-inflammatory properties of the active compound by modulating the immuno-inflammatory response of epithelial conjunctival cells.

The majority of chemical solar filters are cytotoxic, particularly on sensitive ocular cells (corneal and conjunctival cells). Consequently, a non-cytotoxic UV filter would be interesting in dermatology, but more especially in ophthalmology. In fact, light damage to the eye can be avoided thanks to a very efficient ocular antioxidant system; indeed, the chromophores absorb light and dissipate its energy. After middle age, a decrease in the production of antioxidants and antioxidative enzymes appears with accumulation of endogenous molecules that are phototoxic. UV radiations can induce reactive oxygen species formation, leading to various ocular diseases. Because most UV filters are cytotoxic for the eye, we investigated the anti-UV properties of Calophyllum inophyllum oil in order to propose it as a potential vehicle, free of toxicity, with a natural UV filter action in ophthalmic formulation. Calophyllum inophyllum oil, even at low concentration (1/10,000, v/v), exhibited significant UV absorption properties (maximum at 300nm) and was associated with an important sun protection factor (18-22). Oil concentrations up to 1% were not cytotoxic on human conjunctival epithelial cells, and Calophyllum inophyllum oil appeared to act as a cytoprotective agent against oxidative stress and DNA damage (85% of the DNA damage induced by UV radiations were inhibited with 1% Calophyllum oil) and did not induce in vivo ocular irritation (Draize test on New Zealand rabbits). Calophyllum inophyllum oil thus exhibited antioxidant and cytoprotective properties, and therefore might serve, for the first time, as a natural UV filter in ophthalmic preparations.

Modulation of cell surface molecules involved in immune recognition and cellular interactions (class I major histocompatibility complex or MHC-I, B7.1 or CD80, integrin alpha4 or CD49d, tetraspanins CD9, CD81) was examined in modified B16 melanoma cells displaying either inhibited IGF-I expression or transfected OVA encoding gene. It was shown that inhibiting IGF-I expression or inserting OVA encoding gene did not lead to modification relevant to the presence of MHC-I or B7.1. However downregulation of tetraspanin CD9 was observed in modified IGF-I but not in OVA encoding gene inserted melanoma cells. Expression of tetraspanin CD81 and integrin alpha4/CD49d remained unchanged. Inoculated into syngeneic recipients, the modified melanoma cells exhibited significant delayed outgrowth with a reduction in the percentage of lethal tumors observed essentially in hosts injected with inhibited IGF-I expression cells.

Purpose: Lipopolysaccharide (LPS) may act as a key stimulatory agent in ocular surface diseases (OSDs) through TNF-alpha release. We used in vivo confocal microscopy (CM) and ex vivo flow cytometry, two new tools for assessing ocular inflammation induced by LPS., Methods: We investigated a model of acute inflammation in rabbits by subconjunctival injection of LPS and developed new evaluation techniques for animal models: CM, to observe inflammatory infiltrates, and conjunctival impression cytology (IC) specimens processed with in vitro CM and flow cytometry for assessing TNF-alpha and TNF receptor-1 (TNFR-1) expression. A neutralizing anti-TNF-alpha antibody was used to assess the role of TNF-alpha., Results: In vivo CM provided high-resolution images of inflammatory infiltrates and leukocyte rolling in blood vessels. It showed that the LPS group presented strong conjunctival inflammation, reaching its maximum level 4 h after injection. Flow cytometry and immunostaining in IC specimens showed an increased expression of TNF-alpha and TNFR-1 in the epithelium. Immunohistology confirmed these results and showed infiltration of vimentin+, CD4(+), and CD8(+) cells in the conjunctiva. TUNEL-positive cells were found 4 h after injection. Neutralizing anti-TNF-alpha significantly inhibited LPS-induced inflammation and apoptosis evaluated by in vivo CM; and inhibited LPS-induced TNF-alpha and TNFR-1 expression by ex vivo conjunctival IC specimens evaluated by flow cytometry., Conclusions: IC specimens and new-generation in vivo CM were thus in good agreement with immunohistology and appeared to be reliable, effective, and nonharmful methods to investigate experimental models of OSDs. The two new tools applied here evaluate the animal models in vivo on the cellular lever. This study is consistent with the experimental research's strategy by reducing the number of experimental animals used.

