Publications by authors named "Gilles Thuret"

93 Publications

Reproducing diabetic retinopathy features using newly developed human induced-pluripotent stem cell-derived retinal Müller glial cells.

Glia 2021 Mar 8. Epub 2021 Mar 8.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France.

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.
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http://dx.doi.org/10.1002/glia.23983DOI Listing
March 2021

Radial Endothelial Striae Over 360 Degrees in Fuchs Corneal Endothelial Dystrophy: New Pathophysiological Findings.

Cornea 2021 Feb 15. Epub 2021 Feb 15.

Corneal Graft Biology, Engineering and Imaging Laboratory, BiiGC, EA2521, Federative Institute of Research in Sciences and Health Engineering, Faculty of Medicine, Jean Monnet University, Saint-Etienne, France; Department of Ophthalmology, University Hospital, Saint-Etienne, France; and Department of Biomedical Engineering, Doshisha University, Kyotanabe, Japan.

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.
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http://dx.doi.org/10.1097/ICO.0000000000002666DOI Listing
February 2021

Detection of refractive photokeratectomy traces during eye banking: impossible with organ culture but possible with an active storage machine: case report.

Cell Tissue Bank 2021 Jan 4. Epub 2021 Jan 4.

Laboratory for Corneal Graft Biology, engineering and imaging (BiiGC, EA2521), Faculty of Medicine, University Jean Monnet, Health Innovation Campus, 10 rue de la marandière, F-42055, Saint-Etienne, France.

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.
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http://dx.doi.org/10.1007/s10561-020-09895-4DOI Listing
January 2021

New Freeware for Image Analysis of Lissamine Green Conjunctival Staining.

Cornea 2021 Mar;40(3):351-357

Corneal Graft Biology, Engineering and Imaging Laboratory, Health Innovation Campus, Faculty of Medicine, Jean Monnet University, Saint-Etienne, France.

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.
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http://dx.doi.org/10.1097/ICO.0000000000002617DOI Listing
March 2021

One threat, different answers: the impact of COVID-19 pandemic on cornea donation and donor selection across Europe.

Br J Ophthalmol 2020 Nov 26. Epub 2020 Nov 26.

University Eye Clinic Maastricht, Maastricht University Medical Centre+, Maastricht, The Netherlands.

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.
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http://dx.doi.org/10.1136/bjophthalmol-2020-317938DOI Listing
November 2020

Using Optical Quality Analysis System for predicting surgical parameters in age-related cataract patients.

PLoS One 2020 12;15(10):e0240350. Epub 2020 Oct 12.

Ophthalmology Department, University Hospital, Saint-Etienne, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240350PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549767PMC
December 2020

Tolerance to Light of Patients Suffering From Infectious Keratitis.

Cornea 2021 Jan;40(1):5-11

Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Federative Institute of Research in Sciences and Health Engineering, Faculty of Medicine, Jean Monnet University, Saint-Etienne, France.

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.
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http://dx.doi.org/10.1097/ICO.0000000000002516DOI Listing
January 2021

Ex vivo model of herpes simplex virus type I dendritic and geographic keratitis using a corneal active storage machine.

PLoS One 2020 22;15(7):e0236183. Epub 2020 Jul 22.

Corneal Graft Biology, Engineering and Imaging Laboratory, Health Innovation Campus, Faculty of Medicine, Jean Monnet University, Saint-Etienne, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0236183PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375596PMC
September 2020

Corneal donation for research versus for transplantation: A-year prospective study of acceptance rates in a French University Hospital.

PLoS One 2020 21;15(5):e0233392. Epub 2020 May 21.

Corneal Graft Biology, Engineering and Imaging Laboratory, BiiGC, EA, Federative Institute of Research in Sciences and Health Engineering, Faculty of Medicine, Jean Monnet University, Saint-Etienne, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0233392PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241724PMC
August 2020

Three-month Storage of Human Corneas in an Active Storage Machine.

