three-dimensional (3d) reconstruction model  (Medis)

Journal: The British Journal of Ophthalmology

Article Title: Toxicological evaluation of preservative-containing and preservative-free topical prostaglandin analogues on a three-dimensional-reconstituted corneal epithelium system

doi: 10.1136/bjo.2010.189449

Figure Lengend Snippet: Cell-viability MTT 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test: cell viability of phosphate-buffered saline (PBS)-, preservative-free (PF)-tafluprost-, 0.005% benzalkonium chloride (BAC)-bimatoprost-, 0.010% BAC-, 0.015% BAC-travoprost-, 0.020% BAC-latanoprost- and 0.020% BAC-treated 3D-human corneal epithelium after 24 h incubation without or with a 24 h recovery period. Antiglaucoma treatments induced a BAC-dose-dependent decrease in cell viability as assessed by the MTT test. PF-tafluprost showed a higher level of viability than all BAC-containing eye-drops. *p<0.01 compared with PBS at the same time point; # p<0.03 compared with 0.010% BAC at the same time point; $p<0.002 or p<0.03 ($$) compared with 0.020% BAC at the same time point; ◆p<0.05 compared with 0.020% BAC-latanoprost at the same time point; ●p<0.02 compared with 0.015% BAC-travoprost at the same time point.

Article Snippet: Supplied by SkinEthic Laboratories (Nice, France), the reconstructed three-dimensional (3D) model of human corneal epithelium (HCE) is an appropriate alternative toxicological method to the classical Draize test in rabbits.

Techniques: Saline, Incubation, Eye Drops

Journal: The British Journal of Ophthalmology

Article Title: Toxicological evaluation of preservative-containing and preservative-free topical prostaglandin analogues on a three-dimensional-reconstituted corneal epithelium system

doi: 10.1136/bjo.2010.189449

Figure Lengend Snippet: Apoptosis analysis: localisation of terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL)-positive cells (green) on 3D-human corneal epithelium samples after phosphate-buffered saline (PBS) (A), preservative-free (PF)-tafluprost (B), 0.005% benzalkonium chloride (BAC)-bimatoprost (C), 0.010% BAC (D), 0.015% BAC-travoprost (E), 0.020% BAC-latanoprost (F) and 0.020% BAC (G) incubation after 24 h without (left column) or with the 24 h recovery period (right column). Quantification of TUNEL-positive cells (H) was performed after 24 h of treatment with all solutions followed or not by a 24 h recovery period. At 24 h or 24 h+24 h recovery, no or very few apoptotic cells were observed after PBS (A) or PF-tafluprost (B) treatments. Bimatoprost with 0.005% BAC (C) induced moderate expression of apoptosis, and 0.010% BAC (D), 0.015% BAC-travoprost (E), 0.020% BAC-latanoprost (F) 0.020% BAC (G) all induced a great number of TUNEL-positive cells principally in the apical cell layers, and also in the middle epithelial layers. The nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). The quantification of TUNEL-positive cells shows the increase in apoptotic cell number in a BAC dose-dependent manner. BAC at 0.010% and 0.020% significantly increased the number of TUNEL-positive cells at 24 h, and at 24 h+24 h recovery compared with control. The antiglaucoma eye-drops travoprost and latanoprost, which contain higher concentrations of BAC, showed a much higher expression of apoptotic cells than did PF-tafluprost or control. *p<0.02 for 0.010% BAC and p<0.01 for other solutions compared with PBS at the same time point; #p<0.005 compared with 0.010% BAC at the same time point; $p<0.001 compared with 0.020% BAC at the same time point; ◆p<0.003 compared with 0.020% BAC-latanoprost at the same time point; ●p<0.03 compared with 0.015% BAC-travoprost at the same time point.

Article Snippet: Supplied by SkinEthic Laboratories (Nice, France), the reconstructed three-dimensional (3D) model of human corneal epithelium (HCE) is an appropriate alternative toxicological method to the classical Draize test in rabbits.

Techniques: TUNEL Assay, Saline, Incubation, Expressing, Staining, Control, Eye Drops

Journal: The British Journal of Ophthalmology

Article Title: Toxicological evaluation of preservative-containing and preservative-free topical prostaglandin analogues on a three-dimensional-reconstituted corneal epithelium system

