Expanded biochemical analyses of human tear fluid: Polyvalent faces of the schirmer strip.

The tear film forms a protective barrier between the ocular surface and the external environment. Despite its small volume, recent advancements in preanalytical and analytical procedures have enabled its in-depth analysis using multiple approaches. However, the diversity of tear film collection methods and the lack of standardization in pre-analytical methods represent the main obstacles to reproducible results and comparison among different studies. In this study, we first improved the pre-analytical procedures for the extraction of various molecular entities from Schirmer strips (ScS). Subsequently, our investigation focused on analyzing the molecular variances that might occur between two primary tear collection methods: capillary tube (CT) and ScS. Additionally, we examined different parts of the ScS to underscore these variations, which could serve as crucial factors for developing a standardized, optimized protocol for sample processing. Our results show that the inclusion of surfactants in the extraction process enhanced both the yield of protein extraction and the number of proteins identified in ScS, by effectively lysing the cells and improving the solubility of several intracellular proteins. In addition to proteins, nucleic acids could also be recovered for gene expression analyses, particularly from the bulb region of the ScS which is placed in the cul-de-sac. Despite their diluted nature, extracts from ScS remain a suitable material for retrieving tear proteins such as IL-17A at levels as low as the fg/mL range, thanks to highly sensitive immunoassays. Collection methods can affect measured tear protein levels. Lactoferrin is found in higher percentages in capillary electrophoresis analysis of tears collected using ScS compared to tears collected by CT (39.6 ± 4.8% versus 31 ± 4.4%).

Evaluation of neuroprotective and immunomodulatory properties of mesenchymal stem cells in an ex vivo retinal explant model.

BACKGROUND: Glaucoma is a blinding degenerative neuropathy in which the death of retinal ganglion cells (RGCs) causes progressive loss of visual field and eventually vision. Neuroinflammation appears to be a key event in the progression and spread of this disease. Thus, microglial immunomodulation represents a promising therapeutic approach in which mesenchymal stem cells (MSCs) might play a crucial role. Their neuroprotective and regenerative potentials have already raised hope in animal models. Yet no definitive treatment has been developed, and some safety concerns have been reported in human trials. In the present study, we investigated the neuroprotective and immunomodulatory properties as well as the safety of MSCs in an ex vivo neuroretina explant model. METHODS: Labeled rat bone marrow MSCs were placed in coculture with rat retinal explants after optic nerve axotomy. We analyzed the neuroprotective effect of MSCs on RGC survival by immunofluorescence using RBPMS, Brn3a, and NeuN markers. Gliosis and retinal microglial activation were measured by using GFAP, CD68, and ITGAM mRNA quantification and GFAP, CD68, and Iba1 immunofluorescence stainings. We also analyzed the mRNA expression of both 'M1' or classically activated state inflammatory cytokines (TNFα, IL1ß, and IL6), and 'M2' or alternatively activated state microglial markers (Arginase 1, IL10, CD163, and TNFAIP6). RESULTS: The number of RGCs was significantly higher in retinal explants cultured with MSCs compared to the control group at Day 7 following the optic nerve axotomy. Retinal explants cultured with MSCs showed a decrease in mRNA markers of gliosis and microglial activations, and immunostainings revealed that GFAP, Iba1, and CD68 were limited to the inner layers of the retina compared to controls in which microglial activation was observed throughout the retina. In addition, MSCs inhibited the M1 phenotype of the microglia. However, edema of the explants was observed in presence of MSCs, with an increase in fibronectin labeling at the surface of the explant corresponding to an epiretinal membrane-like phenotype. CONCLUSION: Using an ex vivo neuroretina model, we demonstrated a neuroprotective and immunomodulatory effect of MSCs on RGCs. Unfortunately, the presence of MSCs also led to explant edema and epiretinal membrane formation, as described in human trials. Using the MSC secretome might offer the beneficial effects of MSCs without their potential adverse effects, through paracrine signaling.

An overview of current alternative models in the context of ocular surface toxicity.

