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Aim: To investigate the interest of chromatic confocal microscopy (CCM) to characterise guttae in Fuchs endothelial corneal dystrophy (FECD)., Methods: Descemet's membranes (DM) were obtained during endothelial keratoplasty in patients with FECD and pseudophakic bullous keratopathy (PBK). They were compared with healthy samples obtained from body donation to science. Samples were fixed in 0.5% paraformaldehyde and flat mounted. Surface roughness of DMs was quantified using CCM and the AltiMap software that provided the maximum peak (S p ) and valley (S v ) heights, the mean square roughness (R q ) and the asymmetry coefficient (S sk )., Results: The physiological roughness of healthy samples was characterised by an R q of 0.12±0.05 µm, which was two times rougher than in PBK (R q =0.06±0.03 µm), but both were still flat with a symmetrical distribution between peaks and valleys (S sk close to 0, n peaks =n valleys ), smaller than 1 µm. In FECD, the maximum peak height was 5.10±2.40 µm, up to 5.8 and 8.3 times higher than the control and PBK, respectively. The maximum valley depth was half than the peak (2.28±0.89 µm). The surface with guttae was very rough (R q =0.45±0.14 µm) and the S sk =1.84± 0.43 µm, greater than 0, confirms an asymmetric surface with high peaks and low valleys (n peaks >n valleys ). Moreover, the CCM provided quantitative parameters allowing to distinguish different types of guttae from different patients., Conclusions: CCM is an innovative approach to describe and quantify different morphologies of guttae. It could be useful to analyse the different stages of FECD and define subgroups of patients., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To determine whether the clinical and paraclinical course of Fuchs endothelial corneal dystrophy (FECD) over 1 year is related to the extent of triplet repetition in the transcription factor 4 (TCF4) gene., Methods: A prospective study with a 1-year follow-up was conducted. A total of 104 patients (160 eyes) with FECD and an equivalent number of age- and sex-matched control subjects without FECD were included. At inclusion, the corneas were graded using the modified Krachmer grade (KG) and patients were genotyped for the number of trinucleotide repeats (TNRs) in the TCF4 gene by the short tandem repeat assay. Visual acuity, Scheimpflug tomographic features, and the Visual Function and Corneal Health Status using a visual disability instrument were measured on 2 visits at 1-year intervals., Results: KGs ranged from 1 to 6, and 46% of eyes had grades 1 to 4. 71% of the patients harbored TNR expansion (>40) versus 13% in control subjects (P < 0.001). Severity at inclusion was higher in the presence of TNR expansion when considering eyes independently (mean grade ±SD, 4.08 ± 1.42) without TNR expansion and 4.66 ± 1.27 with TNR expansion (P = 0.024). In 1 year, the ETDRS score significantly decreased by -2.97 (95% confidence interval -4.69 to -1.26, P = 0.001) and the ETDRS score with glare by -4.25 (95% confidence interval -6.22 to -2.27, P < 10-5). There was no relationship between the rate of decline and TNR expansion or KG. Central corneal thickness and Visual Function and Corneal Health Status scores did not significantly vary., Conclusions: It is possible to measure a subtle progression of FECD over a period as short as 1 year. We did not find a relationship between the presence of TNR expansion and the speed of deterioration over 1 year. This work should facilitate the design of future clinical trials on FECD.Trial Registration-URL: ClinicalTrials.gov. Identifier: French Fuchs Follow-up Study (F3S): NCT03974230., Competing Interests: Conflicts of interest statement: P. Gain, N. Koizumi, N. Okumura, and G. Thuret were shown using the ICMJE disclosures form. The remaining authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

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.

Purpose: The objective of this study was to analyze the nycthemeral variations in blood pressure (BP) in individuals who presented with non-arteritic anterior ischemic optic neuropathy (NAION)., Methods: BP was recorded for 24 h (ambulatory blood pressure monitoring, ABPM) in 65 patients with acute NAION. Three definitions of nighttime periods were used: definition 1, 1 a.m.-6 a.m.; definition 2, 10 p.m.-7 a.m.; and definition 3, 10 p.m.-8 a.m. For each of these definitions, patients were classified according to the value of nocturnal reduction in BP into dippers (10-20%), mild dippers (0-10%), reverse dippers (< 0%), and extreme dippers (> 20%)., Results: The proportions of dippers, mild dippers, reverse dippers, and extreme dippers varied significantly depending on the definition chosen. We found the highest number of patients with extreme dipping (23%) when using the strictest definition of nighttime period (definition 1, 1 a.m.-6 a.m.), as compared with 6.2% and 1.5% for the other definitions, respectively. Overall, 13 of 33 patients without known systemic hypertension (39%) were diagnosed with hypertension after ABPM. No risk factor for NAION was associated with the extreme-dipping profile. Finally, the prevalence of systemic hypertension was high (69%)., Conclusion: In our population of patients who had an episode of NAION, the proportion of extreme dippers was higher than that usually found in the literature. However, extreme dipping is not a frequent feature of patients with NAION as compared to patients with systemic hypertension. ABPM is recommended for all patients with NAION and unknown history of systemic hypertension., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The bioengineering of corneal endothelial grafts consists of seeding in vitro cultured corneal endothelial cells onto a thin, transparent, biocompatible, and sufficiently robust carrier which can withstand surgical manipulations. This is one of the most realistic alternatives to donor corneas, which are in chronic global shortage. The anterior capsule of the crystalline lens has already been identified as one of the best possible carriers, but its challenging manual preparation has limited its use. In this study, we describe a femtosecond laser cutting process of the anterior capsule of whole lenses in order to obtain capsule discs of 8 mm diameter, similar to conventional endothelial grafts. Circular marks made on the periphery of the disc indicate its orientation. Immersion in water for 3 days is sufficient to completely remove the lens epithelial cells and to enable the seeding of corneal endothelial cells, which remain viable after 27 days of culture. Therefore, this method provides a transparent, decellularized disc ready to form viable tissue engineered endothelial grafts.

Corneal epithelial barrier represents one of the major limitations to ocular drug delivery and can be explored non-invasively through the evaluation of its electrical properties. Human corneas stored in active storage machine (ASM) could represent an interesting physiological model to explore transcorneal drug penetration. We designed a new system adapted to human corneas preserved in ASM to explore corneal epithelial barrier function ex-vivo. A bipolar set-up including Ag/AgCl electrodes adaptors to fit the corneal ASM and a dedicated software was designed and tested on freshly excised porcine corneas ( n  = 59) and human corneas stored 14 days in ASM ( n  = 6). Porcine corneas presented significant and proportional decrease in corneal impedance in response to increasing-size epithelial ulcerations and acute exposure to benzalkonium chloride (BAC) 0.01 and 0.05%. Human corneas stored 14 days in ASM presented a significant increase in corneal impedance associated with the restoration of a multi-layer epithelium and an enhanced expression of tight junctions markers zonula occludens 1, claudin 1 and occludin. These results support the relevance of the developed approach to pursue the exploration and development of human corneas stored in ASM as a physiological pharmacological model., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Philippe Gain reports financial support was provided by 10.13039/501100010115Région Auvergne-Rhône-Alpes (France). Philippe Gain, Gilles Thuret has patent #EP2991479B1 issued to Francais du Sang Ets, Universite Jean Monnet Saint Etienne, Ecole Nationale dIngenieurs de Saint Etienne ENISE., (© 2024 The Authors.)

Introduction: Keratoconus has a significant impact on patients' quality of life (QoL), from diagnosis to the advanced stages of the disease. The aim of this research was to identify domains of QoL affected by this disease and its treatment., Methods: Phone interviews were conducted using a semi-structured interview guide, with patients with keratoconus stratified according to their current treatment. A board of keratoconus experts helped identify the guide's main themes., Results: Thirty-five patients (rigid contact lenses, n = 9; cross-linking, n = 9; corneal ring implants, n = 8; and corneal transplantation, n = 9) were interviewed by qualitative researchers. Phone interviews revealed several QoL domains affected by the disease and its treatments: "psychological", "social life", "professional life", "financial costs" and "student life". All domains were impacted, independently of the treatment history. Few differences were found between treatment regimens and keratoconus stages. Qualitative analysis enabled the development of a conceptual framework based on Wilson and Cleary's model for patient outcomes common to all patients. This conceptual model describes the relationship between patients' characteristics, their symptoms, their environment, their functional visual impairment and the impact on their QoL., Conclusions: These qualitative findings supported the generation of a questionnaire to evaluate the impact of keratoconus and its treatment on patients' QoL. Cognitive debriefings confirmed its content validity. The questionnaire is applicable for all stages of keratoconus and treatments and may help tracking change over time in regular clinical settings. Psychometric validation is yet to be performed before its use in research and clinical practices., (© 2023. The Author(s).)