Transplantation 2020 06;104(6):1159-1165

Corneal Graft Biology, Engineering and Imaging Laboratory, BiiGC, EA2521, Federative Institute of Research in Sciences and Health Engineering, Faculty of Medicine, Jean Monnet University, Saint-Etienne, France.

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.
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http://dx.doi.org/10.1097/TP.0000000000003109DOI Listing
June 2020

Specific PCR and Quantitative Real-Time PCR in Ocular Samples from Acute and Delayed-Onset Postoperative Endophthalmitis.

Am J Ophthalmol 2020 04 23;212:34-42. Epub 2019 Nov 23.

Grenoble Alpes University, Grenoble, France; Department of Ophthalmology, Grenoble Alpes University Hospital, Grenoble, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ajo.2019.11.026DOI Listing
April 2020

Ocular injuries caused by less-lethal weapons in France.

Lancet 2019 11;394(10209):1616-1617

Department of Ophthalmology, Sorbonne Université, Pitié-Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris 75013, France. Electronic address:

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http://dx.doi.org/10.1016/S0140-6736(19)31807-0DOI Listing
November 2019

Reprogramming of Adult Retinal Müller Glial Cells into Human-Induced Pluripotent Stem Cells as an Efficient Source of Retinal Cells.

Stem Cells Int 2019 15;2019:7858796. Epub 2019 Jul 15.

Sorbonne Université, INSERM, CNRS, Institut de la Vision, F-75012 Paris, France.

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.
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http://dx.doi.org/10.1155/2019/7858796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6664555PMC
July 2019

Capabilities of Gabor-domain optical coherence microscopy for the assessment of corneal disease.

J Biomed Opt 2019 04;24(4):1-17

LighTopTech Corp., West Henrietta, New York, United States.

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.
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http://dx.doi.org/10.1117/1.JBO.24.4.046002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479593PMC
April 2019

Innovative corneal active storage machine for long-term eye banking.

Am J Transplant 2019 06 25;19(6):1641-1651. Epub 2019 Jan 25.

Corneal Graft Biology, Engineering and Imaging Laboratory, BiiGC, EA2521, Federative Institute of Research in Sciences and Health Engineering, Faculty of Medicine, Jean Monnet University, Saint-Etienne, France.

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.
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http://dx.doi.org/10.1111/ajt.15238DOI Listing
June 2019

Evaluation of corneal epithelial wound healing after penetrating keratoplasty in patients receiving a new matrix therapy agent (regenerating agent).

Eur J Ophthalmol 2020 Jan 31;30(1):119-124. Epub 2018 Oct 31.

Department of Ophthalmology, University Hospital of Saint-Etienne, Saint-Etienne, France.

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/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.
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http://dx.doi.org/10.1177/1120672118808971DOI Listing
January 2020

How transparent film applied on dermatologic imaging devices in order to prevent infections affects image quality?

Skin Res Technol 2019 Mar 27;25(2):229-233. Epub 2018 Oct 27.

Department of Medical, Surgical and Neurological Science, Dermatology Section, S. Maria alle Scotte Hospital, University of Siena, Siena, Italy.

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.
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http://dx.doi.org/10.1111/srt.12642DOI Listing
March 2019

Endothelial quality of eye bank-prestripped DMEK prepared form organ-cultured corneas with the Muraine technique.

Cell Tissue Bank 2018 Dec 31;19(4):705-716. Epub 2018 Aug 31.

Ophthalmology Department, University Hospital, Charles Nicolles Hospital, Rouen, France.

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 for DMEK and 2471 (2135-2714) for controls (P = 1). Forty-eight hours after stripping, vECD was 2057 (1829-2463) cells/mm 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.
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http://dx.doi.org/10.1007/s10561-018-9723-0DOI Listing
December 2018

Transplantation Blues: Inadvertent Staining of Amyloid Deposits With Trypan Blue.

Cornea 2018 Jul;37(7):824-828

Department of Ophthalmology, University Hospital, Saint-Etienne, France.

Purpose: To describe inadvertent persistent staining of stromal amyloid deposits by trypan blue (TB) after penetrating keratoplasty (PK) and Descemet membrane endothelial keratoplasty (DMEK) performed in patients with corneal amyloidosis.