The 21st century has seen a steadily increasing social awareness of animal suffering, with increased attention to ethical considerations. Developing new integrated approaches to testing and assessment (IATA) strategies is an Organisation for Economic Co-operation and Development (OECD) goal to reduce animal testing. Currently, there is a lack of alternative models to test for ocular surface toxicity (aside from irritation) in lieu of the Draize eye irritation test (OECD guideline No. 405) performed in rabbits. Five alternative in vitro or ex vivo methods have been validated to replace this reference test, but only in combination. However, pathologies like Toxicity-Induced Dry Eye (TIDE), cataract, glaucoma, and neuropathic pain can occur after exposure to a pharmaceutical product or chemical and therefore need to be anticipated. To do so, new models of lacrimal glands, lens, and neurons innervating epithelia are required. These models must take into account real-life exposure (dose, time, and tear film clearance). The scientific community is working hard to develop new, robust, alternative, in silico, and in vitro models, while attempting to balance ethics and availability of biological materials. This review provides a broad overview of the validated methods for analyzing ocular irritation and those still used by some industries, as well as promising models that need to be optimized according to the OECD. Finally, we give an overview of recently developed innovative models, which could become new tools in the evaluation of ocular surface toxicity within the scope of IATAs.

Hidden in the Eyes-Recurrence of Systemic Hemopathies Reportedly "In Remission": Six Cases and Review of Literature.

Background and Objectives: Secondary ocular localizations of hematological malignancies are blinding conditions with a poor prognosis, and often result in a delay in the diagnosis. Materials and Methods: We describe a series of rare cases of ocular involvement in six patients with hematological malignancies, reportedly in remission, who presented secondary ocular localizations, challenging to diagnose. Two patients had an acute lymphoblastic leukemia (ALL) and developed either a posterior scleritis or a pseudo-panuveitis with ciliary process infiltration. One patient had iris plasmacytoma and developed an anterior uveitis as a secondary presentation. Two patients had a current systemic diffuse large B-cell lymphoma (DLBCL) and were referred either for intermediate uveitis or for papilledema and vitritis with secondary retinitis. Finally, one patient with an acute myeloid leukemia (AML) presented a conjunctival localization of a myeloid sarcoma. We herein summarize the current knowledge of ophthalmologic manifestations of extramedullary hematopathies. Results: Inflammatory signs were associated with symptomatic infiltrative lesions well displayed in either the iris, the retina, the choroid, or the cavernous sinus, from the admission of the patients in the ophthalmological department. These findings suggest that patients with ALL, AML, systemic DLBCL, and myeloma can present with ophthalmic involvement, even after having been reported as in remission following an effective systemic treatment and/or allograft. Conclusions: Early detection of hidden recurrence in the eyes may permit effective treatment. Furthermore, oncologists and ophthalmologists should be aware of those rare ocular malignant locations when monitoring patient's progression after initial treatment, and close ophthalmologic examinations should be recommended when detecting patient's ocular symptoms after treatment.

Deepening of lipidome annotation by associating cross-metathesis reaction with mass spectrometry: application to an in vitro model of corneal toxicity.

The in-depth knowledge of lipid biological functions needs a comprehensive structural annotation including a method to locate fatty acid unsaturations, which remains a thorny problem. For this purpose, we have associated Grubbs' cross-metathesis reaction and liquid chromatography hyphenated to tandem mass spectrometry to locate double bond positions in lipid species. The pretreatment of lipid-containing samples by Grubbs' catalyst and an appropriate alkene generates substituted lipids through cross-metathesis reaction under mild, chemoselective, and reproducible conditions. A systematic LC-MS/MS analysis of the reaction mixture allows locating unambiguously the double bonds in fatty acid side chains of phospholipids, glycerolipids, and sphingolipids. This method has been successfully applied at a nanomole scale to commercial standard mixtures consisting of 10 lipid subclasses as well as in lipid extracts of human corneal epithelial (HCE) cell line allowing to pinpoint double bond of more than 90 species. This method has also been useful to investigate the lipid homeostasis alteration in an in vitro model of corneal toxicity, i.e., HCE cells incubated with benzalkonium chloride. The association of cross-metathesis and tandem mass spectrometry appears suitable to locate double bond positions in lipids involved in relevant biological processes.

AOP and IATA applied to ocular surface toxicity.