Purpose: To assess tocilizumab (TCZ) as an emergent treatment for corticosteroid-resistant active Graves' orbitopathy (GO)., Methods: We conducted a single-centre prospective study. We assessed TCZ in patients with active corticosteroid-resistant GO. Each patient received intravenous TCZ every four weeks until symptom stabilization. Our primary outcome was GO activity evaluated by the clinical activity score (CAS). The secondary outcomes included variation in thyroid-stimulating immunoglobulin (TSI)., Results: We included ten patients. Three patients had compressive neuropathy with visual field impairment and vision loss. CAS improved significantly in 100% of the patients included in the analysis, with a decrease in the mean CAS of 4.5 ± 1.2 points ( p  = .003). There was a significant decrease in the TSI after therapy, from 21.7 ± 22.9 at baseline to 4.0 ± 3.3 ( p  = .006). A mean of three infusions was necessary to drastically decrease the TSI amount. The baseline mean before TCZ was 4.7 ± 1.2 and the final mean after TCZ IV infusion was 0.2 ± 0.4., Conclusions: Our study showed the efficiency of TCZ in patients with GO resistant to corticosteroid therapy, as shown in previous studies. Our present work adds two important pieces of information: TCZ might be particularly useful for GO with compressive neuropathy and it is efficient regardless of initial TSI level. Considering the numerous advantages over steroids (high response rate and lower rate of adverse events), further randomized controlled trials should be conducted to assess the possible place of TCZ as a first-line treatment.

Fuchs endothelial corneal dystrophy (FECD) is a slowly evolving, bilateral disease of the corneal endothelium, characterized by an abnormal accumulation of extracellular matrix (ECM) in the basement membrane (Descemet's membrane, DM). This results in the formation of small round excrescences, called guttae, and a progressive disappearance of endothelial cells. In the intermediate stage, the numerous guttae create significant optical aberrations, and in the late stage, the loss of endothelial function leads to permanent corneal edema. The molecular components of guttae have not been fully elucidated. In the current study, we conducted shotgun proteomics of the DMs, including guttae, obtained from patients with FECD and revealed that 32 proteins were expressed only in the FECD-DMs but not in the DMs of control subjects. Subsequent enrichment analyses identified associations with multiple ECM-related pathways. Immunostaining of flat-mounted DMs confirmed that 4 of the top 5 identified proteins (hemoglobin α, SRPX2, tenascin-C, and hemoglobin γδεβ) were expressed in FECD-DMs but not in non-FECD-DMs. Fibrinogen α was strongly expressed in FECD-DMs, but weakly expressed in non-FECD-DMs. We also demonstrated that matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) can display the in situ spatial distribution of biomolecules expressed in the DM, including the guttae., (© 2023. The Author(s).)

Corneal endothelial diseases are the leading cause of corneal transplantation. The global shortage of donor corneas has resulted in the investigation of alternative methods, such as cell therapy and tissue-engineered endothelial keratoplasty (TEEK), using primary cultures of human corneal endothelial cells (hCECs). The main challenge is optimizing the hCEC culture process to increase the endothelial cell density (ECD) and overall yield while preventing endothelial-mesenchymal transition (EndMT). Fetal bovine serum (FBS) is necessary for hCEC expansion but contains TGF-βs, which have been shown to be detrimental to hCECs. Therefore, we investigated various TGF-β signaling pathways using inhibitors to improve hCEC culture. Initially, we confirmed that TGF-β1, 2, and 3 induced EndMT on confluent hCECs without FBS. Using this TGF-β-induced EndMT model, we validated NCAM as a reliable biomarker to assess EndMT. We then demonstrated that, in a culture medium containing 8% FBS for hCEC expansion, TGF-β1 and 3, but not 2, significantly reduced the ECD and caused EndMT. TGF-β receptor inhibition had an anti-EndMT effect. Inhibition of the ROCK pathway, notably that of the P38 MAPK pathway, increased the ECD, while inhibition of the ERK pathway decreased the ECD. In conclusion, the presence of TGF-β1 and 3 in 8% FBS leads to a reduction in ECD and induces EndMT. The use of SB431542 or LY2109761 may prevent EndMT, while Y27632 or Ripasudil, and SB203580 or SB202190, can increase the ECD.

Background: To report the long-term outcomes of round autologous anterior lens capsules (ALCs) cut by a femtosecond laser (FSL) and transplanted onto refractory macular holes (MHs) in a prospective interventional study., Methods: Three eyes of three patients were included for persistent MH after reattached rhegmatogenous retinal detachment (RRD) (n = 2) or RRD recurrence by persistent MH (n = 1), in a university hospital. A 6 mm diameter ALC disc was carefully extracted during FSL-assisted lens extraction, stained with 0.06% trypan blue, decellularised, transplanted using a catheter and unfolded over the MH. Gas or silicone-oil tamponade was used. At 1 year, the main criterion was anatomic success, defined as complete MH closure. Secondary criteria were changes in best corrected visual acuity (BCVA), ellipsoid zone (EZ) and external limiting membrane (ELM) defects, complications., Results: Baseline data were: minimum and maximum diameters, respectively 887, 1079 and 1180 μm; 1260, 1213 and 1350 μm; central posterior staphyloma in two highly myopic eyes; number of prior surgeries 2 ± 1. At 1 year, the three MHs were closed with stable transplanted ALCs. Distant BCVA improved respectively from 3.0, 0.8, 3.0 to 1.0, 0.2, 0.7 logMAR, i.e. all eyes achieved ≥0.3 logMAR improvement. All patients had decreased EZ and ELM defects, without reaching normal profile. No adverse event occurred., Conclusions: FSL-cut ALC helps standardise this challenging surgery: it prevents from tears and facilitates manipulation, so that the ALC disc is perfectly transparent and biocompatible, with a large MH overlap. One-year follow-up highlighted that this technique helps safely close refractory MHs with satisfactory visual recovery., (© 2022. The Author(s), under exclusive licence to The Royal College of Ophthalmologists.)

Evaluating lipid profiles in human tissues and biofluids is critical in identifying lipid metabolites in dysregulated metabolic pathways. Due to various chemical characteristics, single-run lipid analysis has not yet been documented. Such approach is essential for analyzing pathology-related lipid metabolites. Age-related macular degeneration, the leading cause of vision loss in western countries, is emblematic of this limitation. Several studies have identified alterations in individual lipids but the majority are based on targeted approaches. In this study, we analyzed and identified approximately 500 lipid species in human biofluids (plasma and erythrocytes) and ocular tissues (retina and retinal pigment epithelium) using the complementarity of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase chromatography (RPC), coupled to high-resolution mass spectrometry. For that, lipids were extracted from human eye globes and blood from 10 subjects and lipidomic analysis was carried out through analysis in HILIC and RPC, alternately. Furthermore, we illustrate the advantages and disadvantages of both techniques for lipid characterization. RPC showed greater sensitivity in hydrophobicity-based lipid separation, detecting diglycerides, triglycerides, cholesterol, and cholesteryl esters, whereas no signal of these molecules was obtained in HILIC. However, due to coelution, RPC was less effective in separating polar lipids like phospholipids, which were separated effectively in HILIC in both ionization modes. The complementary nature of these analytical approaches was essential for the detection and identification of lipid classes/subclasses, which can then provide distinct insights into lipid metabolism, a determinant of the pathophysiology of several diseases involving lipids, notably age-related macular degeneration., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Retinal melanosome/melanolipofuscin-containing cells (MCCs), clinically visible as hyperreflective foci (HRF) and a highly predictive imaging biomarker for the progression of age-related macular degeneration (AMD), are widely believed to be migrating retinal pigment epithelial (RPE) cells. Using human donor tissue, we identify the vast majority of MCCs as melanophages, melanosome/melanolipofuscin-laden mononuclear phagocytes (MPs). Using serial block-face scanning electron microscopy, RPE flatmounts, bone marrow transplantation and in vitro experiments, we show how retinal melanophages form by the transfer of melanosomes from the RPE to subretinal MPs when the "don't eat me" signal CD47 is blocked. These melanophages give rise to hyperreflective foci in Cd47 -/- -mice in vivo, and are associated with RPE dysmorphia similar to intermediate AMD. Finally, we show that Cd47 expression in human RPE declines with age and in AMD, which likely participates in melanophage formation and RPE decline. Boosting CD47 expression in AMD might protect RPE cells and delay AMD progression., (© 2023. The Author(s).)

