title: Multiple endpoint analysis of BAC-preserved and unpreserved antiallergic eye drops on a 3D-reconstituted corneal epithelial model.
abstract: OBJECTIVE
  To compare the effects of benzalkonium chloride (BAC)-preserved and unpreserved antiallergic eye drops on the human 3D-reconstituted corneal epithelial model (3D-HCE).
  
  
  METHODS
  3D-HCE were treated for 24 h followed or not by a 24 h post-incubation recovery period (24 h+24 h) with phosphate-buffered saline (PBS), 0.01% BAC, unpreserved formulations of ketotifen, N Acetyl-Aspartyl Glutamic Acid (NAAGA), cromoglycate, or BAC-preserved commercial formulations of ketotifen, olopatadine, epinastine, and levocabastine. The 3D-HCE viability was evaluated using the 3-(4,5-Dimethylthiazol-2-yl) -2,5-Diphenyltetrazolium Bromide (MTT) test at 24 h and 24 h+24 h. At 24 h, the numbers of Cluster of Differentiation 54 (CD54)- and Ki67-immunopositive cells as well as the number of apoptotic deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were evaluated on 3D-HCE frozen sections. The expression of the tight junction-associated protein occludin was also assessed using fluorescence confocal microscopy on flat-mounted 3D-HCE epithelia.
  
  
  RESULTS
  The MTT and the TUNEL tests revealed a significant decrease of cell viability and an increased apoptosis in the superficial layers of the 3D-HCE only when treated with BAC-containing formulations and in a BAC concentration-dependent manner. The expression of CD54 and Ki67 in the basal layers was also increased in this group. A concentration-dependent disorganization of occludin distribution in the epithelium treated with BAC-containing solutions was also observed. The unpreserved formulations induced effects comparable to the control.
  
  
  CONCLUSIONS
  BAC-preserved solutions decreased cell viability and induced apoptosis in a concentration-dependent manner. Moreover, they induced CD54 expression, proliferation in the basal layers, and changes in the distribution of occludin, which is consistent with a disorganization of the tight-junctions and suggests the loss of the epithelial barrier function. On the contrary, the unpreserved solutions did not impair cell structures and viability, suggesting a better tolerance for the ocular surface. As allergic patients often exhibit impaired and inflammatory ocular surface, BAC-free compounds should be the first choice when treating allergic conjunctivitis.
authors: INSERM, UMR_S968, Institut de la Vision, Paris, France.
authors: A
authors: A
authors: Pauly
authors: E
authors: E
authors: Brasnu
authors: L
authors: L
authors: Riancho
authors: F
authors: F
authors: Brignole-Baudouin
authors: C
authors: C
authors: Baudouin
mesh_terms: D018926:Anti-Allergic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001548:Benzalkonium Compounds / Q000494:pharmacology*; D015415:Biomarkers / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D023381:Endpoint Determination; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects*; D005455:Fluorescent Antibody Technique; D006801:Humans; D020287:In Situ Nick-End Labeling; D007249:Inflammation / Q000378:metabolism / Q000473:pathology; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D062793:Occludin; D009883:Ophthalmic Solutions / Q000494:pharmacology*; D011310:Preservatives, Pharmaceutical / Q000494:pharmacology*; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism
chemical_list: D018926:Anti-Allergic Agents; D001548:Benzalkonium Compounds; D015415:Biomarkers; D019394:Ki-67 Antigen; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D009883:Ophthalmic Solutions; D011310:Preservatives, Pharmaceutical; D018799:Intercellular Adhesion Molecule-1