Until now, the Draize test on rabbits has been the only test performed to anticipate ocular toxicity of pharmaceutical compounds, mainly irritation. The OECD is urging the scientific community to develop and validate alternative methods to reduce the need for animal testing. Since the models and tests used cannot reflect the entire biologic response, it is necessary to combine them into integrated approaches to testing and assessment (IATA) to obtain robust data. IATAs, along with adverse outcome pathways (AOP) that encompass molecular cascades and key events, require the best combinations of tests. This commentary manuscript describes these OECD tools and proposes original approaches for ocular surface AOP and an IATA for toxicity-induced dry eye (TIDE).

Comparison of Two Experimental Mouse Dry Eye Models through Inflammatory Gene Set Enrichment Analysis Based on a Multiplexed Transcriptomic Approach.

The goal of this study was to explore the specific signaling pathways related to inflammation in two experimental mouse dry eye (EDE) models. Female C57BL/6 mice housed for 10 days in a controlled desiccative environment were either treated with scopolamine (EDE-1; n = 18) or subjected to extraorbital lacrimal gland excision bilaterally (EDE-2; n = 10). Non-induced mice (n = 20) served as healthy controls. A corneal fluorescein staining (CFS) scoring was used at baseline through to day (D) 10 to evaluate epitheliopathy. At D10, corneas and conjunctivas were collected for multiplexed transcriptomic analysis with the NanoString® mouse inflammatory CodeSet. Both EDE-1 and EDE-2 mice presented a change in corneal integrity, with a significant increase in CFS scores at D10. More gene transcripts were identified in EDE-2 compared with EDE-1 (116 vs. 96, respectively), and only a few were common to both models, 13 for the cornea and 6 for the conjunctiva. The gene functional annotation analysis revealed that the same inflammatory pathways were involved in both models. Comparative profiling of gene expression in the two EDE models leads to the identification of various targets and signaling pathways, which can be extrapolated to and confirmed in human disease.

Moraxella keratitis: epidemiology and outcomes.

We aimed to assess the prevalence, risk factors, and visual outcome of Moraxella keratitis. We retrospectively reviewed the medical charts of patients diagnosed with Moraxella spp. keratitis at the Quinze-Vingts National Ophthalmology Hospital, Paris, France, between January 2016 and December 2018. Definitive microbiological identification was performed on archival strains using matrix-assisted laser desorption ionization time of flight coupled to mass spectrometry. One hundred one culture-proven cases of Moraxella keratitis were identified. The most common isolates were Moraxella lacunata (50%) and Moraxella nonliquefasciens (38%). Systemic predisposing factors, principally diabetes mellitus (13%) were identified in 28% of patients, and 87% of patients had ocular surface conditions, including blepharitis (25%), prior ocular surgery (21%), glaucoma (17%), exposure keratopathy (16%), and trauma (16%). Severely affected inpatients were treated empirically with fortified antibiotics including vancomycin, piperacillin, and gentamicin. The presence of hypopyon and being over the age of 60 years were associated with a poorer final visual acuity (p < 0.05). Adjuvant treatment, mostly amniotic membrane transplantation, was required for 31 eyes. The prognostic factors significantly associated with the need for adjuvant treatment were a larger infiltrate and hypoesthesia. The clinical features including ulcer healing, treatment duration, and infiltrate size were not different between Moraxella species. Keratitis caused by Moraxella spp. are rare in France but may threaten sight. The early identification of patients with a poor ocular surface, particularly those with neurotrophic keratopathy and anesthetic cornea, is crucial to prevent delayed healing of ulcers and the need for adjuvant treatment.

Multimodal imaging of Hurler syndrome-related keratopathy treated with deep anterior lamellar keratoplasty.