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

The pathophysiology underlying olfactory dysfunction is still poorly understood, and more efficient biomolecular tools are necessary to explore this aspect. Immunohistochemistry (IHC) on cross sections is one of the major tools to study the olfactory epithelium (OE), but does not allow reliable counting of olfactory sensory neurons (OSNs) or cartography of the OE. In this study, we want to present an easy immunostaining technique to compensate for these defects of IHC. Using the rat model, we first validated and pre-screened the key OSN markers by IHC on cross sections of the OE. Tuj-1, OMP, DCX, PGP9.5, and N-cadherin were selected for immunostaining on flat-mounted OE because of their staining of OSN dendrites. A simple technique for immunostaining on flat-mounted septal OE was developed: fixation of the isolated septum mucosa in 0.5% paraformaldehyde (PFA) preceded by pretreatment of the rat head in 1% PFA for 1 hour. This technique allowed us to correctly reveal the olfactory areas using all the 5 selected markers on septum mucosa. By combining the mature OSN marker (OMP) and an immature OSN marker (Tuj-1), we quantified the mature (OMP+, Tuj-1-), immature (OMP-, Tuj-1+), transitory (OMP+, Tuj-1+) and total OSN density on septal OE. They were respectively 42080 ± 11820, 49384 ± 7134, 14448 ± 5865 and 105912 ± 13899 cells per mm2 (mean ± SD). Finally, the same immunostaining technique described above was performed with Tuj-1 for OE cartography on ethmoid turbinates without flat-mount., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gavid et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The use of ultrafast laser pulses for eye anterior segment surgery has seen a tremendous growth of interest as the technique has revolutionized the field, from the treatment of myopia, hyperopia, and presbyopia in the cornea to laser-assisted cataract surgery of the crystalline lens. For the latter, a comprehensive understanding of the laser-tissue interaction has yet to be achieved, mainly because of the challenge of observing the interaction zone in situ with sufficient spatial and temporal resolution in the complex and multi-layered tissue of the crystalline lens. We report here on the dedicated characterization results of the laser-tissue interaction zone in the ex vivo porcine lens using three different methods: in situ and real-time microscopy, wide-field optical imaging, and phase-contrast microscopy of the histological cross sections. These complementary approaches together revealed new physical and biological consequences of laser irradiation: a low-energy interaction regime (pulse energy below ~1 µJ) with very limited cavitation effects and a stronger photo-disruption regime (pulse energy above 1 µJ) with a long cavitation duration from seconds to minutes, resulting in elongated spots. These advances in the understanding of the ultrafast laser's interactions with the lens are of the utmost importance for the preparation of the next-generation treatments that will be applied to the lens.

Background: Anophthalmia, microphthalmia and coloboma are a genetically heterogenous spectrum of developmental eye disorders. Recently, variants in the Wnt-pathway gene Frizzled Class Receptor 5 (FZD5) have been identified in individuals with coloboma and rarely microphthalmia, sometimes with additional phenotypes and variable penetrance., Materials and Methods: We identified variants in FZD5 in individuals with developmental eye disorders from the UK (including the DDD Study [www.ddduk.org/access.html]), France and Spain using whole genome/exome sequencing or customized NGS panels of ocular development genes., Results: We report eight new families with FZD5 variants and ocular coloboma. Three individuals presented with additional syndromic features, two explicable by additional variants in other genes ( SLC12A2 and DDX3X ). In two families initially showing incomplete penetrance, re-examination of apparently unaffected carrier individuals revealed subtle ocular colobomatous phenotypes. Finally, we report two families with microphthalmia in addition to coloboma, representing the second and third reported cases of this phenotype in conjunction with FZD5 variants., Conclusions: Our findings indicate FZD5 variants are typically associated with isolated ocular coloboma, occasionally microphthalmia, and that extraocular phenotypes are likely to be explained by other gene alterations.

Background: Nonarteritic anterior ischemic optic neuropathy (NAAION) is a major cause of blindness in individuals over 50 years of age, with no available effective treatment. The oral dual endothelin receptor antagonist, bosentan, increases retinal optic nerve head blood flow in healthy humans and glaucoma patients. The objective of this trial is to assess the efficacy of bosentan administered at the acute stage in improving outcomes in NAAION patients., Methods: ENDOTHELION (ENDOTHELin antagonist receptor in Ischemic Optic Neuropathy) is a phase III, interventional, prospective, multicentre, placebo-controlled randomised double-blind clinical trial. The primary outcome is change in the visual field mean deviation (MD) at 3 months (Humphrey 30-2 SITA standard programme). Secondary outcomes include MD and visual acuity changes up to 24 months, changes in peripapillary retinal nerve fibre and macular ganglion cell layer thickness in the affected eye, as measured by optical coherence tomography, rate of NAAION bilateralisation at 2 years, and quality-of-life. Patients over 50 years of age presenting with typical NAAION of recent onset (less than 21 days) are randomly assigned to either 125 mg oral bosentan or placebo, twice a day, during 8 weeks. Besides visits during the treatment phase, patients attend follow-up visits at 2, 3, 6, 12 and 24 months. The inclusion of patients began in August 2015 at five French University hospital ophthalmology departments and two specialised ophthalmology centres. It is planned to include 86 patients in this trial. To date we have included 72 patients and 49 have completed the full follow-up process., Discussion: An endothelin receptor antagonist is a potential approach to improving the anatomical and functional prognosis of patients with NAAION. This multicentre double-blind randomised controlled trial is an opportunity to assess (1) the effect of bosentan on the structure and function of the optic nerve in NAAION, at 3 months, (2) the effect of bosentan on the bilateralisation rate at 24 months and (3) the tolerance profile of bosentan in this population., Trial Registration: ClinicalTrials.gov NCT02377271 . Registered on March 3, 2015., (© 2022. The Author(s).)

Keratoconus (KC) is a multifactorial progressive ectatic disorder characterized by local thinning of the cornea, leading to decreased visual acuity due to irregular astigmatism and opacities. Despite the evolution of advanced imaging methods, the exact etiology of KC remains unknown. Our aim was to investigate the involvement of corneal epithelium in the pathophysiology of the disease. Corneal epithelial samples were collected from 23 controls and from 2 cohorts of patients with KC: 22 undergoing corneal crosslinking (early KC) and 6 patients before penetrating keratoplasty (advanced KC). The expression of genes involved in the epidermal terminal differentiation program and of the oxidative stress pathway was assessed by real time PCR analysis. Presence of some of the differentially expressed transcripts was confirmed at protein level using immunofluorescence on controls and advanced KC additional corneal samples. We found statistically significant under-expression in early KC samples of some genes known to be involved in the mechanical resistance of the epidermis (KRT16, KRT14, SPRR1A, SPRR2A, SPRR3, TGM1 and TGM5) and in oxidative stress pathways (NRF2, HMOX1 and HMOX2), as compared to controls. In advanced KC samples, expression of SPRR2A and HMOX1 was reduced. Decreased expression of keratin (KRT)16 and KRT14 proteins was observed. Moreover, differential localization was noted for involucrin, another protein involved in the epidermis mechanical properties. Finally, we observed an immunofluorescence staining for the active form of NRF2 in control epithelia that was reduced in KC epithelia. These results suggest a defect in the mechanical resistance and the oxidative stress defense possibly mediated via the NRF2 pathway in the corneal keratoconic epithelium., Competing Interests: The authors have declared that no competing interests exist.

Objectives: Corneal transplantation is the most common transplant worldwide and its success critically depends on the management of corneal graft rejection through topical steroid therapy during the first 12 months after surgery. There is currently no published data on adherence after keratoplasty. This pilot study aims to explore the adherence to topical steroid after penetrating keratoplasty using a smart electronic device., Methods: Thirty patients undergoing penetrating keratoplasty were included to evaluate the adherence to topical dexamethasone medication for 12 months after surgery. Patients received the usual post-transplantation treatment (topical dexamethasone) and follow-up after surgery (day 15, months 1, 2, 3, 4, 5, 6, 9, and 12). Adherence to treatment was monitored using the KaliJAR device (Kali Care, Santa Clara, CA, USA), which recorded the number of single-dose units (SDU) discarded. At control visits, data recorded by the device were compared to the manually count of SDU. Adherence ratio and individual adherence curve were explored for all patients., Results: Data from 27 patients showed a high agreement between adherence ratio calculated based on the device data and obtained from manual counting of the discarded SDU (intraclass coefficient correlation of 0.87 [95% CI: 0.738-0.938]). Mean adherence to the treatment over the 12-month study period was 95.2 ± 4%., Conclusions: Adherence to topical dexamethasone for 12 months after corneal transplantation was high. The connected device was able to record accurately the discarded SDU. This approach would be a particular interest in the early identification and personalized follow-up of poorly adherent patients., (© The Author(s) 2022.)

A preclinical study was performed to investigate the efficacy and safety of a new viral inactivated, devitalized, freeze-dried and gamma-sterilized human umbilical cord amniotic membrane ( l hUC-AM) for the treatment of deep scleral and corneal defects with or without perforation. Firstly, l hUC-AM was investigated on experimental deep sclerectomy in rabbit eyes ( n = 12) and compared to autograft ( n = 4) on cross section histology. Secondly, l hUC-AM was studied on a selected series of uncontrolled cases of corneal defects ( n = 18) with or without perforation, in dogs and cats. l hUC-AM tolerance, reconstruction of the deep corneal lesion and recovery of the structural aspect of the tissue were followed post-surgery. In experimental deep sclerectomy, histology showed that the l hUC-AM was well tolerated and degraded completely in 45 days while allowing an overall quality and kinetic of scleral regeneration, similar to autograft. In the clinical situations, l hUC-AM was well tolerated, with ocular inflammatory signs quickly decreasing after surgery. Mean follow-up was 16.40 ± 11.43 months. In 15 out of 18 cases, l hUC-AM allowed ocular surface wound healing. The ocular surface was fully reconstructed three months after surgery. This study suggests a good safety and efficacy profile of l hUC-AM in the treatment of deep corneal or scleral defect in animals. This new tissue should now facilitate the treatment of severe ocular surface diseases in humans.