Introduction: Following material vigilance cases encountered with the hydrophilic acrylic intraocular lens, ACR6D SE preloaded in the Premier shooter, we studied the cytotoxicity of the intraocular lens and its conditioning to identify the cytotoxic element. We proposed medical device modification to improve its biocompatibility., Materials and Methods: Biocompatibility-cytotoxicity assays were carried out according to ISO 10993-5 recommendations. Tests were performed on the SRA 01/04 human lens epithelial cell line. Neutral red, Hoechst 33342, and YO-PRO-1 fluorescent probes were used to assess membrane integrity, total DNA, and membrane fluidity, respectively. Materials samples were prepared in culture medium according to the ISO 10993-5 elution procedure. Pure saline solutions and conditioning liquids were tested directly on cells., Results: The intraocular lens and injector were not cytotoxic. Conditioning liquids induced membrane fluidity perturbation characteristic of apoptosis. Tests performed on new versions of the medical device identified a better adapted conditioning liquid., Conclusion: The results suggest that the cytotoxicity of the conditioning liquid could explain the postoperative complication rate. When we changed the conditioning liquid with sterile irrigating solution (i.e., rich divalent cation marine solution), we eliminated cellular stress. Fluorescent probes are well adapted to assess medical device biocompatibility-cytotoxicity.

Purpose: The aim of this study was to compare, in vivo on a rat model, two different preservatives- benzalkonium chloride (BAC) and polyquaternium-1 (PQ-1)-using new experimental approaches., Methods: Thirty (30) eyes of 15 male Lewis rats were used in this study. Rats were randomly divided into five groups instilled twice a day for 11 days with eye drops containing different concentrations of preservatives, 0.1% BAC, 0.5% BAC, 0.1% PQ-1, 0.5% PQ-1, and balanced salt solution (BSS) as a control. The ocular surface toxicity of these two preservatives was investigated using new in vivo experimental approaches. Slit-lamp examination, the fluorescein test, the red phenol test, impression cytology, and in vivo corneal confocal microscopy were used to evaluate the rat ocular surface after preservative instillation. Histology sections and immunohistochemistry were also examined to confirm these results., Results: Compared to PQ-1, BAC consistently and dramatically altered the corneoconjunctival surface as evaluated by slit-lamp examination, the fluorescein test, impression cytology, in vivo confocal microscopy, and histology. The 0.5% BAC solution also significantly decreased tear production compared to the control. Although 0.5% PQ-1 significantly decreased goblet cell density in comparison to the control, and some abnormalities were observed with in vivo confocal microscopy, no statistically significant differences were observed between these two groups using the tear production test, slit-lamp and fluorescein evaluation, or histology., Conclusion: Using an acute rat model of ocular toxicity by comparing preservatives at high concentrations, we demonstrated in vivo that high doses of PQ-1 were much less toxic than BAC. In vivo confocal microscopy and impression cytology are new promising experimental approaches to studying the rat corneoconjunctival surface, particularly in the field of ocular surface toxicity.

Purpose: To investigate in vitro whether eye toxicity is attributable to the preservative or the fluoroquinolone used in ophthalmic formulations., Methods: Corneal and conjunctival cell lines were incubated with preserved (benzalkonium chloride [BAC]) or unpreserved ofloxacin solutions for 15 minutes. Several concentrations of BAC were also tested (0.0025%-0.01%). Membrane integrity, reactive oxygen species, and superoxide anion production were assessed with the neutral red test, the 2',7'-dichlorofluorescein diacetate test, and the dihydroethidium test, respectively. P2X7 cell death receptor activation was evaluated using the YO-PRO-1 assay and apoptosis (chromatin condensation and translocation of phosphatidylserine) using the Hoechst 33342 and annexin V-FITC dyes. Tests were performed with microplate cytofluorometry, inverted fluorescence microscopy, and flow cytometry., Results: The preserved solution and all tested BAC concentrations induced a significant decrease in membrane integrity, unlike the unpreserved ofloxacin. Reactive oxygen species and superoxide anion productions observed for all solutions were significantly higher for the preserved ofloxacin and BAC solutions, which also induced apoptosis (chromatin condensation and translocation of phosphatidylserine) through P2X7 pore opening, whereas unpreserved ofloxacin did not., Conclusions: The cytotoxicity observed with fluoroquinolone eye drops seems to be caused mainly by the preservative, which induced P2X7 cell death receptor activation associated with oxidative stress and apoptosis. Therefore, it is recommended that fluoroquinolone be used in preservative-free formulations.