BACKGROUND: Hurler syndrome-associated keratopathy is an exceedingly rare corneal disorder that requires corneal transplantation in advanced stages. Precise assessment of the corneal condition is necessary for deciding which type of keratoplasty (i.e., deep anterior lamellar or penetrating) should be proposed. We aimed to confront the results of multimodal imaging with those of histology in a case of Hurler syndrome-associated keratopathy. CASE PRESENTATION: A 16-year-old patient with Hurler's syndrome treated with hematopoietic stem cell transplantation was referred for decreased vision related to advanced keratopathy. The patient was treated with deep anterior lamellar keratoplasty (DALK) in both eyes with uncomplicated outcome. Visual acuity improved from 0.1 (20/200) preoperatively to 0.32 (20/63) and 0.63 (20/32) after transplantation. The corneal endothelial cell density was 2400 cells/mm2 in both eyes 3 years after transplantation. In vivo confocal microscopy (IVCM) and spectral domain optical coherence tomography (SD-OCT) were performed preoperatively. The corneal buttons retrieved during keratoplasty were processed for histology. In SD-OCT scans, corneal opacities appeared as diffuse stromal hyperreflectivity associated with increased corneal thickness. IVCM showed diffuse cytoplasmic granular hyperreflectivity and rounded/ellipsoid aspects of keratocytes, presence of small intracellular vacuoles, and hyperreflective epithelial intercellular spaces. Bowman's layer was thin and irregular. The corneal endothelium was poorly visualized but no endothelial damage was observed. Histology showed irregular orientation and organization of stromal lamellae, with the presence of macrophages whose cytoplasm appeared clear and granular. A perinuclear clear halo was visible within the epithelial basal cells. Bowman's layer featured breaks and irregularities. CONCLUSIONS: The observed corneal multimodal imaging features in mucopolysaccharidosis-related keratopathy were concordant with histology. Compared with standard histology, multimodal imaging allowed additional keratocyte features to be observed. It revealed both morphological and structural changes of all corneal layers but the endothelium. This information is essential for therapeutic management which should include DALK as the first-choice treatment in case of impaired visual acuity.

Proinflammatory Markers, Chemokines, and Enkephalin in Patients Suffering from Dry Eye Disease.

Dry eye symptoms are among the leading complaints in ophthalmology. Dry eye disease (DED) is associated with significant pain affecting quality of life. Cellular and molecular mechanisms underlying ocular pain associated with DED are not fully understood. In this study, we investigated the ocular surface of patients with DED using in vivo confocal microscopy (IVCM) to quantify corneal nerve density and its relation with corneal inflammation. Gene expression of the proinflammatory markers HLA-DR, IL-6, CXCL12, and CCL2 and the receptors CXCR4 and CCR2, as well as PENK (enkephalin precursor), was therefore quantified in conjunctival impression cytology specimens. Thirty-two patients with DED and 15 age-matched controls were included. Subbasal nerve density was significantly lower in DED patients compared to controls. IVCM analysis revealed that DED patients had a significantly higher corneal dendritic cell density compared to controls. Conjunctival impression cytology analysis revealed that HLA-DR, IL-6, CXCR4, and CCL2/CCR2 mRNA levels were significantly increased in DED patients compared to controls, whereas PENK mRNA levels were significantly decreased. Similar results were obtained in vitro on immortalized human conjunctiva-derived epithelial cells challenged with osmotic stress that mimics the DED condition. These results demonstrate that proinflammatory molecules and endogenous enkephalin have opposite gene regulation during DED.

Hyperosmolarity and Benzalkonium Chloride Differently Stimulate Inflammatory Markers in Conjunctiva-Derived Epithelial Cells in vitro.

Tear hyperosmolarity is known to cause ocular surface inflammation in dry eye syndrome. Benzalkonium chloride (BAK), an eyedrop preservative, is known to induce dry eye in long-term-treated patients. Analyzing the modulation of the proinflammatory potential of hyperosmolarity in the presence of BAK on the conjunctiva could give new insights into the effect of this preservative on the disease. In a hyperosmolar model on a conjunctiva-derived cell line, and in the presence of BAK, we evaluated key inflammatory markers [CCL2, IL-8, IL-6, macrophage migration inhibitory factor (MIF) and intercellular adhesion molecule (ICAM)-1] as well as the osmoprotectant element nuclear factor of activated T cells (NFAT)5 using ELISA, RT-qPCR or immunofluorescence staining. Hyperosmolarity highly stimulated CCL2 and NFAT5 in these cells. BAK alone only increased IL-6 expression. The stress-combined condition stimulated CCL2, NFAT5, MIF and IL-8 secretion. ICAM-1 was not modulated by any of the conditions tested. In this model, hyperosmolarity and BAK induced the release of different proinflammatory mediators, and, when combined, they lead to the release of additional inflammatory cytokines. This in vitro study highlights the importance of avoiding long-term ophthalmic treatments containing BAK, as tear film hyperosmolarity can be a result of its detergent action.

Predisposing factors, clinical and microbiological insights of bacterial keratitis: analysis of 354 cases from a leading French academic centre.