Purpose: To report a detailed surgical procedure of tissue engineered endothelial keratoplasty (TEEK) in a rabbit model and its postoperative evaluation., Methods: TEEKs were prepared 7 days before transplantation by seeding human or rabbit corneal endothelial cells on either femtosecond laser-cut ultrathin human stromal lamellae (fs-UTSL) or femtosecond laser-cut human anterior lens capsule (fs-HALC). Thirty transplantations were performed on aphakic eyes. Recombinant tissue plasminogen activator (rTPA) was used throughout the surgery. The native endothelium was removed by full-surface scraping and central descemetorhexis. The transplantation was performed as a human Descemet's membrane endothelial keratoplasty. Controls included Descemetorhexis only and transplantation of carrier alone. Postoperative follow-up was performed by slit lamp and optical coherence tomography, followed by histology., Results: Controls remained oedematous. No fibrin occurred during surgery. All but three TEEKs adhered immediately. One/6 fs-UTSL and 9/16 fs-HALC cleared perfectly (p = 0.161). All failures could be explained by at least one of the following causes intraoperative bleeding, vitreous prolapsus, early partial detachment, postoperative irido corneal synechiea/angle closure. Presumed immune rejection was observed in three rabbits only after 4 weeks. Immunostaining with anti-human CD166 allowed to perfectly differentiate human cells from rabbit cells. In successful TEEK at 3 or 4 weeks, human cells formed a normal endothelium and started migrating outside the carrier., Conclusion: Though the transplantation of a TEEK in rabbits is a complex model with many causes of failure, established procedure including use of rTPA allows reliable preclinical study. In addition, we suggest that fs-HALC might be a potential carrier for TEEK., (© 2021 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Background: Meibomian gland dysfunction is the most common etiology of dry eye disease worldwide and intense pulsed light appears to be a promising treatment with encouraging results. Lacrystim® is a new IPL device (CE marking in 2019) and no studies have yet been published on it. We propose the first study on this device with an objective assessment of its efficacy and an extended follow-up over 6 months., Methods: Patients presenting with a dry eye disease (DED) with stable mild to moderate MGD and having received Lacrystim® treatment between june 2019 and june 2020 were included. 3 IPL sessions were performed at D0, D15 and D45 with 4 shots per side at a fluence of 8 mJ/cm2. DED clinical evaluation was performed at D0, D15, D45, 3rd month and 6th month: Oxford scale and break up time, Schirmer test and Ocular Surface Disease Index (OSDI) questionnaire. Lacrydiag® imaging device carried out an objective examination of tear film: interferometry, meibography, tear meniscus height and non-invasive break up time (NIBUT). The primary endpoint was the evolution in NIBUT between the first visit D0 and 3rd month. Data collection was done retrospectively. Statistical analysis was done using a linear mixed-effects model and a non-parametric linear mixed-effects model (R software)., Results: Forthy five consecutive patients were included. NIBUT significantly increased between D0 and 3rd month: mean difference of 1.63 seconds, IC95% [0.51; 2.62], (p = 0.002) with a prolonged effect at 6th month. OSDI and OXFORD scores and interferometry were also significantly improved at 3rd month and 6th month. There was no significant change in BUT, Schirmer test and tear meniscus height. No adverse event was noted., Conclusions: IPL delivered by Lacrystim® appears effective and safe to treat MGD although a randomized controlled trial is needed to validate its results., Trial Registration: This work was approved by a local ethics committee "Terre d'éthique" (institutional review board number: IRBN672019/CHUSTE) and registered on the clinicaltrial.gov website ( NCT04147962 , 01/11/2019)., (© 2022. The Author(s).)

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, SM ECD was also compared to Hoechst-stained cell nuclei count ( Hoechst ECD)., 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. SM ECD did not differ from Hoechst ECD ( 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.

Background—The COVID-19 pandemic has changed our standard practices: operating rooms were only available for functional emergencies and outpatient visits were drastically reduced in favor of telemedicine. Aim: To report the personalized “one-shot” surgery using absorbable 10-0 Vicryl (V10-0) or polyglactin 910 monofilament in mechanical corneal injuries from February 2020 to December 2021. Methods—Prospective case series with at least 12-months’ follow-up, in a French university hospital. Among the overall population of open or closed-globe emergencies (n = 40), non-penetrating corneal lamellar lacerations (long axis > 2 mm) in zone 1 (OTC group) were treated with V10-0 suture(s) (n = 10), replacing traditional non-absorbable 10-0 nylon suture(s) or medical options in first line. The outpatient visits were performed on day (D)10, month (M)2, M6 then every six months. One interim visit by phone teleconsultation was scheduled between D10 and M2, and other(s) as needed. The main outcome was best-corrected visual acuity (BCVA) at M6. Secondary outcomes included mainly corneal astigmatism (CA) at M6 complications. Results—Among the ten corneal wounds, there were three children (30%), eight domestic accidents (80%), three eyes with metallic foreign bodies (30%), four open-globe injuries (40%), and nine eyes that received high-speed projectiles or sharp objects (90%). The complete V10-0 suture(s) absorption occurred in all eyes between D10 and M2. At M6, mean far and near BCVA decreased from 0.680 ± 0.753 and 0.490 ± 0.338 preoperatively to 0.050 ± 0.071 and 0.220 ± 0.063 logMAR (p = 0.019 and p = 0.025 respectively), mean CA decreased from 4.82 ± 3.86 preoperatively to 1.15 ± 0.66 diopters (p = 0.008). BCVA and CA were unchanged thereafter. No serious adverse event nor repeated surgery occurred. The mean number of teleconsultations was 1.20 ± 0.63 without an additional nonscheduled outpatient visit. Conclusions—The absorbable V10-0 sutures might be a safe and effective alternative for eligible corneal wounds, while reducing the number of outpatient visits, especially for children (no suture removal). The COVID-19 pandemic highlighted that they are ideally suited to logistical challenges.

