Background/aim Rejection is the main cause of graft failure after penetrating keratoplasty (PK). Its prevention by repeated instillation of steroid eye-drops has not evolved in decades. Poor adherence and discontinuous nature of eye-drop treatment may explain some PK failures. In a rabbit model, we previously demonstrated that a subconjunctival dexamethasone implant was well tolerated and prevented rejection efficiently in the first 5–6 weeks. This clinical trial investigates its tolerance and safety after PK. Methods Single-centre, phase II non-randomised tolerance and safety pilot study (NCT02834260). Designed to analyse the risk of elevated intraocular pressure (IOP), discomfort and resorption time. Fourteen patients with a low rejection risk indication of PK were enrolled between January 2017 and August 2018. The implant was injected in the 12 o’clock position, 5 mm from the limbus, at the end of PK. A steroid eye-drop treatment was planned when implant resorption was complete. Patients were monitored regularly for 12 months: IOP (main outcome measure at 1 month), discomfort and redness scores, implant status, rejection episode and central corneal thickness by optical coherence tomography. An independent data safety monitoring committee verified safety aspects. Results No increase in IOP or other adverse event related to the implant was observed. Average resorption time was 6 weeks. The switch to steroid eye-drops was uneventful. One patient, included despite preoperative corneal neovascularisation (unintended protocol deviation) experienced a rejection. Conclusions This is the first proof of concept that dropless immunosuppression is possible after low rejection risk PK. Trial registration number NCT02834260.

Purpose: Unlike corneas stored in cold storage (CS) which remain transparent and thin, corneas stored in organoculture (OC) cannot be assessed by specular microscopy (SM), because edema and posterior folds occur during storage and prevent from specular reflection. We previously developed an active storage machine (ASM) which restores the intraocular pressure while renewing the storage medium, thus preventing major stromal edema. Its transparent windows allow multimodal corneal imaging in a closed system. Aim: to present SM of corneas stored in this ASM. Methods: Ancillary study of two preclinical studies on corneas stored for one and three months in the ASM. A prototype non-contact SM was developed (CMOS camera, ×10 objective, collimated LED source, micrometric stage). Five non-overlapping fields (935 × 748 μm) were acquired in exactly the same areas at regular intervals. Image quality was graded according to defined categories (American Cornea Donor Study). The endothelial cell density (ECD) was measured with a center method. Finally, _SMECD was also compared to Hoechst-stained cell nuclei count (_HoechstECD). Results: The 62 corneas remained thin during storage, allowing SM at all time points without corneal deconditioning. Image quality varied depending on donors and days of control but, overall, in the 1100 images, we observed 55% of excellent and 30% of good quality images. _SMECD did not differ from _HoechstECD (p = 0.084). Conclusions: The ASM combines the advantages of CS (closed system) and OC (long-term storage). Specular microscopy is possible at any time in the ASM with a large field of view, making endothelial controls easy and safe.

Aim: The human corneal endothelium is a monolayer of flat hexagonal cells. It is a nearly regular hexagonal tessellation during the first years of life, but with age, becomes less regular in shape and size. The aim is to evaluate geometrically the age of an endothelial mosaic. Material and methods Segmented endothelial mosaics of healthy subjects of different age groups are compared by morphological criteria. The mosaics are studied according to their age group (decades), their age and their location (center or mid-periphery of the cornea). The measures used are: the cell density, the Ripley's L function and the cell area and perimeter density. Results: These measures point out the endothelial cell density decrease, the cell area, perimeter and diameter increase, the cell heterogeneity increase, and the differences between central and mid-peripheral cells increases with age. Conclusion: These measures are able to characterize healthy mosaics.

Eye drops containing steroids and antibiotics are widely used to treat a large range of ocular diseases of the ocular surface. They require frequent instillation or a high dosage, which can affect quality of life. We developed a biomaterial from human umbilical cord that can be loaded with drugs before being placed in the inferior conjunctival fornix. In the present work, this viro-inactivated, freeze-dried, and sterile foam was loaded with dexamethasone phosphate. We studied the release kinetic of 100 mg of biomaterial loaded with 100 µg of dexamethasone phosphate. Assays have shown that the product can be loaded with 100 µg of dexamethasone and allows a progressive release over time for at least 48 h. In addition, when compared with the instillation of the same dexamethasone quantity (100 µg), instilled regularly via eye-drop solution at 0.79 mg/mL, the drug penetration through corneal tissues was better with the dexamethasone-loaded biomaterial.

