Mélody Dutot

In vitro modulation of preservative toxicity: High molecular weight hyaluronan decreases apoptosis and oxidative stress induced by benzalkonium chloride

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.

Multipurpose solutions and contact lens: modulation of cytotoxicity and apoptosis on the ocular surface.

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 solutions 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.

P2X7-pannexin-1 and amyloid β-induced oxysterol input in human retinal cell: Role in age-related macular degeneration?

Age-related macular degeneration (AMD) is the most common cause of severe vision loss worldwide. Amyloid β involvement in degenerative diseases such as AMD is well known and its toxicity has been related to P2X7 receptor-pannexin-1. Recently, oxysterols (oxidized derivatives of cholesterol) have been implicated in AMD pathogenesis. The aim of our study was to highlight amyloid β/oxysterols relationship and to describe P2X7 receptor-pannexin-1 role in oxysterols toxicity. Using retinal epithelial cells, we first quantified sterols levels after amyloid β incubation and second we investigated the cytotoxic effects induced by oxysterols. For the first time, our results showed that amyloid β induced oxysterols formation in human retinal pigmented epithelial cells. We showed that oxysterol toxicity is mediated by P2X7 receptor activation. This activation was dependent on pannexin-1 with 25-hydroxycholesterol whereas P2X7 receptor signaling pathway was pannexin-1-independent for 7-ketocholesterol. Taken together our data suggest a pivotal role of P2X7 receptor-pannexin-1 in oxysterols toxicity in retinal cells which could be an important target to develop new treatments for AMD.

Corneal protection with high-molecular-weight hyaluronan against in vitro and in vivo sodium lauryl sulfate-induced toxic effects.

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.

Ocular Burn: Rinsing and Healing with Ionic Marine Solutions and Vegetable Oils

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.

Formulation and cytotoxicity evaluation of new self-emulsifying multiple W/O/W nanoemulsions

Three multiple water-in-oil-in-water (W/O/W) nanoemulsions have been designed for potential inclusion of either lipophilic or hydrophilic drugs using a two-step emulsification process exclusively based on low-energy self-emulsification. The W/O primary emulsion was constituted by a blend of oil (medium chain triglyceride), a mixture (7:3) of two surfactants, and a 10% water phase. The surfactants were a mixture of Polysorbate-85/Labrasol®, Polysorbate-85/Cremophor® EL or glycerol/Polysorbate-85. The final W/O/W nanoemulsions were obtained by the addition of water, with a weight ratio nanoemulsion/water of 1:2. The multiple emulsion stability was found to increase from 24 hours to 2 and 6 months with Labrasol, glycerol, and Cremophor, respectively. Cytotoxicity was found for formulations including Labrasol and Cremophor EL. The concentration of emulsion inhibiting 50% cell viability (IC50) was determined using the alamarBlue® test, giving after 24 hours of incubation, IC50 = 10.2 mg/mL for the Labrasol formulation and IC50 = 11.8 mg/mL for the Cremophor EL formulation. Corresponding calculated IC50 values for surfactants were 0.51 mg/mL for Labrasol and 0.59 mg/mL for Cremophor EL. In both cases, cytotoxicity was due to an apoptotic mechanism, evidenced by chromatin condensation and P2X7 cell death receptor activation. The formulation including glycerol, investigated between 1 and 100 mg/mL concentration of nanoemulsion, did not affect cell viability. Moreover, neither chromatin condensation nor P2X7 activation was found between the 10 and 30 mg/mL final concentration of the emulsion. This last formulation would therefore be of major interest for further developments.

Lipid deregulation in UV irradiated skin cells: Role of 25-hydroxycholesterol in keratinocyte differentiation during photoaging

Skin photoaging due to UV irradiation is a degenerative process that appears more and more as a growing concern. Lipids, including oxysterols, are involved in degenerative processes; as skin cells contain various lipids, the aim of our study was to evaluate first, changes in keratinocyte lipid levels induced by UV exposure and second, cellular effects of oxysterols in cell morphology and several hallmarks of keratinocyte differentiation. Our mass spectrometry results demonstrated that UV irradiation induces changes in lipid profile of cultured keratinocytes; in particular, ceramides and oxysterols, specifically 25-hydroxycholesterol (25-OH), were increased. Using holography and confocal microscopy analyses, we highlighted cell thickening and cytoskeletal disruption after incubation of keratinocytes with 25-OH. These alterations were associated with keratinocyte differentiation patterns: autophagy stimulation and intracellular calcium increase as measured by cytofluorometry, and increased involucrin level detected by immunocytochemistry. To conclude, oxysterol deregulation could be considered as a common marker of degenerative disorders. During photoaging, 25-OH seems to play a key role inducing morphological changes and keratinocyte differentiation.

New approach to modulate retinal cellular toxic effects of high glucose using marine epa and dha

BackgroundProtective effects of omega-3 fatty acids against cellular damages of high glucose were studied on retinal pigmented epithelial (RPE) cells.MethodsRetinal epithelial cells were incubated with omega-3 marine oils rich in EPA and DHA and then with high glucose (25 mM) for 48 hours. Cellular responses were compared to normal glucose (5 mM): intracellular redox status, reactive oxygen species (ROS), mitochondrial succinate deshydrogenase activity, inflammatory cytokines release and caveolin-1 expression were evaluated using microplate cytometry, ELISA and flow cytometry techniques. Fatty acids incorporation in retinal cell membranes was analysed using chromatography.ResultsPreincubation of the cells with fish oil decreased ROS overproduction, mitochondrial alterations and TNFα release. These protective effects could be attributed to an increase in caveolin-1 expression induced by marine oil.ConclusionMarine formulations rich in omega-3 fatty acids represent a promising therapeutic approach for diabetic retinopathy.

Per os administered refined olive oil and marine PUFA-rich oils reach the cornea: possible role on oxidative stress through caveolin-1 modulation.

BackgroundOlive 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.MethodsIn 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 μM, 1 hour). Effects on reactive oxygen species generation, mitochondria and caveolin-1 expression were studied using microcytofluorometry, flow cytometry and confocal microscopy.ResultsOur 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.ConclusionOleic acid, a potential potent modulator of oxidative stress, could be added to PUFA-rich oils to prevent oxidative stress-linked corneal pathology.

The Role of the P2X7 Receptor in Ocular Stresses: A Potential Therapeutic Target

The P2X7 receptor is expressed in both anterior and posterior segments of the eyeball. In the ocular surface, the P2X7 receptor is activated in case of external aggressions: preservatives and surfactants induce the activation of P2X7 receptors, leading to either apoptosis, inflammation, or cell proliferation. In the retina, the key endogenous actors of age-related macular degeneration, diabetic retinopathy, and glaucoma act through P2X7 receptors’ activation and/or upregulation of P2X7 receptors’ expression. Different therapeutic strategies aimed at the P2X7 receptor exist. P2X7 receptor antagonists, such as divalent cations and Brilliant Blue G (BBG) could be used to target either the ocular surface or the retina, as long as polyunsaturated fatty acids may exert their effects through the disruption of plasma membrane lipid rafts or saffron that reduces the response evoked by P2X7 receptor stimulation. Treatments against P2X7 receptor activation are proposed by using either eye drops or food supplements.