Objectives: To assess to which extent the COVID-19 pandemic affected corneal transplantation by virtue of donor selection algorithms in different European countries., Design: Survey., Setting: 110 eye banks in 26 European countries., Participants: 64 eye banks covering 95% of European corneal transplantation activity., Interventions: A questionnaire listing the number of corneas procured and distributed from February to May 2018-2020 was circulated to eye banks., Main Outcome Measures: The primary outcome was the number of corneal procurements. Additional outcomes were national algorithms for donor selection, classified according to their stringency (donors with COVID-19 history, suspected for COVID-19, asymptomatic, PCR testing) and the pandemic severity in each country. We calculated Spearman's correlation coefficient to determine, two by two, the relationship between the 3-month decline in eye banking activity (procurement), the stringency of donor selection algorithm and the grading of pandemic severity (cases and deaths). A partial correlation was run to determine the relationship between decline and stringency while controlling for pandemic severity., Results: Procurements decreased by 38%, 68% and 41%, respectively, in March, April and May 2020 compared with the mean of the previous 2 years, while grafts decreased, respectively, by 28%, 68% and 56% corresponding to 3866 untreated patients in 3 months. Significant disparities between countries and the decrease in activity correlated with stringency in donor selection independent of pandemic severity., Conclusions: Our data demonstrate significant differences between countries regarding donor screening algorithms based on precautionary principles and, consequently, a decrease in the donor pool, already constrained by a long list of contraindications. Fundamental studies are needed to determine the risk of SARS-CoV-2 transmission by corneal transplantation and guide evidence-based recommendations for donor selection to justify their substantial medical and economic impact., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To report the long-term outcomes of large diameter epiretinal lyophilized amniotic membranes (lAMs) in recurrent or persistent macular holes (MHs) with or without rhegmatogenous retinal detachment (RRD), in a prospective interventional case series., Methods: Ten eyes of 10 patients underwent pars plana vitrectomy for MH-associated RRD (n = 5) or persistent MH without RRD (n = 5), in a university Hospital. A 3 or 4 mm diameter disc of lAM, stained with 0.06% trypan blue, was inserted with a catheter through a sclerotomy and positioned over the MH. Gas or silicone-oil tamponade was used. At 1 year, the main outcome was anatomic success defined as complete MH closure. Secondary outcomes were best corrected visual acuity (BCVA) recovery, changes in ellipsoid zone (EZ) and external limiting membrane (ELM) defects, complications. Mean follow-up was 13.8 ± 2.9 months (range, 12-18)., Results: Mean baseline data were minimum and maximum diameters, respectively, 945 ± 330 and 1507 ± 717 μm; axial length 26.58 ± 3.38 mm; and number of prior surgeries 1.4 ± 0.96. At 1 year, anatomic success was achieved in eight eyes (80%), and two had reduced diameter of MH. All RRDs were reattached without recurrence. Mean logMAR BCVA improved from 1.92 ± 0.58 to 1.17 ± 0.57 (p < 0.001), with nine eyes (90%) achieving ≥0.3 logMAR improvement. Mean EZ and ELM defects decreased (p = 0.004, p = 0.003, respectively). Postoperative complications were RRD (n = 1) reattached by subsequent surgery, lAM slightly retracted under silicone (n = 1), foveal atrophy after early lAM displacement (n = 1)., Conclusion: A 1-year follow-up highlighted that epiretinal large discs of blue-stained lAM can help safely close refractory MHs, and provide satisfactory visual recovery., (© 2021 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Background: The risk of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission through corneal graft is an ongoing debate and leads to strict restrictions in corneas procurement, leading to a major decrease in eye banking activity. The aims of this study are to specifically assess the capacity of human cornea to be infected by SARS-CoV-2 and promote its replication ex vivo, and to evaluate the real-life risk of corneal contamination by detecting SARS-CoV-2 RNA in corneas retrieved in donors diagnosed with Coronavirus Disease 2019 (COVID-19) and nonaffected donors., Methods and Findings: To assess the capacity of human cornea to be infected by SARS-CoV-2, the expression pattern of SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE-2) and activators TMPRSS2 and Cathepsins B and L in ocular surface tissues from nonaffected donors was explored by immunohistochemistry (n = 10 corneas, 78 ± 11 years, 40% female) and qPCR (n = 5 corneas, 80 ± 12 years, 40% female). Additionally, 5 freshly excised corneas (80 ± 12 years, 40% female) were infected ex vivo with highly concentrated SARS-CoV-2 solution (106 median tissue culture infectious dose (TCID50)/mL). Viral RNA was extracted from tissues and culture media and quantified by reverse transcription quantitative PCR (RT-qPCR) (viral RNA copies) 30 minutes (H0) and 24 hours (H24) after infection. To assess the risk of corneal contamination by SARS-CoV-2, viral RNA was tested by RT-qPCR (Ct value) in both corneas and organ culture media from 14 donors diagnosed with COVID-19 (74 ± 10 years, 29% female) and 26 healthy donors (79 ± 13 years, 57% female), and in organ culture media only from 133 consecutive nonaffected donors from 2 eye banks (73 ± 13 years, 29% female). The expression of receptor and activators was variable among samples at both protein and mRNA level. Based on immunohistochemistry findings, ACE-2 was localized mainly in the most superficial epithelial cells of peripheral cornea, limbus, and conjunctiva, whereas TMPRSS2 was mostly expressed in all layers of bulbar conjunctiva. A significant increase in total and positive strands of IP4 RNA sequence (RdRp viral gene) was observed from 30 minutes to 24 hours postinfection in central cornea (1.1 × 108 [95% CI: 6.4 × 107 to 2.4 × 108] to 3.0 × 109 [1.4 × 109 to 5.3 × 109], p = 0.0039 and 2.2 × 107 [1.4 × 107 to 3.6 × 107] to 5.1 × 107 [2.9 × 107 to 7.5 × 107], p = 0.0117, respectively) and in corneoscleral rim (4.5 × 109 [2.7 × 109 to 9.6 × 109] to 3.9 × 1010 [2.6 × 1010 to 4.4 × 1010], p = 0.0039 and 3.1 × 108 [1.2 × 108 to 5.3 × 108] to 7.8 × 108 [3.9 × 108 to 9.9 × 108], p = 0.0391, respectively). Viral RNA copies in ex vivo corneas were highly variable from one donor to another. Finally, viral RNA was detected in 3 out of 28 corneas (11%) from donors diagnosed with COVID-19. All samples from the 159 nonaffected donors were negative for SARS-CoV-2 RNA. The main limitation of this study relates to the limited sample size, due to limited access to donors diagnosed with COVID-19 and concomitant decrease in the procurement corneas from nonaffected donors., Conclusions: In this study, we observed the expression of SARS-CoV-2 receptors and activators at the human ocular surface and a variable increase in viral RNA copies 24 hours after experimental infection of freshly excised human corneas. We also found viral RNA only in a very limited percentage of donors with positive nasopharyngeal PCR. The low rate of positivity in donors diagnosed with COVID-19 calls into question the utility of donor selection algorithms., Trial Registration: Agence de la Biomédecine, PFS-20-011 https://www.agence-biomedecine.fr/., Competing Interests: The authors have declared that no competing interests exist.

The state of the art therapy for treating corneal endothelial disease is transplantation. Advances in the reproducibility and accessibility of surgical techniques are increasing the number of corneal transplants, thereby causing a global deficit of donor corneas and leaving 12.7 million patients with addressable visual impairment. Approaches to regenerate the corneal endothelium offer a solution to the current tissue scarcity and a treatment to those in need. Methods for generating corneal endothelial cells into numbers that could address the current tissue shortage and the possible strategies used to deliver them have now become a therapeutic reality with clinical trials taking place in Japan, Singapore and Mexico. Nevertheless, there is still a long way before such therapies are approved by regulatory bodies and become clinical practice. Moreover, acellular corneal endothelial graft equivalents and certain drugs could provide a treatment option for specific disease conditions without the need of donor tissue or cells. Finally, with the emergence of gene modulation therapies to treat corneal endothelial disease, it would be possible to treat presymptomatic patients or those presenting early symptoms, drastically reducing the need for donor tissue. It is necessary to understand the most recent developments in this rapidly evolving field to know which conditions could be treated with which approach. This article provides an overview of the current and developing regenerative medicine therapies to treat corneal endothelial disease and provides the necessary guidance and understanding towards the treatment of corneal endothelial disease., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: To report evidences that the abnormal endothelium of some Fuchs endothelial corneal dystrophy (FECD) present centripetal radial lines over 360 degrees., Methods: A case report of retroilluminated pictures of 2 patients with FECD and flat mounts of isolated Descemet membranes of 1 patient with FECD and of 1 healthy donor. Interpretation and development of a new pathophysiological theory., Results: The 3 FECD images unequivocally demonstrate the existence of very numerous radial centripetal lines over 360 degrees, in the central 8 to 9 mm of the cornea and ending in the area of maximum guttae concentration. These lines resemble, in a much longer length, the physiological striae that we described in 2012 at the periphery of the endothelium of normal corneas., Conclusions: We suppose that these lines reflect an accelerated migration of a population of pathological endothelial cells that deposit collagen on their path before being slowed down and then blocked in the center, explaining the progressive accumulation of guttae in this area. This new migration theory assumes that FECD behaves as a corneal endothelial stem-cell disease., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

In 2013, a clinical trial was initiated to investigate cell therapy for the treatment of corneal endothelial decompensation. Cultivating human corneal endothelial cells (CECs) while maintaining their functional phenotype is challenging; therefore, establishment of a confirmed protocol is pivotal for obtaining approval from regulatory authorities for use of cellular therapy products. In this study, we evaluated organ culture (OC) as a storage method for donor corneas used as a raw material for establishing CEC cultures. OC allows storage of corneal tissue for conventional corneal transplantation at 31-37 °C for up to 5 weeks, whereas storage at 4 °C is limited to 2 weeks. We investigated 20 pairs of corneas: one cornea of each pair was stored in OC and the other in cold storage for one week before CEC culture. In 15/20 cases, the CECs assumed a hexagonal sheet-like monolayer structure and expressed endothelial function-related markers. CECs were also obtained from OC corneas that had been stored for 1 (n = 19) and 2 (n = 7) months. As a further test, CECs were cultivated from 5 OC corneas that had been transported from France to Japan. In all cases, these corneas, even after international transport, generated CECs that formed hexagonal monolayers with clinically applicable and sufficiently high cell densities. In conclusion, the CEC cultures required for endothelial cell therapy can be obtained from OC corneas without changing the standard storage operating procedures of the eye banks., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Objectives: Staphylococcus aureus is one of the main causes of bacterial keratitis in humans. This study was aimed at investigating the mechanisms of S. aureus adhesion to the human corneal epithelium involved during the initial stage of infectious keratitis., Methods: Human corneas stored in a specific active storage machine that restores a normal pluristratified epithelium were used to assess S. aureus adhesion level to intact and injured tissues using immunostaining. S. aureus adhesion to immobilized fibronectin was measured in microtiter plate. Internalization of S. aureus clinical isolates recovered from keratitis was assessed on human corneal epithelial HCE-2 cells., Results: Superficial corneal injury unmasked fibronectin molecules expressed within the human corneal epithelium. S. aureus adhesion level was increased by 117-fold in the area of injured epithelium ( p < 0.0001). The deletion of staphylococcal fnbA/B genes decreased by 71% the adhesion level to immobilized fibronectin ( p < 0.001). The deletion of fnbA/B genes and the incubation of the corneas with anti-fibronectin blocking antibodies prior to the infection significantly reduced the S. aureus adhesion level to injured corneal epithelium ( p < 0.001). Finally, S. aureus clinical isolates triggered its internalization in human corneal epithelial cells as efficiently as the 8325-4 wt., Conclusion: S. aureus was almost unable to bind the intact corneal epithelium, whereas a superficial epithelial injury of the corneal epithelium strongly increased S. aureus adhesion, which is mainly driven by the interaction between staphylococcal fibronectin-binding proteins and unmasked fibronectin molecules located underneath the most superficial layer of the corneal epithelium., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Maurin, Courrier, He, Rigaill, Josse, Laurent, Gain, Thuret and Verhoeven.)