The age-related difference in fluoroquinolone-induced tendon toxicity was investigated. In vitro tendon cells from juvenile and young adult rabbits, respectively, were incubated with quinolone (nalidixic acid, NA) or fluoroquinolone (ofloxacin, OFX or pefloxacin, PEF) at 0.01 microM to 1 mM for 72 h. Redox status, glutathione (GSH), reactive oxygen species (ROS), and mitochondrial activity were assessed using intracellular fluorescent probes. Fluorescence signal was detected on living adherent tenocytes in microplates using cold-light cytofluorometry. Tendon toxicity differed significantly between the two cell groups and the difference was greatest with highest dose (1 mM). For 72 h, significant (p < 0.001) differences between immature and young adult primary tenocytes were observed for redox status decrease, GSH decrease, and ROS production increase. Mitochondrial activity remained unaltered in immature tenocytes. We confirm two groups of intrinsic tendon toxicity (OFX/NA vs. PEF) associated to oxidative stress (GSH decrease). Our in vitro experimental model confirms the clinical observations of age dependent tenotoxicity. First group (NA, OFX) showed greater intrinsic tenotoxicity for young adult than immature tenocytes, second group (PEF) was highly toxic for immature and young adult cells. The three quinolones do not altered mitochondrial activity in immature tenocytes whereas alteration was observed in young adult tenocytes.

Purpose: The aim of the current study was to compare the corneas of three commonly used laboratory animals with a new in vivo confocal microscope., Methods: Six eyes of three adult male New Zealand albino rabbits, six eyes of three adult male Lewis rats, and six eyes of three adult male Swiss mice were used in this study. Corneas were analyzed in vivo using the Rostock Cornea Module of the Heidelberg Retina Tomograph (HRT)-II. For all eyes, 20 confocal microscopic images of each layer, that is, the superficial and basal corneal epithelia, the Bowman layer, the anterior and posterior stroma, and the endothelium, were recorded. The images were then analyzed qualitatively and compared among animals. Cellular densities of anterior and posterior stroma keratocytes of rabbits and endothelium density of the three different animals were also measured and compared., Results: The Rostock Cornea Module of the HRT II was successfully used to analyze all corneal layers of these three commonly used laboratory animals. Although the cellular patterns of the corneal layers of these three animals, as observed with in vivo confocal microscopy, were quite similar, some differences were seen in terms of endothelial cell density and stroma appearance. Superficial cells were seen as hyper- and hyporeflective polygonal cells. Basal cells had dark cytoplasm without visible nuclei and were closely organized. A Bowman layer was observed in all three animals as an amorphous tissue containing fine subepithelial nerve plexus. In rabbits, the stroma consisted of an amorphous ground substance with hyper-reflective structures corresponding with keratocyte nuclei. In rats and mice, numerous reflective stellate structures with no clearly visible nuclei were observed within the stroma. Besides endothelial cell density, the endothelium was similar among the three animals and was seen as hyper-reflective cells with dark limits organized in a honeycomb pattern., Conclusions: The Rostock Cornea Module of the HRT II can provide high-resolution images of all corneal layers of rabbits, rats, and mice without sacrificing animals or preparing tissue. This new device may be useful for evaluating the cornea during experimental animal studies.

Introduction: Cyclosporine is a molecule used in ophthalmology for the prevention of corneal graft rejection. The systemic use of this product can lead to serious adverse side effects that can be avoided using the topical formulation of cyclosporine. However, cyclosporine application can induce ocular irritation., Material and Methods: The aim of this study is to evaluate the cytotoxicity of four formulations of 2% cyclosporine eye drops: Sandimmum intravenous solution diluted with NaCl 0.9%, Sandimmun oral solution diluted in castor oil or corn oil after ethanol evaporation, and Sandimmun oral solution diluted in castor oil without previous ethanol evaporation. Two tests--the Draize test and the evaluation of cytotoxicity of adherent alive cells with cold light cytofluorimetry on microplates--were used in this study., Results: These tests demonstrated that the aqueous solution shows more toxicity than the other formulations, and the type of oil and ethanol concentration influence cell viability., Conclusion: These results helped the Pharmacy unit choose the vehicles for a safe cyclosporine eye drop formulation and thus decrease the side effects of cyclosporine eye drop instillation with a decrease in ethanol concentration compared to published formulations.

