Caroline Debbasch

Flow cytometric analysis of conjunctival epithelium in Ocular rosacea and keratoconjunctivitis sicca

PURPOSE To investigate by flow cytometry and impression cytology (IC) specimens the inflammatory status of the conjunctival epithelium and goblet cell density in two series of patients with rosacea and dry eye syndrome compared with a population of healthy subjects. DESIGN Nonrandomized, prospective, comparative case series. PARTICIPANTS Twenty-six eyes of 13 patients with rosacea, 26 eyes of 13 patients with dry eye syndrome, and 24 eyes of 12 control subjects were included in this study. METHODS IC specimens were collected after clinical examination of the ocular surface and analyzed by flow cytometry, using antibodies directed to human lymphocyte antigen-DR (HLA-DR), intercellular adhesion molecule-1 (ICAM-1) (CD 54), and the peptidic core of the conjunctival mucin (M1/MUC5AC). The percentage of positive cells was calculated and levels of fluorescence expression quantified and compared with those obtained in a series of 12 healthy subjects. MAIN OUTCOME MEASURES Tear break-up time (TBUT), Schirmer test, fluorescein and lissamin green stainings, and IC were realized in this study. RESULTS A significant increase of HLA-DR and ICAM-1 expressions by epithelial cells was consistently found in the two pathologic groups compared with levels calculated in normal eyes. The two markers were well correlated with each other and inversely with TBUT and Schirmer test. The percentage of goblet cells was significantly decreased in rosacea patients and in dry eye patients compared with the normal group with a significant negative correlation with both HLA DR and ICAM-1 markers. CONCLUSIONS Ocular rosacea and keratoconjunctivitis sicca were associated with severe ocular surface changes, such as an overexpression of inflammatory markers and a significant decrease in the number of goblet cells.

Comparison of the Effects of Preserved and Unpreserved Formulations of Timolol on the Ocular Surface of Albino Rabbits

Thirty-six albino rabbits, randomly divided into six groups, were used to study their ocular tolerance to (a) 0.25 and 0.50% Timoptol® preserved with 0.01% benzalkonium chloride, (b) 0.25 and 0.50% Timoptol-LP®, a gel-forming solution preserved with 0.012% benzododecinium bromide, and (c) 0.25 and 0.50% Timabak® unpreserved in the ABAK® eyedrops dispenser. All eyedrops were applied in the right eye for 60 days. A clinical follow-up with slitlamp examination and break-up time evaluation was performed for 2 months. At the end of the experimentation, the animals were sacrificed and their eyes enucleated for histological analyses of the conjunctiva and cornea. There was no significant difference in the clinical examination between each group, except for the break-up time evaluation between Timoptol and Timabak at each concentration which was better with the unpreserved timolol. Histological results showed a significant difference in the corneal stroma edema between preserved and unpreserved timolol. This study confirms that using unpreserved timolol may be beneficial for the long-term treatment of glaucomatous patients as it increases tear film stability and decreases epithelial permeability and stromal aggression of the cornea.

Evaluation of corneal stromal changes in vivo after laser in situ keratomileusis with confocal microscopy

PURPOSE To assess by in vivo confocal microscopy the modifications of the corneal stroma after laser in situ keratomileusis (LASIK) for myopia. DESIGN Nonrandomized comparative (self-controlled) trial. PARTICIPANTS Sixteen eyes of 13 patients were examined before surgery and at days 8, 30, and 90, and 9 eyes were examined at 6 months postoperatively using an in vivo confocal microscope. TESTING/INTERVENTION: Stromal morphologic changes, keratocyte density, flap thickness, and subclinical haze were evaluated and compared at different time points. LASIK was performed with a Flapmaker microkeratome (Solan Ophthalmic products, Jacksonville, FL) and a Lasersight LSX excimer laser (LaserSight Technologies Inc., Winter Park, FL). MAIN OUTCOME MEASURE Confocal microscopy results. RESULTS Microfolds at the Bowmans layer were found in most eyes, as well as variable reflectivity particles (pa) located at the interface level in all eyes examined postoperatively. The density of these particles significantly decreased with time with, respectively, 504 +/- 101 pa/mm2 at day 8 and 380 +/- 111 pa/mm2 at day 30 (P = 0.003), 332 +/- 100 pa/mm2 at month 3 and 312 +/- 40 pa/mm2 at month 6. The mean flap and the activated-cells area thicknesses were, respectively, 102 +/- 26 microm and 61 +/- 19 microm and showed significant negative correlation (P < 0.0001). The intensity of the added peak (47.3 microm 8.6%), corresponding to the subclinical haze, realized by Z-scan measure, was also negatively correlated with flap thickness (P = 0.01). Keratocyte (k) density quantified in the posterior stroma significantly increased from day 0 (480 +/- 67 k/mm2) to day 8 (701 +/- 41 k/mm2, P < 0.0001 compared with day 0) and day 30 (917 +/- 143 k/mm2, P = 0.0006, compared with day 0) but significantly decreased at 3 months postoperatively (597 +/- 56 k/mm2, P < 0.0001 compared with day 30) to reach the initial level at month 6 (502 +/- 41 k/mm2, nonsignificant compared with day 0). There was no correlation between preoperative or postoperative spherical equivalent and the density of particles, keratocytes, and the haze intensity. CONCLUSIONS This study confirms the presence of microfolds and particles at the interface level, as well as subclinical impairment. Evaluation of keratocyte density constitutes a major contribution of confocal microscopy toward an understanding of the keratocyte response to corneal wound healing after corneal refractive surgery. Moreover, flap thickness seems to be involved in the postoperative cellular activation with a higher response when thin.