AIMS: To report an epidemiological update of bacterial keratitis (BK) in a tertiary ophthalmology centre over 20 months compared with a previous study on the same timeframe from 1998 to 1999. METHODS: 354 patients with BK documented by microbiological corneal scraping or resolutive under antibiotics treatment from January 2020 to September 2021 were analysed retrospectively. RESULTS: One or several risk factors were found in 95.2% of patients: contact lens wear (45.2%), ocular surface disease (25.0%), systemic disease (21.8%), ocular trauma (11.9%) and ocular surgery (8.8%). The positivity rate of corneal scrapings was 82.5%, with 18.2% polybacterial. One hundred seventy-five (59.9%) bacteria were Gram-negative, and 117 (40.1%) were Gram-positive. The most common bacteria were Pseudomonas aeruginosa (32.5%), Moraxella spp (18.1%) and Staphylococcus aureus (8.2%). Final visual acuity (logarithm of the minimum angle of resolution) was associated with age (r=+0.48; p=0.0001), infiltrate size (r=+0.32; p<0.0001), ocular surface disease (r=+0.13; p=0.03), ocular trauma (r=-0.14; p=0.02) and contact lens wear (r=-0.26; p<0.0001). Gram-negative bacteria were responsible for deeper (r=+0.18; p=0.004) and more extensive infiltrates (r=+0.18; p=0.004) in younger patients (r=-0.19; p=0.003). Compared with the previous period, the positivity rate of corneal scrapings and the proportion of Gram-negative bacteria, especially Moraxella spp, increased. All P. aeruginosa and Moraxella spp were sensitive to quinolones, and all S. aureus were sensitive to both quinolones and methicillin. CONCLUSION: Contact lens wear remained the leading risk factor. The bacteria distribution was reversed, with a predominance of Gram-negative bacteria and increased Moraxella spp.

Localisation and quantification of benzalkonium chloride in eye tissue by TOF-SIMS imaging and liquid chromatography mass spectrometry.

Benzalkonium (BAK) chloride is the most commonly used preservative in eye drops. It is generally composed of benzyldimethyldodecylammonium C12 and benzyldimethyltetradecylammonium C14 and is supposed to increase penetration of active compounds. However, numerous studies have reported its toxic effect to ocular surface especially in long-term treatments like against glaucoma, a sight-threatening disease. Albino rabbits were treated with a hyperosmolar solution and a high concentration of BAK solution for 1 month. Enucleated eyes were cryo-sectioned and analysed by mass spectrometry. Mass spectrometry imaging using time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been used to characterize the spatial distribution and to determine the relative quantity of BAK at the surface of rabbit eye sections. Liquid chromatography coupled with mass spectrometry (LC-MS) using a hybrid linear ion trap-Orbitrap® mass spectrometer was used to obtain relative quantification of BAK at the sample surface. TOF-SIMS images of BAK ions indicated a distribution at the ocular surface and in deeper structures. Didecyldimethylammonium (DDMAC), which is used in hospitals as a substitute for BAK, was also detected and showed an accumulation around the eyes. After extraction with acetonitrile and chromatographic separation using a Gemini C18 column and an original elution gradient, the relative quantities of BAK and DDMAC present in the whole eye section surface were determined. This LC-MS method was validated in terms of limits of quantification, linearity, repeatability and reproducibility and its feasibility was evaluated in surgically obtained human samples. Specimens of iris, lens capsule or trabecular meshwork were found with significant levels of BAK and DDMAC, thus confirming the penetration of BAK in deep ocular structures, with potential deleterious effects induced by this cytotoxic compound. The analytical method developed here could therefore be of primary interest in the field of pharmaco-toxicology in order to localise, identify and quantify drugs or xenobiotic compounds present at biological sample surfaces.

An Overview of Current Glaucomatous Trabecular Meshwork Models.