Methods: Case series of patients with corneal amyloidosis in whom intraoperative TB was used.

Results: One patient, hospitalized for acute rejection 6 weeks after DMEK, presented with an intense blue staining of small, spindle-shaped structures in the anterior half of the cornea. DMEK had been performed for endothelial failure of a previous PK procedure done 13 years earlier for advanced lattice corneal dystrophy (LCD). After 6 months, the stromal blue tattoo persisted with impaired visual acuity, and PK was performed. Blue-stained structures were amyloid deposits characteristic of LCD recurrence. In parallel, among 85 consecutive triple procedures (PK + cataract + intraocular lens [IOL]) performed over 7 years, in which TB was used, only patients with LCD (n = 18 eyes in 17 patients) or presumed secondary amyloidosis due to chronic inflammation (n = 1), presented an isolated intense blue ring of the graft-host interface. This persisted up to 7 years with no clinical consequence.

Conclusions: TB can stain corneal amyloid deposits. After PK, staining is limited to the recipient peripheral cornea and has no apparent clinical consequence. However, during DMEK performed after a failed PK, TB stains fibrils accumulated during slow LCD recurrence and scattered on the whole graft. The long-term staining duration indicates strong interactions between TB and amyloid.
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http://dx.doi.org/10.1097/ICO.0000000000001591DOI Listing
July 2018

Immunosuppression by a subconjunctival implant releasing dexamethasone in a rabbit model of penetrating keratoplasty.

Br J Ophthalmol 2018 05 3;102(5):692-699. Epub 2018 Feb 3.

Corneal Graft Biology, Engineering and Imaging Laboratory (BiiGC), EA2521, SFR143, Universite Jean Monnet, Saint-Etienne, France.

Aims: To evaluate the efficacy of a subconjunctival dexamethasone-releasing implant in preventing rejection of penetrating keratoplasty (PK) in an animal model.

Methods: Twenty-two rabbits underwent allogenic PK. After randomisation, they received either a 700 µg dexamethasone implant under the conjunctiva at the end of surgery (n=10), one dexamethasone 1 mg/mL eye-drop thrice daily (n=6) or a placebo thrice daily (n=6). The suture was left in place. Animals were observed weekly by slit-lamp and optical coherence tomography with quantification of transparency, neovascularisation and central corneal thickness (CCT). At 5-6 weeks, they were euthanised for histology. The residual dexamethasone concentration in ocular tissues was measured with an ultra-performance liquid chromatography-tandem mass spectrometer.

Results: Placebo group: early neovascularisation was systematic, penetrating the graft by 270-360° at 5-6 weeks. Rejection occurred in 50% of cases. Eye-drop and implant groups: similar course without rejection at 6 weeks and normal CCT. Neovascularisation was observed in 5/6 rabbits in the eye-drop group and in 6/8 in the implant group, with two cases of new vessels penetrating the graft from week 3. Neovascularisation scores did not differ significantly between the two treatments and were significantly lower than for the placebo. Histology was in agreement in all cases. Implants disappeared after 3-5 weeks. No local side effect was observed. Tissue concentrations were all higher at day 8 (n=2) in the implant group than in the eye drop group and lower at 6 weeks (n=8).

Conclusions: In this PK model characterised by a high rejection rate, a subconjunctival dexamethasone implant was for 6 weeks as effective as the topical form in preventing allograft rejection.
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http://dx.doi.org/10.1136/bjophthalmol-2017-310734DOI Listing
May 2018

Comparison of four methods of surface roughness assessment of corneal stromal bed after lamellar cutting.

Biomed Opt Express 2017 Nov 12;8(11):4974-4986. Epub 2017 Oct 12.

Corneal Graft Biology, Engineering and Imaging Laboratory, BiiGC, EA2521, Federative Institute of Research in Sciences and Health Engineering, Faculty of Medicine, Jean Monnet University, 10 rue de la Marandière, 42055 Saint-Etienne Cédex 02, France.

