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

International audiencePurpose: Using an established three-dimensional (3D) toxicological model based on reconstituted human corneal epithelium (HCE), this study investigated the tolerability of four topical intraocular pressure (IOP)-lowering agents: the commercial solutions of benzalkonium chloride (BAC)-containing 0.005% latanoprost, 0.004% travoprost, 0.03% bimatoprost containing 0.02%, 0.015%, and 0.005% BAC, respectively and the preservative-free (PF) tafluprost. Solutions of 0.01% and 0.02% BAC alone were also evaluated for comparison. Methods: The 3D-HCEs were treated with either solutions for 24 hours followed or not by a 24-hour recovery period. We used a modified MTT procedure to assess cell viability in the HCE. Frozen sections of HCE were analyzed using fluorescence microscopy for the evaluation of apoptosis (TUNEL), inflammation (ICAM-1), and proliferation (Ki67). Corneal epithelial tight junctions (occludin and ZO-1) were also assessed by en-face confocal microscopy in response to the different eye drops. Results: The MTT test revealed that the cytotoxicity of antiglaucoma eye drops was primarily related to the concentration of their common BAC preservative (0.02%BAC-latanoprost > 0.015%BAC-travoprost > 0.005%BAC-bimatoprost). PF-tafluprost did not induce obvious cytotoxicity and showed the least expression of inflammatory or apoptotic markers and revealed preservation of membrane immunostaining of tight junction proteins in comparison with BAC-containing solutions. Conclusion: The toxicological model of 3D reconstructed corneal epithelia model confirmed the ocular surface cytotoxicity of BAC-containing antiglaucomatous solutions. Compared to the formulations containing the toxic preservative BAC, PF-tafluprost was well tolerated without inducing significant corneal epithelium deterioration

Effets inflammatoires et toxiques des préparations ophtalmiques topiques sur la surface oculaire (développement et standardisation de modèles d'évaluation in vitro et in vivo)

Le programme REACH et l interdiction d utiliser les animaux pour tester les produits cosmétiques en 2013 imposent de revoir les outils classiques d évaluation de la toxicité oculaire et de développer de nouvelles approches. D abord, nous avons utilisé des systèmes in vitro classiques pour explorer la toxicité de collyres antiallergiques grâce à des tests de stress oxydant, d apoptose et d inflammation et nous avons ainsi pu relier leur toxicité à la présence de chlorure de benzalkonium (BAC), conservateur et irritant de référence des collyres dans nos études. Ensuite, nous avons enrichi le test classique de Draize, de méthodes sensibles et non invasives telles que l analyse en cytométrie en flux des cellules de la conjonctive, le dosage de cytokines dans les larmes et l analyse en microscopie confocale in vivo (IVCM) de la cornée. Dans le but d une standardisation indispensable à la conduite d études toxicologiques multicentriques, nous avons développé une classification précise des différentes altérations cytologiques et histologiques, allant de la cellule épithéliale, aux kératocytes, aux cellules endothéliales et vasculaires. Associée à la mesure de l épaisseur cornéenne, elle a permis de construire une échelle de cotation de gravité des lésions cornéennes, délivrant un score, valeur numérique objective, associé à un profil de sévérité toxique. In vitro, sur des épithéliums cornéens humains reconstruits, nous avons mis au point une nouvelle procédure de test MTT, plus sensible pour l évaluation de la cytotoxicité et l avons corrélé à une batterie de tests immunohistologiques couvrant des mécanismes d inflammation, d apoptose et de proliférationThe REACH program and the future ban to use animals for the toxic evaluation of cosmetics in 2013 impose to revise the classic tools of eye toxicity testing and to develop new approaches. We first used classic in vitro systems to investigate the toxicity of antiallergic eye drops by evaluating the induction of oxidative stress, apoptosis and inflammation. We were so able to connect their toxicity to the presence of benzalkonium chloride (BAC), the common eyedrop preservative used as the irritant of reference in our studies. Then we used flow cytometric analysis of conjunctival brushings, in conjunction with multiple cytokine assays in tears and in vivo confocal microscopy (IVCM) of the cornea as sensitive and noninvasive tools to refine the classical Draize eye test. In the perspective of future multicenter studies, we standardized the use of IVCM by developing a scoring system, based on the corneal thickness measurement and the precise classification of cytological and histological changes, at the level of the epithelial cells, the keratocytes, the endothelial and the vascular cells. This system enables to deliver a gravity score, as an objective numerical value for the assessment of the toxic-induced corneal damage severity. In vitro, using a reconstituted human corneal epithelium model, we developed a new, more sensitive MTT procedure for the evaluation of cytotoxicity, and correlated this method with a panel of immunohistological tests covering inflammatory, as well as apoptotic and proliferative mechanismsPARIS-BIUP (751062107) / SudocSudocFranceF