PURPOSE: To provide an overview of the existing alternative models for studying trabecular meshwork (TM). METHODS: Literature review. RESULTS: The TM is a complex tissue that regulates aqueous humor outflow from the eye. Dysfunction of the TM is a major contributor to the pathogenesis of open-angle glaucoma, a leading cause of irreversible blindness worldwide. The TM is a porous structure composed of trabecular meshwork cells (TMC) within a multi-layered extracellular matrix (ECM). Although dysregulation of the outflow throughout the TM represents the first step in the disease process, the underlying mechanisms of TM degeneration associate cell loss and accumulation of ECM, but remain incompletely understood, and drugs targeting the TM are limited. Therefore, experimental models of glaucomatous trabeculopathy are necessary for preclinical screening, to advance research on this disease's pathophysiology, and to develop new therapeutic strategies targeting the TM. Traditional animal models have been used extensively, albeit with inherent limitations, including ethical concerns and limited translatability to humans. Consequently, there has been an increasing focus on developing alternative in vitro models to study the TM. Recent advancements in three-dimensional cell culture and tissue engineering are still in their early stages and do not yet fully reflect the complexity of the outflow pathway. However, they have shown promise in reducing reliance on animal experimentation in certain aspects of glaucoma research. CONCLUSION: This review provides an overview of the existing alternative models for studying TM and their potential for advancing research on the pathophysiology of open-angle glaucoma and developing new therapeutic strategies.

Profiling tear film enzymes reveals major metabolic pathways involved in the homeostasis of the ocular surface.

The ocular surface (OS) enzymes are of great interest due to their potential for novel ocular drug development. We aimed first to profile and classify the enzymes of the OS to describe major biological processes and pathways that are involved in the maintenance of homeostasis. Second, we aimed to compare the enzymatic profiles between the two most common tear collection methods, capillary tubes (CT) and Schirmer strips (ScS). A comprehensive tear proteomic dataset was generated by pooling all enzymes identified from nine tear proteomic analyses of healthy subjects using mass spectrometry. In these studies, tear fluid was collected using CT (n = 4), ScS (n = 4) or both collection methods (n = 1). Classification and functional analysis of the enzymes was performed using a combination of bioinformatic tools. The dataset generated identified 1010 enzymes. The most representative classes were hydrolases (EC 3) and transferases (EC 2). Phosphotransferases, esterases and peptidases were the most represented subclasses. A large portion of the identified enzymes was common to both collection methods (n = 499). More enzymes were specifically detected in the ScS-extracted proteome. The major pathways in which the identified enzymes participate are related to the immune system and protein, carbohydrate and lipid metabolism. Metabolic processes for nucleosides, cellular amides, sugars and sulfur compounds constituted the most enriched biological processes. Knowledge of these molecules highly susceptible to pharmacological manipulation might help to predict the metabolism of ophthalmic medications and develop novel prodrug strategies as well as new drug delivery systems. Combining such extensive knowledge of the OS enzymes with new analytical approaches and techniques might create new prospects for understanding, predicting and manipulating the metabolism of ocular pharmaceuticals. Our study reports new, essential data on OS enzymes while also comparing the enzyme profiles obtained via the two most popular methods of tear collection, capillary tubes and Schirmer strips.

Corneal neuroepithelial compartmentalized microfluidic chip model for evaluation of toxicity-induced dry eye.

Part of the lacrimal functional unit, the cornea protects the ocular surface from numerous environmental aggressions and xenobiotics. Toxicological evaluation of compounds remains a challenge due to complex interactions between corneal nerve endings and epithelial cells. To this day, models do not integrate the physiological specificity of corneal nerve endings and are insufficient for the detection of low toxic effects essential to anticipate Toxicity-Induced Dry Eye (TIDE). Using high-content imaging tool, we here characterize toxicity-induced cellular alterations using primary cultures of mouse trigeminal sensory neurons and corneal epithelial cells in a compartmentalized microfluidic chip. We validate this model through the analysis of benzalkonium chloride (BAC) toxicity, a well-known preservative in eyedrops, after a single (6h) or repeated (twice a day for 15 min over 5 days) topical 5.10-4% BAC applications on the corneal epithelial cells and nerve terminals. In combination with high-content image analysis, this advanced microfluidic protocol reveal specific and tiny changes in the epithelial cells and axonal network as well as in trigeminal cells, not directly exposed to BAC, with ATF3/6 stress markers and phospho-p44/42 cell activation marker. Altogether, this corneal neuroepithelial chip enables the evaluation of toxic effects of ocular xenobiotics, distinguishing the impact on corneal sensory innervation and epithelial cells. The combination of compartmentalized co-culture/high-content imaging/multiparameter analysis opens the way for the systematic analysis of toxicants but also neuroprotective compounds.

Ocular safety of cationic emulsion of cyclosporine in an in vitro corneal wound-healing model and an acute in vivo rabbit model.