Corneal lamellar cutting with a blade or femtosecond laser (FSL) is commonly used during refractive surgery and corneal grafts. Surface roughness of the cutting plane influences postoperative visual acuity but is difficult to assess reliably. For the first time, we compared chromatic confocal microscopy (CCM) with scanning electron microscopy, atomic force microscopy (AFM) and focus-variation microscopy (FVM) to characterize surfaces of variable roughness after FSL cutting. The small area allowed by AFM hinders conclusive roughness analysis, especially with irregular cuts. FVM does not always differentiate between smooth and rough surfaces. Finally, CCM allows analysis of large surfaces and differentiates between surface states.
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http://dx.doi.org/10.1364/BOE.8.004974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695945PMC
November 2017

Storage of Porcine Cornea in an Innovative Bioreactor.

Invest Ophthalmol Vis Sci 2017 11;58(13):5907-5917

Corneal Graft Biology, Engineering and Imaging Laboratory, BiiGC, EA2521, Faculty of Medicine, Jean Monnet University, Saint-Etienne, France.

Purpose: To quantify cell survival and tissue structure preservation of porcine cornea stored in a bioreactor (BR) that recreates a transcorneal pressure gradient equivalent to intraocular pressure (IOP) and renews the medium.

Methods: A BR comprising endothelial and epithelial chambers was machined in a biocompatible material. The porcine cornea, securely held, separated the chambers. Medium flow and pressure inside the endothelial chamber were maintained by a peristaltic pump. In the epithelial chamber, the corneal surface was alternatively exposed to air and a specific medium. Two transparent windows allowed thickness measurement by optical coherence tomography without opening the BR. Porcine corneas stored in the BR-on (pressure 20 mm Hg, flow 5 μL/min, temperature 31°C) were compared with (1) BR-off (no pressure or flow); (2) organ culture; and (3) Petri dish with agar on the endothelial side. Epithelial and limbal structure and differentiation, corneal transparency and thickness, and endothelial viability were compared after 7 days of storage and with fresh corneas.

Results: Corneas stored in the BR-on were thinner and more transparent than those stored with the other methods. The BR-on preserved a stratified and differentiated (K3/K12+) corneal epithelium and undifferentiated basal limbal cells with stemness markers (K3/K12-; ABCB5, K14, p75+), as well as endothelial integrity.

Conclusions: By recreating equivalent IOP and medium renewal, the BR obtained unprecedented storage quality of porcine corneas and preserved their main epithelial, limbal, and endothelial characteristics.
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http://dx.doi.org/10.1167/iovs.17-22218DOI Listing
November 2017

Micro-instillation of fluorescein with an inoculation loop for ocular surface staining in dry eye syndrome.

Acta Ophthalmol 2018 Mar 25;96(2):e140-e146. Epub 2017 Oct 25.

Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Federative Institute of Research in Sciences and Health Engineering, Faculty of Medicine, Jean Monnet University, Saint-Etienne, France.

Purpose: To describe and validate the micro-instillation of fluorescein on the ocular surface by a disposable calibrated inoculation loop to improve corneal and conjunctival staining quality.

Methods: Accuracy and precision of the volume of 0.5% sodium fluorescein collected by a single use 1 μl-calibrated inoculation loop were measured using a precision balance. Twenty patients (40 eyes) suffering from dry eye syndrome were enrolled in a prospective interventional nonrandomized study. Fluorescein was instilled with the loop, and slit-lamp images were taken within 30 seconds using cobalt blue light with and without a yellow barrier filter. For comparison, after a washout period, the same images were retaken after instillation of one drop of fluorescein from a single-dose unit. The main outcome measure was the staining quality assessed by three experts, blind to the instillation method. Patient discomfort (tolerance, by a questionnaire) was also compared.

Results: The mean volume collected by the loop was 1.18 ± 0.12 μl, compared with 33.70 ± 6.10 μl using the single-dose unit. The loop avoided excess dye responsible for unpleasant tearing, masking of lesions and rapid diffusion into the stroma. Micro-instillation greatly improved image quality without losing information. The yellow filter further improved image contrast. Tolerance was excellent.