Correlation between the retroversion of the humeral head and the orientation of the intertubercular sulcus: a CT scan anatomical study

International audienceThe purpose was to study the anatomy of the humeral head, more specifically the retroversion of the humeral head and the orientation of the intertubercular sulcus, using CT scan, and to make correlations between those two entities of the proximal humerus. Sixty dry adult humeri from an osteological collection underwent CT scan from the proximal to the distal extremity. The measurements obtained by CT-reformation were recorded by two independent radiologists. We determined the humeral head axis, the transepicondylar axis, the retroversion of the humeral head, and the orientation of the intertubercular sulcus (ITS). Statistical analysis using SPSS determined the Pearson correlation coefficient. The CT scan measurements were similar to those in the literature, and thus allowed us to validate CT scan assessment. Statistical analysis showed a significant reverse correlation [the coefficient of correlation was -0.37 (p = 0.004)] between the retroversion of the humeral head and the orientation of the intertubercular sulcus: the more the retroversion of the humeral head increases, the more the angle of the orientation of the ITS decreases. To the best of our knowledge, this reverse correlation between the retroversion of the humeral head and the orientation of the intertubercular sulcus has never been described. This new anatomical data might be helpful for orthopedic surgery

Th1- and Th2-related chemokine and chemokine receptor expression on the ocular surface in endotoxin-induced uveitis.

International audienceTo determine whether the ocular surface inflammation in uveitis mimics or counteracts intraocular inflammatory pathways by directly comparing T-helper (Th) lymphocytes Th1 and Th2 markers in conjunctival and ciliary body expression in endotoxin-induced uveitis (EIU). This study used the following inflammatory markers: chemokine receptor, CC chemokine receptor 4 (CCR4), and its ligand, macrophage-derived chemokine (MDC), to evaluate Th2 participation; chemokine receptor, CCR5, to evaluate the Th1 system; and its ligand, regulated on activation normal T cell expressed and secreted (RANTES), to evaluate both Th1 and Th2 systems

Comparative study of topical anti-allergic eye drops on human conjunctiva-derived cells: responses to histamine and IFNγ and toxicological profiles

International audienc

A dynamic study of the anterior cruciate ligament of the knee using an open MRI.

Place: GermanyRecent anatomical and radiological studies of the anterior cruciate ligament (ACL) suggest the ACL length and orientation change during knee flexion, and an open MRI sequencing during knee flexion enables a dynamic ACL analysis. This study's goal is to describe a normal ACL using a 1T open MRI and, in particular, variations in length and insertion angles at different degrees of flexion. Twenty-one volunteers with clinically healthy knees received a dynamic MRI with their knees in hyperextension, neutral position, and flexed at 45° and 90° angles. For each position, two radiologists measured the ACL lengths and angles of the proximal insertion between the ACL's anterior edge and the roof of the inter-condylar notch. Additionally, we measured the ACL's and the tibial plateau's distal angle insertion between their anterior edges and then compared these with the nonparametric Wilcoxon test. The ACL had a significant extension between the 90° flexion and all other positions (hyperextension: 31.75 ± 2.5 mm, neutral position: 32.5 ± 2.6 mm, 45°: 35.6 ± 1.6 mm, 90°: 35.6 ± 1.6 mm). There was also a significant increase of the angle insertion between the proximal 90° flexion and all other positions, as well as between hyperextension and bending to 45° (hyperextension: 2.45° ± 3.7°, neutral: 13.4° ± 9.7°, 45°: 33 25 ± 9.3, 90: 51.85° ± 9.3°). Additionally, there is a significant increase in the distal angle insertion for all positions (hyperextension: 133.2° ± 5.4°, neutral position: 134.95° ± 4.4°, 45°: 138.35° ± 5.9°, 90°: 149.15° ± 8.6°). Our study is the first to exhibit that a dynamic MRI has a significant ACL extension in vivo during bending. This concept opens the way for further studies to improve the diagnosis of traumatic ACL injuries using a dynamic MRI