The detection of corneas operated on for refractive surgery [LASIK or photorefractive keratectomy (PRK)] will become a major concern for eye banks in the coming years because this surgery is often forgotten during the interview with the deceased's relatives. We present here 2 corneas operated on with PKR and stored successively in organ culture (OC) and in the active storage machine (ASM) that restores intraocular pressure, restores the cornea to its original shape, respects transparency and incorporates non-invasive controls. The 2 corneas of a 49-year-old donor operated 17 years earlier by PRK for -2 and -3 diopters myopia were stored in OC for 14 days and then placed in ASM for 48 h. Thickness map and OCT topography were performed under the 2 storage conditions, histology and electron microscopy were then performed. Traces of PRK remained unnoticed in OC while they were evident in ASM with central epithelial anomaly, central thinning and flattening of central keratometry shown by OCT. Histology and ultrastructure confirmed the absence of Bowman's membrane in the center. By placing the cornea under physiological conditions, and in particular by triggering its deswelling and by restoring its natural curvature, the ASM allows effective detection of subtle refractive surgery traces like those present after PRK., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature.)

Purpose: To develop a method to label proliferating corneal endothelial cells (ECs) in rabbits in vivo and track their migration over time., Methods: We compared intraperitoneal (IP) and intracameral (IC) administration of 5-ethynyl-2'-deoxyuridine (EdU) in two experiments: (1) six rabbits received IP or IC EdU. Blood and aqueous humor (AH) samples were incubated with HL-60 cells. Flow cytometry detected the EdU incorporation, representing the bioavailability of EdU. (2) In vivo EdU labeling was investigated in pulse-chase study: 48 rabbits received EdU IP or IC. The corneas were flat-mounted after 1, 2, 5, or 40 days and imaged using fluorescence microscopy. EdU+ and Ki67+ ECs were quantified and their distance from the peripheral endothelial edge was measured., Results: EdU was bioavailable in the AH up to 4 hours after IC injection. No EdU was detected in the blood or the AH after IP injection. High quality EdU labeling of EC was obtained only after IC injection, achieving 2047 ± 702 labeled ECs. Proliferating ECs were located exclusively in the periphery within 1458 ± 146 µm from the endothelial edge. After 40 days, 1490 ± 397 label-retaining ECs (LRCs) were detected, reaching 2219 ± 141 µm from the edge, indicating that LRCs migrated centripetally., Conclusions: IC EdU injection enables the labeling and tracking of proliferating ECs. LRCs seem to be involved in endothelial homeostasis, yet it remains to be investigated whether they represent endothelial progenitor cells., Translational Relevance: EdU labeling in animal models can aid the search for progenitor cells and the development of cell therapy for corneal endothelial dysfunction.

Muller glial cells (MGCs) are responsible for the homeostatic and metabolic support of the retina. Despite the importance of MGCs in retinal disorders, reliable and accessible human cell sources to be used to model MGC-associated diseases are lacking. Although primary human MGCs (pMGCs) can be purified from post-mortem retinal tissues, the donor scarcity limits their use. To overcome this problem, we developed a protocol to generate and bank human induced pluripotent stem cell-derived MGCs (hiMGCs). Using a transcriptome analysis, we showed that the three genetically independent hiMGCs generated were homogeneous and showed phenotypic characteristics and transcriptomic profile of pMGCs. These cells expressed key MGC markers, including Vimentin, CLU, DKK3, SOX9, SOX2, S100A16, ITGB1, and CD44 and could be cultured up to passage 8. Under our culture conditions, hiMGCs and pMGCs expressed low transcript levels of RLPB1, AQP4, KCNJ1, KCJN10, and SLC1A3. Using a disease modeling approach, we showed that hiMGCs could be used to model the features of diabetic retinopathy (DR)-associated dyslipidemia. Indeed, palmitate, a major free fatty acid with elevated plasma levels in diabetic patients, induced the expression of inflammatory cytokines found in the ocular fluid of DR patients such as CXCL8 (IL-8) and ANGPTL4. Moreover, the analysis of palmitate-treated hiMGC secretome showed an upregulation of proangiogenic factors strongly related to DR, including ANG2, Endoglin, IL-1β, CXCL8, MMP-9, PDGF-AA, and VEGF. Thus, hiMGCs could be an alternative to pMGCs and an extremely valuable tool to help to understand and model glial cell involvement in retinal disorders, including DR., (© 2021 The Authors. GLIA published by Wiley Periodicals LLC.)

Purpose: Lissamine green (LG) is often used in addition to fluorescein to assess the severity of conjunctival damage in dry eye syndrome, which is graded manually. Our purpose was to describe an algorithm designed for image analysis of LG conjunctival staining., Methods: Twenty pictures of patients suffering from dry eye with visible LG conjunctival staining were selected. The images were taken by 2 different digital slit lamps with a white light source and a red filter transmitting over the wavelengths absorbed by LG. Conjunctival staining appeared in black on a red background. The red channel was extracted from the original image. Stained areas were then detected using a Laplacian of Gaussian filter and applying a threshold whose value was determined manually on a subset of images. The same algorithm parameters remained constant thereafter. LG-stained areas were also drawn manually by 2 experts as a reference., Results: The delineation obtained by the algorithm closely matched the actual contours of the punctate dots. In 19 cases of 20 (95%), the algorithm found the same Oxford grade as the experts, even for confluent staining that was detected as a multitude of dots by the algorithm but not by the experts, resulting in a high overestimation of the total number of dots (without mismatching the Oxford grade estimated by the experts). The results were similar for the 2 slit-lamp imaging systems., Conclusions: This efficient new image-analysis algorithm yields results consistent with subjective grading and may offer advantages of automation and scalability in clinical trials., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: To characterize the corneal epithelium (CE) and limbal epithelium (LE) of human corneas stored in an innovative active storage machine (ASM) after a period of organ culture (OC)., Methods: Corneas unsuitable for graft and stored in a standard commercial OC medium for 2 to 5 weeks were transferred into our ASM for 14 days. The ASM actively maintained an overpressure on the endothelial side (20 mm Hg) while ensuring medium renewal. We compared three modalities of storage in the ASM's epithelial chamber: (1) alternating exposure to a supplemental hormonal epithelial medium (SHEM) and air (air-lifting), (2) continuous immersion in SHEM, and (3) continuous immersion in OC medium. Passive immersion of the whole cornea in OC medium or of the CE in SHEM with or without airlifting served as controls. Paired corneas were used for better comparability. Histology, differentiation (by immunolabeling), and ultrastructure were analyzed at the end., Results: The ASM with air-lifting was most effective in regenerating a pluristratified and differentiated CE (apical ZO-1 and MUC16 staining and regeneration of the glycocalyx). In addition, the LE was stratified with preserved expression of ABCB5. The ASM with immersion in SHEM or OC medium gave a less stratified and differentiated CE. In the three control groups, the epithelia, when present, were paucistratified and less differentiated., Conclusions: In human corneas previously stored in OC, the ASM regenerates a CE with differentiation characteristics close to normal., Translational Relevance: Regeneration of the epithelium of human corneas discarded by eye banks will increase tissue availability for research.

Purpose: With very photophobic patients, the advantages of red or near infrared light to develop new ophthalmology imaging devices seem obvious: no or little glare, possibility of long signal integration, no phototoxicity, and lesser autofluorescence of ocular tissues. Nevertheless, in this range, the shortest possible wavelength facilitates signal detection. The aim of this study was, thus, to determine the maximal irradiance tolerated with 6 wavelengths: 2 red, 2 far red, and 1 near infrared lights to determine the shortest wavelength well tolerated by patients, in comparison with the standard cobalt blue light of ophthalmology slitlamp., Methods: An interventional, monocentric, single-group assignment study was conducted on 30 eyes of 30 patients with infectious keratitis. Thanks to a customized machine, the photophobic eye was exposed to the 6 lights with increasing intensity. The patients switched off the light when the discomfort was too elevated. The maximal cumulative irradiance possible at 482, 650, 675, 700, 750, and 800 nm were 171, 689, 759, 862, 920, and 889 mW/cm, respectively., Results: The maximal cumulative irradiance tolerated by patients increased significantly with wavelength (P < 0.001), but the difference was not significant between each increment: red at 675 nm gave a significantly higher cumulative irradiance than blue at 482 nm; red at 700 nm did not provide significant gain compared with 675 nm; and far red at 750 nm still provided additional gain compared with 700 nm, but no significant gain was observed between 750 and 800 nm. The shortest wavelengths were stopped more quickly, and more than 50% of patients reached the maximum irradiance delivered by the source at 750 and 800 nm., Conclusions: We demonstrate that a light source at 750 and 800 nm can be used for ophthalmic imaging with good tolerance in photophobic patients., Clinical Trial Registration: NCT03586505.

