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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.

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 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.

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: Chang's 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.