La cornée est le hublot transparent de l’oeil. Bien que de nombreux modèles alternatifs utilisant des cornées animales ex vivo aient vu le jour durant ces 30 dernières années, les recherches préclinique (étude de nouvelles stratégies diagnostiques et thérapeutiques) et fondamentale sur la cornée ont toujours besoin de l’expérimentation animale in vivo. Elle fait aujourd’hui l’objet d’une réglementation stricte afin d’éviter tout abus et maltraitance. Les animaux les plus fréquemment utilisés en recherche cornéenne sont des mammifères (souris, rat, lapin, chat, chien, cochon, boeuf et primate non humain). Malgré leur proximité phylogénétique de l’Homme, ces animaux peuvent présenter des différences notables avec la cornée humaines qui doivent être connues pour ne pas induire de biais dans l’expérimentation. Les objectifs de cette thèse sont de mettre au point les modèles animaux et les méthodes alternatives nécessaires aux travaux du laboratoire BiiGC (EA 2521, Université de Saint-Étienne, France). Ils sont illustrés par 3 projets innovants : 1/une étude préclinique utilisant un modèle de kératoplastie transfixiante chez le lapin pour évaluer la prévention du rejet d’allogreffe de cornée par implant sous conjonctival de déxamethasone ; 2/Le développement d’un bioréacteur cornéen porcin pour l’analyse de la cicatrisation épithéliale ; 3/ l’utilisation d’un modèle lapin de lésion endothéliale pour l’étude de la régénération endothéliale. Ces 3 travaux innovant démontrent la diversité des modèles animaux nécessaires en recherche fondamentale et translationnelle.

Purpose: The corneal bioreactor (BR) in our laboratory, with an intraocular pressure (IOP) of 21 mmHg and the renewal of the culture medium, allows to maintain the homeostasis of the 3 layers of the cornea. We showed that an IOP reduced to 10 mmHg allowed to simulate a corneal edema (760 ± 57 µm) with a healthy epithelium.Aim: To compare 2 hyperosmolar eye drops in the evolution of corneal deturgescence and epithelial ulcer healing: ODM5 composed mainly of NaCl 5% and hyaluronic acid (Horus Pharma, Saint‐Laurent‐du‐Var, France) and Muro 128 composed mainly of NaCl 5% and various excipients (Bausch+Lomb, Bridgewater, NJ)Methods: Beforehand, 3 pairs of human corneas were preserved for 2 weeks in the BR filled with CorneaMax (Eurobio, France), with a pressure maintained at 10 mmHg. A central epithelial ulcer of 6.64 ± 0.04 mm in diameter was made (D0). Each cornea received a drop of Muro 128 or ODM5 four times a day. Corneal central volume measurement (Casia 1, Tomey) was performed daily at the beginning and end of the day. Ulcer healing was monitored daily by fluorescein labelling and quantified by image analysis (Fiji) taken with a macroscope. At D14, the corneas were removed and fixed for immunostaining of E‐Cad (cell architecture), Zo‐1 (tight junctions), MUC16 (mucins), K3K12 (cell maturity) and for histology (Saffron Eosin Hemathein).Results: Mean daily volume loss was comparable: 2.43 ± 1.55 mm3 with ODM5 vs. 2.18 ± 1.71 mm3 with Muro 128 (p = 0.698 3). Corneal ulcers in all 3 pairs healed at D4, D5, D5 with ODM5 vs. D5, D13, and 1 cornea never healed with Muro 128. In IHC and histology, the epithelium was of better quality with ODM5.Conclusions: The efficacy of the 2 eye drops is comparable. Epithelial preservation is partly better with ODM5, possibly related to the presence of hyaluronic acid.