The Optical Quality Analysis System (OQAS, Visiometrics) provides objective measurements of image formed onto retina, by combining quantification of ocular media transparency and of optical aberrations. In order to evaluate its contribution in the assessment of age-related cataract, we conducted a monocentric clinical study to determine the relationships between clinical grading of lens opacity, OQAS parameters, and parameters required for cataract surgery by phacoemulsification with ultrasound (called "phacodynamics"). Clinical parameters were: best-corrected visual acuity (BCVA, expressed as Log of minimal angle resolution (logMAR)) and the lens opacity classification system III (LOCS III) as a gold standard determined by two independent observers who graded total cataract and nuclear, cortical and posterior sub capsular components. The OQAS provided an objective scatter index (OSI), a modulation transfer function (MTF, expressed in cycle per degree (cpd)) and a Strehl ratio (SR) used as an aberration marker. Patients were operated on by the same surgeon using a phacoemulsification machine that provided the cumulative dissipated energy (CDE) and total ultrasound time (US time) necessary to extract the lens. Patients with poor compliance, corneal or retinal diseases impairing OSI, or who required surgical settings variation, were excluded. Twenty-one eyes of 21 patients aged 76±8 years were analyzed. They were 11 pure nuclear, 3 pure cortical, and 7 mixed cataracts. Mean LOCS III and OSI were respectively: 4.86 ±2.03 and 6.12 ±3.07 (mean±SD). Medians (10°-90° percentiles) were: for BCVA 0.30 (0.10-0.70) logMAR, for MTF cutoff 9.31 (1.54-30.57) cpd, for SR 0.071 (0.042-0.146), for CDE 8.04 (5.74-23.29) and for US time 58 (39-116) seconds. LOCS III was significantly correlated (spearman r, rs) with BCVA (rs = 0.561, p = 0.008), CDE (rs = 0.457, p = 0.038) and US time (rs = 0.647, p = 0.002). The three OQAS parameters significantly correlated (all rs ≥ 0.526, p<0.05) with BCVA, and LOCS III grading, but the strongest correlations were found with OSI for cortical components and with MTF for nuclear components: only OSI may be used objectively to assess the effect of cortical components on optical quality, and MTF cutoff-integrating scattering and aberrations-seems the best objective parameter for clinical assessment of nuclear cataracts. The three OQAS parameters were also significantly correlated (rs) with CDE, and with US time only for pure nuclear cataracts: OSI had the strongest correlations with phacodynamics (rs = 0.693, p = 0.022 with CDE and rs = 0.703, p = 0.019 US time). OSI increased with cortical components not requiring higher CDE. When measured in optimal conditions (good compliance, no retinal or ocular surface or tear film diseases), the three OQAS parameters are complementary for objective grading of cataract. In the future, they may help to optimize surgical parameters, especially energy distribution, in femtosecond laser assisted cataract surgery., Competing Interests: The authors have declared that no competing interests exist.

Background: Herpetic keratitis (HK) models using whole human corneas are essential for studying virus-host relationships, because of high species specificity and the role of interactions between corneal cell populations that cell culture cannot reproduce. Nevertheless, the two current corneal storage methods (hypothermia and organ culture (OC)) do not preserve corneas in good physiological condition, as they are characterized by epithelial abrasion, stromal oedema, and excessive endothelial mortality., Methods: To rehabilitate human corneas intended for scientific use, we used an active storage machine (ASM) that restores two physiological parameters that are essential for corneal homeostasis: intraocular pressure and storage medium renewal (21mmHg and 2.6 μL/min, respectively). ASM storage regenerates a normal multilayer epithelium in 2 weeks. We infected six pairs of corneas unsuitable for graft by inoculating the epithelium with herpes simplex virus type 1 (HSV-1), and compared each ASM-stored cornea with the other cornea stored in the same medium using the conventional OC method., Results: Only corneas in the ASM developed a dendritic (n = 3) or geographic (n = 2) epithelial ulcer reproducing typical HSV-1-induced clinical lesions. Corneas in OC showed only extensive desquamations. None of the uninfected controls showed epithelial damage. Histology, immunohistochemistry, transmission electron microscopy and polymerase chain reaction on corneal tissue confirmed infection in all cases (excluding negative controls)., Conclusions: The ASM provides an innovative ex vivo model of HK in whole human cornea that reproduces typical epithelial lesions., Competing Interests: Please note that PG and GT are inventors of the active storage machine on “patent US 20160029618A1” submitted by Jean Monnet University that covers “Medical device intended for long-term storage of a cornea, or for ex vivo experimentation on a human or animal cornea”. The other authors have no proprietary or commercial interest in any materials discussed in this article."I can confirm that this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Corneal storage for the very long term, without degradation, would make it possible to optimize a very limited resource worldwide. We previously demonstrated the superiority, compared to conventional 4-week passive organ culture (OC), of an active storage machine (ASM) that restores intraocular pressure and medium renewal. Here, we investigate eye banking for up to 3 months., Methods: In a randomized preclinical trial with 24 paired corneas, 1 was stored in OC and the other in ASM, using the same medium. Assessments were done on the second day and at 3 months: endothelial cell density (ECD in cells/mm), corneal transparency and thickness. At day 86, OC corneas were deswelled in a common hyperosmotic medium, but not the ASM corneas, which had remained thin. In addition, at day 88, viable ECD was measured using a live/dead assay, and endothelial expression of Na/K ATPase, Cox IV, ZO-1, N-CAM, and CD166 was observed., Results: The ASM extended storage to 3 months with unprecedented endothelial cell quality: no OC corneas remained suitable for transplantation, but one-third of ASM corneas were compliant (ECD > 2000/mm). Given that corneas with ECD > 1600/mm were also usable for emergency, 58% of ASM corneas were usable versus 33% in OC. EC survival was 53% higher in ASM (P < 0.001), structural and functional proteins of ECs were much better preserved in ASM, and it prevented the constant major edema of OC., Conclusions: By extending graft survival to 3 months, the ASM will optimize eye banking and open up new perspectives in experimental research.

Fresh corneal donation is essential for basic and preclinical research, but more unknown to public and the medical teams than donation for transplantation: it may raise concerns. We prospectively compared the acceptance rates and the characteristics of targeted corneal donation for research versus donation for transplantation during one year. The Agence de la Biomédecine authorized us to procure fresh corneas targeted for research, only from the donors with medical contraindications for transplantation, in order not to increase grafts shortage. Three nurses from the hospital coordination team of Saint-Etienne University Hospital, obtained consent for research and transplantation in parallel, screening all intra-hospital deaths cases, following standard protocol to check no refusal from families, despite the French opt-out system. They contacted 127 families for research and 244 for transplantation, in 71% of cases by telephone. Consent was obtained in 62% of cases for research and 54% for transplantation (P = 0.135). The main contraindication for transplantation was the cognitive disorders (66%) followed by the blood cancers (8%). This new specific activity, providing new source of fresh corneas for research immediately usable without any eyebank storage steps, didn't reduce the number of corneas procured for transplantation versus previous years (P = 0.998). Donors in the research group were 10 years older (P<0.001) without difference regarding endothelial cell quality (P = 0.071), allowing maximal clinical relevance for protocols using these fresh human scientific corneas provided by targeted donation., Competing Interests: GAIN, S ACQUART and G THURET are inventors on “patent US 20160029618A1” submitted by University Jean Monnet that covers “Medical device intended for long-term storage of a cornea, or for ex vivo experimentation on a human or animal cornea.” The patent does not alter our adherence to Plos One policies on sharing data and materials.