Conclusion: The 1 μl-calibrated inoculation loop is a safe, convenient, inexpensive, disposable, sterile, well-tolerated tool for reproducible micro-instillation of commercial fluorescein. By greatly improving staining quality, it will help standardize assessment of dry eye severity.
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http://dx.doi.org/10.1111/aos.13559DOI Listing
March 2018

Handheld In Vivo Reflectance Confocal Microscopy for the Diagnosis of Eyelid Margin and Conjunctival Tumors.

JAMA Ophthalmol 2017 08;135(8):845-851

Department of Dermatology, University Hospital of Saint-Etienne, Saint-Etienne, France.

Importance: The clinical diagnosis of conjunctival and eyelid margin tumors is challenging, and new noninvasive imaging techniques could be valuable in this field.

Objective: To assess the diagnostic accuracy of handheld in vivo reflectance confocal microscopy (IVCM) for the diagnosis of eyelid margin and conjunctival tumors.

Design: A prospective observational study was conducted at University Hospital of Saint-Etienne from January 2, 2011, to December 31, 2016 (inclusion of patients until December 31, 2015, and follow-up until December 31, 2016). A total of 278 consecutive patients with eyelid margin or conjunctival lesions were included. Conjunctival lesions were diagnosed with a conventional clinical examination using a slitlamp and by handheld IVCM. Final diagnoses were established by histopathologic examination for 155 neoformations suspicious for being malignant through clinical and/or IVCM examination that were excised and on follow-up of 12 months or longer for the remaining 140 lesions.

Main Outcomes And Measures: Sensitivity, specificity, and positive and negative predictive values for malignant tumors of the conjunctiva and eyelid margin were calculated using clinical examination with slitlamp and handheld IVCM.

Results: In the 278 patients (136 [48.9%] females; mean [SD] age, 59 [21] years), a total of 166 eyelid margin and 129 conjunctival lesions were included in the analysis. Of the 155 excised neoformations with a histopathologic diagnosis, IVCM showed higher sensitivity compared with clinical examination conducted with the slitlamp for malignant tumors of the eyelid margin (98% vs 92%) and conjunctiva (100% vs 88%). The specificity for malignant eyelid margin tumors was higher for IVCM than for slitlamp examination (74% vs 46%), but slightly less for malignant conjunctival tumors (78% vs 88%). Analysis of all neoformations (155 excised and 140 in follow-up) confirmed these differences in the diagnostic accuracy of the clinical examination and IVCM. The presence of hyperreflective Langerhans cells mimicking malignant melanocytes was the main cause for misdiagnosis of malignant conjunctival tumors with IVCM.

Conclusions And Relevance: Handheld IVCM could be a useful tool for the identification of malignant conjunctival tumors. Further studies are required to confirm the usefulness of this device and identify possible features that can differentiate Langerhans cells from malignant melanocytes to prevent the misdiagnosis of melanoma using IVCM.
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http://dx.doi.org/10.1001/jamaophthalmol.2017.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5710297PMC
August 2017

Considering 3D topography of endothelial folds to improve cell count of organ cultured corneas.

Cell Tissue Bank 2017 Jun 10;18(2):185-191. Epub 2017 Apr 10.

Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Federative Institute of Research in Sciences and Health Engineering, Jean Monnet University, 10, Rue de la Marandiere, 42023, Saint-Étienne Cedex 2, France.