PURPOSE: Topical preparations of cyclosporine (CsA) are common therapeutics for the treatment of dry eye. However, they are not devoid of side effects, such as allergy and irritation. The present study aimed at evaluating the safety profile of a new CsA formulation in cationic emulsion (CEm) in vitro with a dynamic corneal wound healing assay using human corneal epithelial (HCE) cells, and in vivo in a rabbit acute toxicity model. METHODS: Three different csa formulations were tested: 1) 0.05%CsA-CEm, 2) commercial 0.05%CsA-Anionic emulsion (CsA-AEm, Restasis®), and 3) 0.05%CsA-Oil solution. Phosphate buffered saline (PBS) was used as negative control and 0.02% benzalkonium chloride (BAK) as the toxic control. In vitro, a wound was created by scratching through a confluent HCE cell layer and exposed 30 min to 1/10 dilutions of the different formulations. Cytotoxicity, cell migration, and proliferation were performed to analyze the recovery at days 1, 2, and 3. In vivo, the eye drops were applied to rabbit eyes 15 times at 5-min intervals. The ocular surface structures were examined with a slit-lamp and by corneal in vivo confocal microscopy (IVCM) for detailed examination of corneal epithelium, stroma, limbus, and conjunctiva-associated lymphoid tissue (CALT) structures. RESULTS: The in vitro study confirmed that a 0.02% BAK solution delayed the corneal healing process (-57%) by severely damaging the remaining HCE cells. The other formulations maintained a normal healing rate with a similar behavior for CsA-CEm, CsA-AEm, and PBS with no significant differences (at D3, 66%-74% closure). In the rabbit, 0.02%BAK showed the highest toxicity, inducing redness, chemosis with damaged corneal epithelium, and inflammatory cell infiltrations. CsA-AEm and CsA-Oil induced moderate infiltrations of inflammatory cells around the CALT. CsA-CEm presented the lowest toxicity with patterns similar to PBS. CONCLUSIONS: The combination of these in vitro and in vivo models evaluated the tolerance/cytotoxicity and the dynamic wound healing potential of CsA in different formulations. While CsA-AEm, CsA-CEm, and CsA-Oil are generally well tolerated, only CsA-CEm appeared to maintain the HCE cells' normal healing rate in vitro and low levels of inflammation in vivo.

Hyperosmolarity potentiates toxic effects of benzalkonium chloride on conjunctival epithelial cells in vitro.

PURPOSE: Benzalkonium chloride (BAK), the most commonly used preservative in eye drops, is known to induce ocular irritation symptoms and dry eye in long-term treated patients and animal models. As tear film hyperosmolarity is diagnostic of some types of dry eye disease, we determined in vitro on conjunctival epithelial cells the cytoxicity of BAK in hyperosmolar conditions through cell viability, apoptosis, and oxidative stress assays. METHODS: The Wong Kilbourne derivative of Chang conjunctival epithelial cells were cultured for 24 h or 48 h either in NaCl-induced hyperosmolar conditions (400-425-500 mOsM), in low concentrations of BAK (10(-4)%, 3.10(-4)%, and 5.10(-4)%), or in combination of both. We investigated cell viability through lysosomal integrity evaluation, cell death (cell membrane permeability and chromatin condensation), and oxidative stress (reactive oxygen species, superoxide anion) using spectrofluorimetry. Immunohistochemistry was performed for cytoskeleton shrinkage (phalloidin staining), mitochondrial permeability transition pore (cytochrome c release), the apoptosis effector active caspase-3, and the caspase-independent apoptosis factor AIF. We also observed early effects induced by the experimental conditions on the conjunctival cell layers using phase contrast imaging of live cells. RESULTS: As compared to standard culture solutions, hyperosmolar stress potentiated BAK cytotoxicity on conjunctival cells through the induction of oxidative stress; reduction of cell viability; cell membrane permeability increase; cell shrinkage with cell blebbing, as shown in phase contrast imaging of live cells; and chromatin condensation. Like BAK, but to a much lesser extent, hyperosmolarity increased cell death in a concentration-dependent manner through a caspase-dependent apoptosis characterized by a release of cytochrome c in the cytoplasm from mitochondria and the activation of caspase-3. Moreover, the caspase-independent apoptosis factor AIF was found translocated from mitochondria to the nucleus in both conditions. CONCLUSIONS: This study showed increased cytotoxic effects of BAK in hyperosmotic conditions, with characteristic cell death processes, namely caspase-dependent and independent apoptosis and oxidative stress. As BAK is known to disrupt tear film, which could promote evaporative dry eye and tear hyperosmolarity, BAK could promote the conditions enhancing its own cytotoxicity. This in vitro hyperosmolarity model thus highlights the risk of inducing a vicious cycle and the importance of avoiding BAK in patients with dry eye conditions.

