Multiple endpoint analysis of BAC-preserved and unpreserved antiallergic eye drops on a 3D-reconstituted corneal epithelial model

[摘要] Purpose: To compare the effects ofbenzalkonium chloride (BAC)-preserved and unpreserved antiallergic eyedrops on the human 3D-reconstituted corneal epithelial model (3D-HCE). Methods: 3D-HCE were treated for 24 hfollowed or not by a 24 h post-incubation recovery period (24 h+24 h)with phosphate-buffered saline (PBS), 0.01% BAC, unpreservedformulations of ketotifen, N Acetyl-Aspartyl Glutamic Acid (NAAGA),cromoglycate, or BAC-preserved commercial formulations of ketotifen,olopatadine, epinastine, and levocabastine. The 3D-HCE viability wasevaluated using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) test at 24 h and 24 h+24 h. At24 h, the numbers of Cluster of Differentiation 54 (CD54)- andKi67-immunopositive cells as well as the number of apoptoticdeoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)-positive cells were evaluated on 3D-HCE frozen sections. Theexpression of the tight junction-associated protein occludin was alsoassessed using fluorescence confocal microscopy on flat-mounted 3D-HCEepithelia. Results: The MTT and the TUNEL testsrevealed a significant decrease of cell viability and an increasedapoptosis in the superficial layers of the 3D-HCE only when treatedwith BAC-containing formulations and in a BAC concentration-dependentmanner. The expression of CD54 and Ki67 in the basal layers was alsoincreased in this group. A concentration-dependent disorganization ofoccludin distribution in the epithelium treated with BAC-containingsolutions was also observed. The unpreserved formulations inducedeffects comparable to the control. Conclusions: BAC-preserved solutionsdecreased cell viability and induced apoptosis in aconcentration-dependent manner. Moreover, they induced CD54 expression,proliferation in the basal layers, and changes in the distribution ofoccludin, which is consistent with a disorganization of thetight-junctions and suggests the loss of the epithelial barrierfunction. On the contrary, the unpreserved solutions did not impaircell structures and viability, suggesting a better tolerance for theocular surface. As allergic patients often exhibit impaired andinflammatory ocular surface, BAC-free compounds should be the firstchoice when treating allergic conjunctivitis.

[学科分类] 生物化学/生物物理