The posterior side of the cornea is covered by the endothelial monolayer, which governs corneal transparency but cannot proliferate. Determination of endothelial cell density (ECD) is therefore the minimal and mandatory quality control in all eye banks. It avoids primary graft failures caused by endothelial insufficiency, and allows allocation of corneas to surgical techniques requiring different numbers of endothelial cells (ECs). Corneas stored in organ culture (17% of grafts worldwide), are characterized by heavy stromal swelling and numerous deep endothelial folds, up to 200 µm high. During microscopic en face observation, flat surfaces are thus exceptional and EC counting is biased by parallax errors, resulting in overestimated eye bank ECD (ebECD). We used a motorized transmitted light microscope to acquire Z-stacks of images every 10 µm, and processed them to reconstruct the 3D surface of the folded endothelium. This method (3D-ECD) takes into account the local point-by-point slope in order to correct ECD. On a set of 30 corneas, we compared 3D-ECD and ebECD determined on five identical zones at the center of the cornea. 3D reconstruction allowed us to visualize twice as many cells, and ebECD was 8.1 ± 4.5% (95%CI 6.4-9.7) higher than 3D-ECD, with 1744 ± 488 versus 1606 ± 473 cells/mm. 3D counting makes it possible to increase cell sampling and to correct overestimation by the conventional en face counting still routinely performed in eye banks.
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http://dx.doi.org/10.1007/s10561-017-9624-7DOI Listing
June 2017

Longitudinal study of retinal status using optical coherence tomography after acute onset endophthalmitis following cataract surgery.

Br J Ophthalmol 2017 09 24;101(9):1211-1216. Epub 2017 Jan 24.

Grenoble Alpes University, Grenoble, France.

Purpose: To analyse the macula imaged with optical coherence tomography (OCT) in patients treated for acute postcataract endophthalmitis.

Methods: Patients presenting with acute postcataract endophthalmitis were included in this observational and multicentre study from January 2008 to December 2011. We recorded the following OCT data at the 3, 6 and 12-month visits: the central macular thickness, the perifoveal macular thickness, the central foveal point thickness and abnormalities of the outer retina, the macula and vitreoretinal interface.

Results: 46 patients were included in the OCT analysis. From month 3 to 12, epiretinal membrane (ERM) prevalence increased from 26% to 39%, vitreomacular traction prevalence decreased from 12% to 6%, non-tractional macular oedema (ME) prevalence varied between 7% and 13%. Only macular thinning remained stable at 10%. At month 12, a significant correlation was found between non-tractional ME and capsular rupture (at the time of cataract extraction, p=0.03). Eyes with an ERM exhibited increased central macular thickness (p=0.001) and lower visual acuity (VA) (p=0.02) at M12 in comparison to the group with normal macula. OCT analysis showed a significant association between ERM and the alteration of the ellipsoid band (p=0.02), as well as the external limiting membrane (ELM, p=0.07) at M12.

Conclusions: ERM and ME were the main macular abnormalities diagnosed after 1 year of follow-up, associated with VA less than or equal to 20/40 in 50% of the cases. Ultrastructural abnormalities of the ELM and the ellipsoid band were frequently observed in those patients.
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http://dx.doi.org/10.1136/bjophthalmol-2016-309542DOI Listing
September 2017

Ganglioside Profiling of the Human Retina: Comparison with Other Ocular Structures, Brain and Plasma Reveals Tissue Specificities.

PLoS One 2016 20;11(12):e0168794. Epub 2016 Dec 20.

Centre des Sciences du Goût et de l'Alimentation, CNRS, INRA, University Bourgogne Franche-Comté, Dijon, France.

Gangliosides make a wide family of glycosphingolipids, highly heterogeneous in both the ceramide moiety and the oligosaccharide chain. While ubiquitously expressed in mammalian tissues, they are particularly abundant in the brain and the peripheral nervous system. Gangliosides are known to play a crucial role in the development, maintenance and functional integrity of the nervous system. However, the expression and roles of gangliosides in the retina, although often considered as a window on the brain, has been far less studied. We performed an in-depth analysis of gangliosides of the human retina, especially using powerful LC/MS methods. We compared the pattern of ganglioside classes and ceramide molecular species of this tissue with other ocular structures and with brain and plasma in elderly human individuals. About a hundred of ganglioside molecular species among 15 distinct classes were detected illustrating the huge structural diversity of these compounds. The retina exhibited a very diverse ganglioside profile and shared several common features with the brain (prominence of tetraosylgangliosides, abundance of d20:1 long chain base and 18:0 fatty acid…). However, the retina stood out with the specific expression of GD3, GT3 and AcGT3, which further presented a peculiar molecular species distribution. The unique ganglioside pattern we observed in the human retina suggests that these ganglioside species play a specific role in the structure and function of this tissue. This lipidomic study, by highlighting retina specific ganglioside species, opens up novel research directions for a better understanding of the biological role of gangliosides in the retina.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0168794PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5173345PMC
June 2017

'En face' ex vivo reflectance confocal microscopy to help the surgery of basal cell carcinoma of the eyelid.