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.

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.

Relationship between in Vitro Toxicity of Benzalkonium Chloride (BAC) and Preservative-Induced Dry Eye

The term “dry eye syndrome” encompasses various ocular surface defects with a common element: deterioration in quantity and/or quality of lacrymal film. A constant step in the evolution of dry eye is an increase in tear osmolarity, leading in turn to other anomalies of the ocular surface. These include decreased conjunctival goblet cell density, increased corneal epithelial desquamation and destabilization of the cornea-tear interface. Iatrogenically induced dry eye is commonly encountered in clinical practice, especially while long-term treatments are employed for chronic ophthalmic affections such as glaucoma or allergy.1–4 On the cellular level, long-term use of topical, preserved drugs is associated with conjunctival metaplasia, stromal infiltrates, and epithelial expression of inflammation-dependent molecules (HLA DR), as well as apoptotic markers.5,6 The toxic and pro-inflammatory actions of preservatives on surface epithelia, as well as their detergent effect on the lipid phase of lacrymal film, seem to be responsible for these alterations.7,8 The role of active compounds of topical drugs in these pathological processes is not well established, although some in vivo and in vitro studies have demonstrated the lack of any noxious, tissue or cellular side effects.9,10 The aim of the present study was to investigate the relationship between the possible in vitro toxic effects of benzalkonium chloride (BAC), the most commonly used ophthalmic preservative, and the pathology of dry eye.

Influence of Preservatives on Conjunctival Cells in Vitro

Abstract Benzalkonium chloride (BAC), which is widely used in ophthalmic preparations for its preservative properties, has been shown to cause conjunctival toxicity. The purpose of this study was to examine the effects of BAC on a continuous human conjunctival cell line (Wong Kilbourne-derived human conjunctiva). Cytotoxicity tests were assessed according to ECVAM recommendations using microplate cold light

Toxicity of Preserved and Unpreserved Antiglaucoma Topical Drugs in an In Vitro Model of Conjunctival Cells

PURPOSE To compare the toxicity of a short-time application of timolol with benzalkonium chloride (timolol-BAC+) and unpreserved timolol (timolol-BAC-) in a human conjunctival cell line. METHODS Changs conjunctival cell line (ATCC CCL 20.2) was treated for 15min. with 0.1%, 0.25% or 0.4% timolol-BAC(+) or BAC(-) and then examined immediately or 24h later. Cell viability, chromatin condensation and free radicals production were studied by microplate cold light cytometry. Moreover, relative cell number was evaluated by crystal violet colorimetric test. The comparison was done with an oxidative stress model of cells treated with 0.001-0.000001% hydrogen peroxide (H(2)O(2)). In addition, cell size and the expression of an apoptotic marker Apo2.7 were evaluated by flow cytometry. RESULTS Timolol-BAC(+) induced a rapid decrease in cell viability ranging from 40% immediately after treatment to 85% 24h later. A small initial decrease in cell viability was also observed with all tested concentrations of timolol-BAC(-) but, 24h later, cell viability either tended to remain constant or cells completely recovered. Cell viability fell down after 24h exposure to 0.001% H(2)O( 2) whereas it was not modified at lower concentrations. 24h after treatment with 0.25% timolol-BAC(+), the relative cell number was reduced by 55% whereas it did not vary after 0.25% timolol-BAC(-) treatment. Only timolol-BAC(+) induced chromatin condensation and cell size reduction. Moreover, cells treated with timolol-BAC(+) overexpressed the apoptotic marker Apo2.7. Both timolol-BAC(+) and BAC(-) induced reactive oxygen species (ROS) production which was significantly more important when 0.25% or 0.4% timolol-BAC(+) were applied. Only 0.001% and 0.0001% H(2)O(2) generated a significant free radicals production. CONCLUSION In our model of conjunctival cells in vitro timolol-BAC(+) induced irreversible cytotoxic damage with some characteristics of apoptosis. The active compound of timolol-BAC(-) could be responsible for reactive oxygen species production and for cell viability variations. The role of oxidative stress in timolol-BAC(+)-induced toxicity seems not to be predominant. in vitro toxic effects of antiglaucoma drugs could, in part, explain some ocular surface disorders in long-term treated patients.