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http://www.ncbi.nlm.nih.gov/pubmed/21437201
Mol Vis. 2011 Mar 16;17:745-55.
Multiple endpoint analysis of BAC-preserved and unpreserved antiallergic eye drops on a 3D-reconstituted corneal epithelial model.
Pauly A, Brasnu E, Riancho L, Brignole-Baudouin F, Baudouin C.
Abstract
PURPOSE: 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.
PMID: 21437201 [PubMed - in process]PMCID: PMC3062522
Mol Vis. 2011 Mar 16;17:745-55.
Multiple endpoint analysis of BAC-preserved and unpreserved antiallergic eye drops on a 3D-reconstituted corneal epithelial model.
Pauly A, Brasnu E, Riancho L, Brignole-Baudouin F, Baudouin C.
Abstract
PURPOSE: 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.
PMID: 21437201 [PubMed - in process]PMCID: PMC3062522