Reduced in vivo ocular surface toxicity with polyquad-preserved travoprost versus benzalkonium-preserved travoprost or latanoprost ophthalmic solutions.

The study used a validated acute in vivo model to compare a new formulation of travoprost 0.004% ophthalmic solution(travoprost PQ), preserved with polyquaternium-1 (PQ), with commercially available formulations of benzalkonium-chloride(BAK)-preserved travoprost 0.004% ophthalmic solution(travoprost BAK) and BAK-preserved latanoprost 0.005%ophthalmic solution (latanoprost BAK). Adult male New Zealand albino rabbits (n = 36) were randomly divided into 6 groups. Phosphate-buffered saline (PBS), 0.001% PQ, 0.015% BAK, travoprost PQ, travoprost BAK or latanoprost BAK were applied onto rabbit eyes as 1 drop, for 15 times at 5-min intervals.The ocular surface reactions were investigated at hour 4 and day 1 using slitlamp examination; in vivo confocal microscopy (IVCM) for cornea, limbus and conjunctiva/conjunctiva-associated lymphoid tissue, conjunctival impression cytology and standard immunohistology in cryosections for detecting CD45+ infiltrating cells and MUC-5AC-labeled cells. PBS, PQ and travoprost PQ did not induce obvious irritation by clinical observation, changes in microstructures of the whole ocular surface as measured by IVCM analysis,inflammatory infiltration or cell damage as measured by impression cytology, altered levels of goblet cell counts or numerous CD45+ cells in the cornea. In contrast, all BAK-containing products induced diffuse conjunctival hyperemia and chemosis, abnormal changes in the ocular surface microstructure,significant total ocular surface toxicity scores,damaged epithelial cells, inflammatory cell infiltration and decreased goblet cell density. Travoprost PQ did not elicitocular surface toxicity when administered to rabbit eyes.These results suggest a greater safety advantage for the ocular surface of patients receiving chronic glaucoma treatment with PQ-preserved drugs.

In Vitro Corneal and Conjunctival Wound-Healing Assays as a Tool for Antiglaucoma Prostaglandin Formulation Characterization.

BACKGROUND: Benzalkonium chloride (BAK)-containing antiglaucoma therapies alter the ocular surface over the long term. We used an in vitro scraping model to compare the effects of preserved and unpreserved topical commercial prostaglandins (PGs) in a wound-healing model. METHODS: Standardized mechanical scraping was performed in confluent immortalized human corneal/conjunctival epithelial cell layers. Cytotoxicity, cell migration and proliferation, as well as the percentage of closure, were analyzed 2 h and 1/2/3/6 days after a 30-min exposure to 1/10 dilutions in phosphate buffered saline (PBS) used also as control, BAK solutions at concentrations ranging from 0.0001% to 0.1%, latanoprost-0.02%BAK, travoprost-0.015%BAK, bimatoprost-0.005%BAK, BAK-free Tafluprost, latanoprost in cationic emulsion, and travoprost (Polyquad® and SofZia®). RESULTS: PG eyedrop preparations with BAK preservative delayed corneal healing, which is primarily related to the presence of BAK, in a dose-dependent manner, especially at day 1, as evidenced through actin disorganization and decreased Ki-67-positive cell numbers. The PGs (BAK-free tafluprost, latanoprost in cationic emulsion,travoprost (Polyquad® and SofZia®)) maintained a normal healing process with results similar to those of control. Conjunctiva-derived cell layers healed more slowly than corneal cell layers and were more sensitive in in vitro cytotoxicity tests. CONCLUSIONS: This novel in vitro scraping model mimics the damaged ocular surface epithelia observed in glaucoma patients affected by ocular surface disease, such as toxic-induced dry eye (TIDE) and offers a tool to assess the potential cytotoxic effects of PG formulations with or without BAK.