Purpose: Rapid identification of virulent pathogens is essential to strengthen the therapeutic strategy of acute endophthalmitis., Objectives: This study sought to compare the contribution of a combination of polymerase chain reaction (PCR)-based tests to culture methods, in patients with postoperative endophthalmitis., Design: Prospective multicenter study diagnostic evaluation., Methods: Setting: university referral centers., Participants: 153 consecutive patients presenting with acute or delayed-onset postoperative endophthalmitis, between 2008 and 2015. There were a total of 284 aqueous humor (AH) and/or vitreous fluid (VF) samples. Outcomes and measurements: microbiological tests of intraocular samples included bacterial culturing of pediatric blood culture bottles; 16SrDNA amplification and sequencing (panbacterial PCR) for detection and identification of all bacterial species; real-time PCR (qPCR) assays targeting the femA or lytA gene for detection of Staphylococcus aureus (S. aureus) or Streptococcus pneumoniae (S. pneumoniae), respectively; and a qPCR assay targeting the tuf gene for detection and quantification of Staphylococcus epidermidis (S. epidermidis)., Results: At the time of admission, the rate of detection of microorganisms by PCR-based tests was not significantly different than that by culturing (38% versus 30% in AH samples [n = 69]; 66% versus 63% in VF samples [n = 82], respectively). In contrast, after 1 intravitreal injection (IVI) of antibiotics, the identification rate by PCR-based tests was higher than that in VF by culturing (62% vs 48%, respectively; n = 94; P = 0.05). Bacteria were identified in 70% of patients, with a predominance of Gram-positive bacteria (93%). Specific qPCR tests targeting S. aureus and S. pneumoniae did not provide additional diagnoses but provided earlier results. The S. epidermidis load in vitreous at the time of patients' admission was higher in cases of final visual acuity (VA) of <20/40 (127,118 ± 125,848 DNA copies/mL) in patients with a VA of ≥20/40 (40350,000 ± 46,912 DNA copies/mL; P = 0.09). No significant changes in S. epidermidis load was found after one IVI., Conclusions: Patients with acute or delayed-onset endophthalmitis should benefit from microbiological identification in vitreous samples by combined analysis using bacterial cultures in pediatric blood culture bottles and panbacterial PCR. The last test was more effective than cultures in vitreous samples collected after an IVI of antibiotics. The qPCR tests targeting S. aureus and S. pneumoniae gave earlier results than culture and panbacterial PCR but did not provide additional diagnoses. As for S. epidermidis infections, determination of bacterial load using the qPCR test targeting the tuf gene could help evaluation of the visual prognosis of patients. Its role in the follow-up of patients after antibiotic treatment needs further investigation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objectives: Complete epithelial wound healing is a milestone in early postoperative care after penetrating keratoplasty. The re-epithelialization rate after penetrating keratoplasty was measured in patients receiving a new matrix therapy agent (regenerating agent, Cacicol ® ) that mimics heparan sulphates., Methods: This was a prospective, open-label, uncontrolled, single-centre observational study. A total of 33 consecutive patients (33 eyes) who underwent an 8.25-mm diameter penetrating keratoplasty were treated with regenerating agent eye drops: one drop in the operating theatre immediately after graft, then on alternate days. Patients were divided into those at low risk (13 patients) and high risk (20 patients) of delayed wound healing, and follow-up was performed by digital slit lamp with fluorescein-dye testing repeated daily at a fixed time. Dye area was measured using ImageJ freeware. The main endpoint was epithelial healing after regenerating agent therapy., Results: The mean ± standard deviation time to complete healing for all patients was 2.7 ± 1.1 (median: 3, range: 1-6) days. This was obtained on Day 1 for 15% of patients, Day 2 for 33%, Day 3 for 88%, Day 4 for 94% and Day 6 for 100%. There was no significant difference between low- and high-risk patients. The area of epithelial defect decreased by a mean ± standard deviation of 75% ± 22% between Day 1 and Day 2, corresponding to a mean ± standard deviation wound-healing rate of 11.5 ± 6.5 mm 2 /D. There were no systemic or local side effects related to regenerating agent., Conclusion: These preliminary data suggest that regenerating agent could be a useful, non-invasive therapeutic approach in postoperative management of penetrating keratoplasty with the potential to accelerate re-epithelialization.

The reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) has broad applications in regenerative medicine. The generation of self-organized retinal structures from these iPSCs offers the opportunity to study retinal development and model-specific retinal disease with patient-specific iPSCs and provides the basis for cell replacement strategies. In this study, we demonstrated that the major type of glial cells of the human retina, Müller cells, can be reprogrammed into iPSCs that acquire classical signature of pluripotent stem cells. These Müller glial cell-derived iPSCs were able to differentiate toward retinal fate and generate concomitantly retinal pigmented epithelial cells and self-forming retinal organoid structures containing retinal progenitor cells. Retinal organoids recapitulated retinal neurogenesis with differentiation of retinal progenitor cells into all retinal cell types in a sequential overlapping order. With a modified retinal maturation protocol characterized by the presence of serum and high glucose levels, our study revealed that the retinal organoids contained pseudolaminated neural retina with important features reminiscent of mature photoreceptors, both rod and cone subtypes. This advanced maturation of photoreceptors not only supports the possibility to use 3D retinal organoids for studying photoreceptor development but also offers a novel opportunity for disease modeling, particularly for inherited retinal diseases., Competing Interests: The authors declare that they have no conflicts of interest.

Optimal ex vivo corneal storage in eye banks is crucial to increase both the number of corneas suitable for graft and their intrinsic quality, mainly the number of viable endothelial cells, which dictates graft survival in recipients. With both passive storage methods used worldwide (short-term cold storage in the United States, long-term organ culture in Europe), significant endothelial cell loss is inevitable. Here we show that, with an active storage machine, also called a bioreactor, which restores 2 fundamental physiological parameters, intraocular pressure and medium renewal, endothelial cell survival is improved by 23% compared with organ culture after 4 weeks' storage. Also observed in the bioreactor is a 4-fold higher expression of Na + /K + ATPase, which supports one of the major endothelial cell pumping functions. In addition, corneas remain thin and transparent, so they are suitable for surgery at any time. This new active eye banking method may help to reduce the severe global scarcity of donor corneas., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

To identify the microstructural modification of the corneal layers during the course of the disease, optical technologies have been pushing the boundary of innovation to achieve cellular resolution of deep layers of the cornea. Gabor-domain optical coherence microscopy (GD-OCM), an optical coherence tomography-based technique that can achieve an isotropic of ∼2-μm resolution over a volume of 1  mm  ×  1  mm  ×  1.2  mm, was developed to investigate the microstructural modifications of corneal layers in four common corneal diseases. Since individual layer visualization without cutting through several layers is challenging due to corneal curvature, a flattening algorithm was developed to remove the global curvature of the endothelial layer and display the full view of the endothelium and Descemet's membrane in single en face images. As a result, GD-OCM revealed the qualitative changes in size and reflectivity of keratocytes in Fuchs endothelial corneal dystrophy (FECD), which varied by the degree of disease. More importantly, elongated shape and hyperactivation characteristics of keratocytes, associated with the early development of guttae, appeared to start in the posterior stroma very early in the disease process and move toward the anterior stroma during disease progression. This work opens a venue into the pathogenesis of FECD.

Background: In the clinical practice, transparent films are used as sterile interfaces in in vivo dermatologic imaging in order to prevent the transmissions of infections. However, in our experience, the use of a transparent film can alter skin images. Our study aimed to compare the optical quality of a series of different plastic films used as interfaces in order to understand if some might be more suitable for imaging., Materials and Methods: We tested the optical properties of 11 different protective transparent films that are marketed in France with a transparency meter and a spectrophotometer., Results: Transmission, minimal diffusion, amount of gray, and contrast were obtained for each transparent film. Transmission ranged from 93.24% to 96.88% (mean 95.36; standard deviation SD 1.02), minimal diffusion from 88.28% to 123.87% (mean 101.04; standard deviation SD 10.02) and contrast from 11.01 to 15.88 (mean 13.93 and SD 1.3). For some films, the transmission was lower at lower wavelengths., Conclusion: All tested films had excellent optical properties. However, some of them had better optical qualities and seemed more suitable for their use in dermatologic imaging., (© 2018 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd.)

Our aim was to measure the endothelial quality of prestripped Descemet membrane endothelial keratoplasty (DMEK) 48 h after preparation in an eye bank with the Muraine technique and shipping in a distant center. Ten pairs of human corneas with similar eye bank endothelial cell density (ebECD) were stored in organ-culture (OC) for 25 days (20, 28) [median (10-90 percentiles)]. One cornea was then randomized to DMEK preparation using the Moria Muraine trephine, the other served as control. The grafts were left attached to the center of the cornea, immersed in the OC medium (without Dextran) and shipped to a distant center. After 48 h, the viable ECD (vECD) was assessed by image analysis after staining with Hoechst/Ethidium/Calcein-AM. In addition, immunostaining was performed on flat mounted tissues for structural (ZO-1, NCAM, CD166) and functional (Na + /K + ATPase) proteins of ECs, and for collagen I. Just before stripping, ebECD was 2428 (2268-2669) cells/mm 2 for DMEK and 2471 (2135-2714) for controls (P = 1). Forty-eight hours after stripping, vECD was 2057 (1829-2463) cells/mm 2 for DMEK and 2119 (1496-2525) for controls (P = 0.508). The expression patterns of the 5 proteins were similar in ECs of both groups. Notably, the deep posterior folds observed in OC controls almost disappeared in prestripped DMEK due to the lack of a link between Descemet membrane and stroma. As a consequence of the elimination of mechanical stress in these zones, EC evenly covered the whole graft. In conclusion, DMEK prestripping with the Muraine technique and shipping away can be used safely by eye banks.