Patrice Rat

Ocular surface changes induced by contact lens wear.

Purpose. To evaluate subclinical inflammation and mucus production of the conjunctiva in asymptomatic contact lens (CL) wearers, and to obtain an estimation of the chronologic variations in each group. Methods. Eighteen eyes fitted with rigid CL (RCL) and 28 eyes with soft CL (SCL) worn daily were compared with 10 eyes from five healthy non-CL wearers. Impression cytology (IC) specimens were collected after clinical examination and were analyzed by flow cytometry using antibodies directed to HLA DR and intercellular adhesion molecule type 1 (ICAM-1) (CD 54), as inflammatory markers, and to the peptidic core of the conjunctival mucin (M1/MUC5AC) for mucus and goblet cell detection. The percentage of positive cells was calculated, and levels of fluorescence expression were quantified and compared between each group. Results. A significant increase of HLA DR and ICAM-1 was observed in the SCL group in comparison with the control group. The two inflammatory markers were highly positively correlated with each other. Mucin detection with M1/MUC5AC did not find a significant difference between each group in terms of percentage of positive cells, but analyses of mean levels of fluorescence showed a significant decrease in the two CL groups. Evolution in time was different for each group, with a regular low level of inflammation in the RCL group in the first 10 years in comparison with the SCL group. In the SCL group, inflammation seemed to be higher before 2 years and after 10 years of wear. Mucin expression was variable in time, but without significant difference at any time. Conclusion. This study confirms difference in expression of subclinical conjunctival inflammation in asymptomatic CL wearers, with lower levels for RCL than SCL wearers with daily or extended wear. The mucin system is also modified by this low but chronic aggression of the ocular surface, with a tendency to decrease with time in the RCL and SCL groups.

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.

A New Nondestructive Cytometric Assay Based on Resazurin Metabolism and an Organ Culture Model for the Assessment of Corneal Viability

Three‐dimensional culture or human corneal equivalents for safety testing are difficult to investigate with classic cytometric or biochemical methods. So a fluorometric method is proposed using resazurin probe.

Evaluation of the Toxicity of Benzalkonium Chloride on the Ocular Surface

Abstract Benzalkonium chloride (BAC), the most widely used preservative in ophthalmic solutions, acts on the ocular surface through toxic or im-munoallergic reactions. Due to its surfactant properties, this quaternary ammonium strongly decreases lachrymal fluid stability. It also causes toxic effects to epithelial cells and increases corneal permeability. In vivo, strong histopathological changes are observed after topical treatments with preservatives, including infiltration by inflammatory cells, similar to those induced in humans by long-term topical treatments. We designed a series of in vitro experiments confirming the toxicity of BAC, even at very low concentrations. In vitro, BAC induces cell necrosis at the concentrations found in most eye drops after a few minutes, and apoptosis when applied at lower concentrations. This apoptotic phenomenon is confirmed by 4′,6-diamidimo-2-phenylindole, dilactate (DAPI) coloration and with the use of flow cytometry. A decrease in cell size is observed with BAC at 0.001%% and is confirmed by morphological assay. An overexpression of Apo 2.7 associated with an increase of sub G1 phase cells is also shown. BAC induces irreversible cytotoxic damages with some characteristics of apoptosis in a concentration-dependent manner.

Effect of age and photoperiodic conditions on metabolism and oxidative stress related markers at different circadian stages in rat liver and kidney

It has been shown that some cytochrome P450-dependent enzyme activities could present daily fluctuations, particularly CYP3A isoenzymes which are enhanced during the dark period. The aim of this study was to investigate whether age and photoperiodic conditions at different circadian stages could influence these fluctuations. Young mature (10 weeks) and old (22 months) Wistar rats were initially exposed to light-dark cycles 12:12 during 4 weeks, and secondly 18:6 for either one week or six weeks. Erythromycin N-demethylase (CYP3A-dependent), 7-ethoxycoumarin O-deethylase (CYP1A-dependent) and aniline 4-hydroxylase (CYP2E-dependent) activities were determined in liver and kidney microsomes at different hours after darkness onset (HADO). In addition, liver and kidney GSH, GSHPx, ATP, TBARS were determined. During the LD 12:12 cycle, while no significant modification was observed in CYP1A- and 2E-dependent enzyme activities as functions of HADO, erythromycin N-demethylase activity (CYP3A-dependent) showed a significant increase during the second third of the dark period in both young and old rats. After switching to a LD 18:6 cycle, this variation was still observed during second third of the dark period, to a lesser but still significant degree, with no difference between one week and six weeks exposure to the new photoperiod. It can be noted that the old rats showed a significantly lower level of erythromycin N-demethylase activity than the young rats, in parallel to a decrease in GSH, GSHPx and ATP, and an increase in TBARS. These results confirm the lower resistance of old animals to oxidative stress. The observed variations in metabolism parameters underline the need for study designs in pharmaco-toxicology taking into account the possible risks induced by circadian changes, especially in aged subjects.