Purpose: The aim of this work was to study, using flow cytometry, the expression of two chemokine receptors, CCR4 and CCR5, known to be related to the TH2 and TH1 systems, respectively, and the expression of HLA-DR, a hallmark of inflammation, on conjunctival impression cytology specimens (ICS) of glaucomatous patients treated over the long term., Patients and Methods: In this case-control study, ICS were taken in a series of 35 glaucomatous patients treated with topical antiglaucoma drugs for more than 1 year (seven with beta-blockers, ten with prostaglandins, and 18 receiving multiple treatments), and 20 normal subjects. Conjunctival cells were collected and incubated with specific monoclonal antibodies directed against CCR4, CCR5, and HLA-DR, in order to measure, in a masked manner using flow cytometry, the percentage of cells positive to each marker in the conjunctival epithelium., Results: Compared to normal subjects, HLA DR expression was significantly elevated in glaucomatous patients, with a tendency toward higher levels in the multitreatment group and lower levels in patients treated with prostaglandins, which did not differ significantly from control values. Both CCR4 and CCR5 significantly increased in glaucoma patients on multitreatment or monotherapy compared with normal subjects., Conclusion: This study demonstrates the overexpression of these two chemokine receptors in the conjunctival epithelium of patients treated for more than 1 year. Our results showing the simultaneous overexpression of CCR4 and CCR5 thus suggest that the chronic use of topical treatments may concurrently stimulate the TH1 and TH2 systems. These results evoke inflammatory mechanisms, combining allergy and toxicity, and confirm the complexity of inflammatory reactions occurring in the ocular surface of glaucoma patients.

Introduction: To evaluate the tolerance and efficacy of heavy silicone oil as internal tamponade for retinal detachment surgery., Patients and Methods: Sixty-six eyes requiring heavy silicone oil for retinal detachment, with at least 1 month follow-up, were retrospectively studied. Preoperative status, surgical technique, tolerance, and anatomical and functional results were analyzed from the patient's file. Indications for heavy silicone injection were inferior retinotomy or inferior retraction in 65% of cases. PVR grade C was present in at least 63% of cases. Retinotomy was performed in 45% of cases. An exchange procedure was performed versus DKline in 65% of cases. Mean follow-up was 7 +/- 4 months., Results: At the end of follow-up, 59% of eyes had a completely reattached retina, 32% without internal tamponade. Another surgery was necessary in 54% of cases. During follow-up, mean intraocular pressure was normal, and there was a significant intraocular inflammation in three cases (4.5%). In seven cases of the 44 ablations of heavy silicone oil, an adherence of residual bubbles was present. Redetachment occurred after ablation for anatomical success in 41% of cases. BCVA was better than 0.05 (20/400) in 54% of cases at the end of follow-up., Conclusion: Heavy silicone was well tolerated and seems not to be pro-inflammatory in our study. It is a good alternative to standard silicone for inferior retinotomy and inferior breaks without PVR. It is not a treatment of inferior retraction, and is not a long-term internal tamponade. During the ablation of heavy silicone oil, adherence of residual bubbles is possible, in which case a coaxial light or an endoillumination could be needed during ablation.

Purpose: In a previous study, it was demonstrated that in vitro in a human conjunctiva-derived cell line, latanoprost in its commercial presentation appeared to be less toxic than the benzalkonium chloride (BAC) it contains as a preservative. Through a microplate cytometry technique, the investigation was furthered by study of whether the three commercially available antiglaucoma prostaglandin analogs could protect the same cell line in vitro against BAC toxicity and whether an antioxidative mechanism could be involved in such prostaglandin effects., Methods: Human conjunctiva-derived epithelial cells from the Chang cell line were exposed to three prostaglandins in their commercial presentation (latanoprost, travoprost, and bimatoprost) and to three concentrations of BAC (0.02%, 0.015%, and 0.005%), corresponding to the concentrations contained in the three prostaglandin eyedrops. Each solution was diluted to 1/10 and was applied for 30 minutes Cellular membrane integrity, cytosolic H2O2, cytosolic O2*- and apoptosis were evaluated using neutral red, H2DCF-DA, hydroethidine, and Yopro-1 probes, respectively., Results: Cellular viability decreased as BAC concentration increased, but it was accompanied by concentration-dependent toxicity. Toxicity of latanoprost and travoprost commercial solutions was statistically significantly lower than their respective BAC concentrations (P < 0.01), whereas bimatoprost induced no significant effects. There was a statistically significant decrease in H2O2 detection with cells exposed to latanoprost (P < 0.01) and travoprost (P < 0.01) and a lower detection of O2*- with cells exposed to latanoprost (P < 0.01) compared with the corresponding BAC concentration alone. The Yopro-1 test showed a BAC-induced apoptotic effect that increased with its concentration. Latanoprost and travoprost produced proapoptotic effects compared with control (P < 0.01), but these were lower than their respective preservative concentrations (statistically significant difference; P < 0.01)., Conclusions: Latanoprost and travoprost were responsible for significant protective effects against BAC toxicity on conjunctiva-derived epithelial cells in vitro, probably related to their antioxidative properties. The low toxicity of the bimatoprost solution did not reveal a possible antioxidative effect. Reduced reactive oxygen species production could be the main mechanism by which prostaglandin analogs protect epithelial cells from the proapoptotic effects of BAC. Further studies will be useful to confirm this hypothesis.