Clin Exp Ophthalmol 2017 Jul 31;45(5):442-447. Epub 2017 Jan 31.

Department of Dermatology, University Hospital of St-Etienne, Saint Etienne, France.

Background: Ex vivo confocal microscopy is a recent imaging technique for the perioperative control of skin tumour margins. Up to date, it has been used in the fluorescence mode and with vertical sections of the specimen margins. The aim of this study was to evaluate its use in the reflectance mode and with a horizontal ('en face') scanning of the surgical specimen in a series of basal cell carcinoma of the eyelid.

Design: Prospective consecutive cohort study was performed at the University Hospital of Saint-Etienne, France.

Participants: Forty-one patients with 42 basal cell carcinoma of the eyelid participated in this study.

Methods: Basal cell carcinomas were excised with a 2-mm-wide clinically safe margin. The surgical specimens were analysed under ex vivo confocal microscopy in the reflectance mode and with an en face scanning in order to control at a microscopic level if the margins were free from tumour invasion. Histopathogical examination was later performed in order to compare the results.

Main Outcome Measures: Sensitivity and specificity of ex vivo confocal microscopy for the presence of tumour-free margins.

Results: Ex vivo confocal microscopy results were consistent with histopathology in all cases (tumour-free margins in 40 out of 42 samples; sensitivity and specificity of 100%).

Conclusions: Ex vivo confocal microscopy in the reflectance mode with an 'en face' scanning can control tumour margins of eyelid basal cell carcinomas and optimize their surgical management. This procedure has the advantage on the fluorescent mode of not needing any contrast agent to examine the samples.
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http://dx.doi.org/10.1111/ceo.12904DOI Listing
July 2017

Very early endothelial cell loss after penetrating keratoplasty with organ-cultured corneas.

Br J Ophthalmol 2017 08 13;101(8):1113-1118. Epub 2016 Dec 13.

Corneal Graft Biology, Engineering and Imaging Laboratory, EA 2521, Faculty of Medicine, Jean Monnet University, Saint-Etienne, France.

Aims: After keratoplasty, postoperative endothelial cell loss is calculated between the eye bank endothelial cell density (ebECD) and the postoperative specular microscopy (SM). To elucidate the very early cell loss, always described after penetrating keratoplasty (PK), we designed two complementary studies.

Methods: (1) Clinical prospective study of 90 consecutive PKs (keratoconus, Fuchs' corneal dystrophy, lattice dystrophy, bullous keratopathy) with organ-cultured corneas and postoperative follow-up by SM at day 5 (D5), D15, month 1 (M1) and M3. This series provided a quantification of the difference between ebECD performed 2 days before graft and very early postoperative ECD. (2) Ten pairs of corneas with comparable ebECD in both corneas and same organ-culture (OC) duration were randomised: one cornea was grafted, and, at the same time, the viable ECD (vECD) of the other was measured after labelling with Hoechst/ethidium/calcein-AM. The relationship between vECD and very early postoperative ECD was studied.

Results: vECD at the time of graft did not differ from ECD 5 days after PK, with a difference of 39 (-356; 355) cells/mm (median (10°; 90° percentile, p=0.799)), whereas a significant difference of 755 (359; 1146) cells/mm, corresponding to 28% (95% CI 26 to 30) of cells, was measured between ebECD and ECD 5 days after PK (p<0.001).

Conclusions: In OC, ebECD provided to surgeons significantly overestimate the number of viable ECs grafted to patients. The absence of difference between the vECD at D0 and ECD at D5 indicates that the very early endothelial cell loss is almost negligible in recipients.
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http://dx.doi.org/10.1136/bjophthalmol-2016-309615DOI Listing
August 2017