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.

Lens epithelial cell protection by aminothiol WR-1065 and anetholedithiolethione from ionizing radiation.

Lens epithelium disorganization, glutathione (GSH) depletion, and epithelial cell death have been incriminated in the cytopathogenic mechanisms that lead to cataract formation following UVB and x‐ray exposures. The objective of this study was to determine the in vitro capacity of the aminothiol WR‐1065, the active metabolite of amifostine, and anetholedithiolethione (ADT or Sulfarlem®) to protect bovine lens epithelial cells against x‐ray irradiation. WR‐1065 and ADT were used at a concentration of 20 μM. A single dose of 10 Gy was delivered at a rate of 2 Gy/min. Fluorimetric assays were then performed using a neutral red probe to evaluate cell viability, a Hoechst 33342 probe (HO) to evaluate nuclear condensation and apoptosis, and a monobromobimane probe to estimate the intracellular GSH pool. Twenty‐four hours after x‐ray exposure, cells pretreated with WR‐1065 showed increased GSH levels, improved cell viability, and decreased HO fluorescence in addition to a lesser proportion of cells with apoptotic nuclear modifications. Between 72 and 120 hr postirradiation, ADT‐pretreated cells also showed increased intracellular GSH levels and cell viability and decreased HO fluorescence and apoptotic cell morphology. This in vitro study demonstrates that WR‐1065 and ADT protects lens epithelial cells from x‐ray injury; thus, ADT and amifostine are appropriate candidates for clinical trials in humans. They are currently used in preventing radiation‐induced xerostomia and should be further tested in the prevention of late radiation‐induced ocular complications such as sicca syndrome and cataract.

[34] Cold light fluorimetry: A microtitration technology for cell culture to evaluate anethole dithiolethione and other biothiols

Publisher Summary Biothiols are compounds of growing biological interest. This chapter describes screening these drugs using a new microtitration technology, cold light fluorimetry (CLF), and comparing it to classic fluorimetric methods. Until now, in vitro screening of such molecules was done using photometric or fluorimetric microtitration methods which are not sufficiently sensitive and/or specific. The only fluorimetric methodology available for cell culture is flow cytometry, which is not adapted to screening because the equipment is too heavy and expensive. The classic microtitration fluorimetric methods are reviewed and the cold light concept is discussed. The three main microplate fluorescence readers have been tested. These fluorimeters read correctly in ultraviolet (UV) or visible spectra but rarely in both, and they have low sensitivity. Therefore a new fluorimetric technology has been developed which proved to be sensitive and could be adapted to direct screening in 96-well microplates. To improve the fluorescence signal, the choice of plastic microplates is decisive. Black microplates used for fluorimetric biochemistry assays are not adapted for cell biology, because they are neither treated for cell culture nor sterilized.

Ionizing radiation-induced death in bovine lens epithelial cells: Mechanisms and influence of irradiation dose rate

We recently reported, in a series of patients receiving total body irradiation before transplant, an influence of dose rate (DR) on cataract formation. The aim of our present in vitro study was to investigate the influence of DR and the mechanisms of lens cell death in a bovine model. After a single fraction of 10 Gy, delivered using low (0.05 Gy/min) or high (2 Gy/min) DR (LDR and HDR, respectively), cells were incubated in media supplemented with two different fetal calf serum (FCS) concentrations (1% and 10%). Cell proliferation was evaluated using Hoechst 33342 (HO) probe and cell viability, with neutral red probe. These fluorimetric assays used a cold light cytofluorimeter. After HO assay, stained cells were examined with fluorescence microscopy to evaluate the nuclear changes related to apoptosis. Global comparison of the mean HO fluorescent values observed with LDR/controls (c) vs. HDR/c revealed a significant difference only after 96 hr (P = 0.036). In 1% FCS conditions, the difference between HDR/c and LDR/c was also statistically significant at 96 hr (P = 0.04). Pairwise multiple comparison using values observed in 1% FCS conditions after 96 hr incubation showed significant difference between HDR vs. c (P = 0.001) and HDR vs. LDR (P = 0.007). This difference, in terms of fluorescence, was correlated to the proportion of cells with nuclear apoptotic morphology. In contrast, cell viability was not influenced by DR whatever the FCS concentration used, from 24 to 96 hr after irradiation. We conclude that our fluorimetric methodology is adapted to evaluate intracellular DNA modifications and cell viability after x‐ray irradiation. We observed that a single fraction of 10 Gy induces in vitro lens epithelial cell apoptosis, which is influenced by DR. In humans, HDR is considered more cataractogenic than LDR. Thus, we speculate that lens cell apoptosis could be one of the major mechanisms of radiation‐induced cataract. Further investigations are necessary to study the other possible mechanisms of cataractogenesis. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 138–144 (2000).