doi: 10.1136/bjo.2010.189449

Figure Lengend Snippet: Inflammation analysis: immunolocalisation of ICAM-1 (CD54) positive cells (green) on 3D-human corneal epithelium samples after phosphate-buffered saline (PBS) (A), preservative-free (PF)-tafluprost (B), 0.005% benzalkonium chloride (BAC)-bimatoprost (C), 0.010% BAC (D), 0.015% BAC-travoprost (E), 0.02% BAC-latanoprost (F) or 0.020% BAC (G) treatments after 24 h of treatment without or with the 24 h postincubation period. Quantification of ICAM-1 (H) was performed after 24 h of treatment with all solutions followed or not by a 24 h postincubation period. ICAM-1-expressing cells were already found in the PBS (A) and PF-tafluprost (B) groups with the same level of expression after 24 h or 24 h+24 h. The increase in ICAM-1 expressions was observed after the treatments with 0.005% BAC-bimatoprost (C) and 0.010% BAC (D) with and without the 24 h postincubation period. However, few remaining ICAM-1 positive cells were observed after treatments with 0.015% BAC-travoprost (E), 0.020% BAC-latanoprost (F) and 0.020% BAC (G) on the 3D-human corneal epithelium samples owing to the erosion. The nuclei were stained with propidium iodide (red). The quantification of terminal deoxynucleotidyl transferase mediated dUTP nick end labelling-positive cells confirmed the observations. *p<0.001 compared with PBS at the same time point; #p<0.005 compared with 0.010% BAC at the same time point; $p<0.0001 compared with 0.020% BAC at the same time point; ◆p<0.002 compared with 0.020% BAC-latanoprost at the same time point; ●p<0.008 compared with 0.015% BAC-travoprost at the same time point.

Article Snippet: Supplied by SkinEthic Laboratories (Nice, France), the reconstructed three-dimensional (3D) model of human corneal epithelium (HCE) is an appropriate alternative toxicological method to the classical Draize test in rabbits.

Techniques: Saline, Expressing, Staining

Journal: The British Journal of Ophthalmology

Article Title: Toxicological evaluation of preservative-containing and preservative-free topical prostaglandin analogues on a three-dimensional-reconstituted corneal epithelium system

doi: 10.1136/bjo.2010.189449

Figure Lengend Snippet: Proliferation analysis: immunolocalisation of Ki-67 positive cells (green) on 3D-human corneal epithelium samples after phosphate-buffered saline (PBS) (A), preservative-free (PF)-tafluprost (B), 0.005% benzalkonium chloride (BAC)-bimatoprost (C), 0.01% BAC (D), 0.015% BAC-travoprost (E), 0.020% BAC-latanoprost (F) or 0.020% BAC (G) treatments after 24 h of treatment without (left column) or with the 24 h recovery period (right column). Ki67 immunostaining showed an epithelium with proliferating cells not restricted to the basal layer after PBS treatment (A) at 24 h. The same aspect was found after the treatment of PF-tafluprost (B). Numerous proliferating cells, with a great number located in all the layers, were found after the treatments of 0.005% BAC-bimatoprost (C), 0.010% BAC (D), 0.015% BAC-travoprost (E), 0.020% BAC-latanoprost (F) and 0.020% BAC (G) at 24 h. At 24 h+24 h recovery, we observed the persistence of some proliferative cells after the treatments of PBS or PF-tafluprost. However, at this time, there were no more proliferative cells after the treatments with all BAC-containing solutions.

Article Snippet: Supplied by SkinEthic Laboratories (Nice, France), the reconstructed three-dimensional (3D) model of human corneal epithelium (HCE) is an appropriate alternative toxicological method to the classical Draize test in rabbits.

Techniques: Saline, Immunostaining

Journal: The British Journal of Ophthalmology

Article Title: Toxicological evaluation of preservative-containing and preservative-free topical prostaglandin analogues on a three-dimensional-reconstituted corneal epithelium system

doi: 10.1136/bjo.2010.189449

Figure Lengend Snippet: Tight junction: occludin and tight junction protein 1 (zona occludens 1, ZO-1). Immunofluorescence analysis of occludin (green: A to G) and ZO-1 (green: H to N) expressions using en face confocal microscopy after treatment with phosphate-buffered saline (PBS) (A), preservative-free (PF)-tafluprost (B), 0.005% benzalkonium chloride (BAC)-bimatoprost (C), 0.010% BAC (D), 0.015% BAC-travoprost (E), 0.020% BAC-latanoprost (F) and 0.020% BAC (G) treatments after 24 h (left column) or 24 h+24 h recovery (right column). The nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). After the treatment with PBS or PF-tafluprost, the 3D-human corneal epithelium revealed a thin occludin/ZO-1 immunostaining in the most superficial cells of samples by forming a green ring around, leaving a diffuse cytoplasmic staining. We still found a slight tight junction expression in the apical cells with 0.005% BAC-bimatoprost (C). The expressions disappeared completely after the treatments with 0.010% BAC (D), 0.015% BAC-travoprost (E), 0.020% BAC-latanoprost (F) and 0.020% BAC (G) at 24 h or 24 h+24 h recovery, leaving dense green patches. Note that the nuclei became smaller after treatments with high-dose BAC-containing solutions.

Article Snippet: Supplied by SkinEthic Laboratories (Nice, France), the reconstructed three-dimensional (3D) model of human corneal epithelium (HCE) is an appropriate alternative toxicological method to the classical Draize test in rabbits.

Techniques: Immunofluorescence, Confocal Microscopy, Saline, Staining, Immunostaining, Expressing