Comprehensive mass spectrometry lipidomics of human biofluids and ocular tissues.

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.

Melanophages give rise to hyperreflective foci in AMD, a disease-progression marker.

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.

Treatment of corticosteroid-resistant Graves' orbitopathy with tocilizumab: a single-centre prospective study.

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.

Exploration of the ocular surface infection by SARS-CoV-2 and implications for corneal donation: An ex vivo study.

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/.

A retrospective study of the efficacy of intense pulsed light delivered by the Lacrystim® for meibomian gland dysfunction therapy.

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).

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

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-1ß, 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.

Corneal endothelial cell therapy: feasibility of cell culture from corneas stored in organ culture.

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.

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

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.

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

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.

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

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.

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

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/mm2 for DMEK and 2471 (2135-2714) for controls (P = 1). Forty-eight hours after stripping, vECD was 2057 (1829-2463) cells/mm2 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.

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

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.

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

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/mm2. 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.

Corneal endothelial cells possess an elaborate multipolar shape to maximize the basolateral to apical membrane area.

PURPOSE: The corneal endothelium is widely believed to consist of geometrically regular cells interconnected by junctional complexes. However, while en face visualization of the endothelial apical surface reveals characteristic polygonal borders, the overall form of the component cells has rarely been observed. METHODS: To visualize the shape of individual endothelial cells within the native monolayer, two independent Cre/LoxP-based cell labeling approaches were used. In the first, a P0-Cre mouse driver strain was bred to an R26-tdTomato reporter line to map neural crest-derived endothelial cells with cytosolic red fluorescent protein. In the second, HPRT-Cre induction of small numbers of green and red fluorescent protein-filled cells within a background of unlabeled cells was achieved using a dual-color reporter system, mosaic analysis with double markers (MADM). Selective imaging of the endothelial lateral membranes at different apicobasal levels was accomplished after staining with antibodies to ZO-1 and the neural cell adhesion molecule (NCAM). RESULTS: When viewed in their entirety in whole-mount preparations, fluorescent protein-filled cells appear star-shaped, extending multiple dendritic processes that radiate outward in the plane of the monolayer. Examination of rare cases where cells expressing different fluorescent proteins lie directly adjacent to one another reveals that these long processes undergo extensive interdigitation. The resulting overlap allows individual cells to extend over a greater area than if the cell boundaries were mutually exclusive. Anti-NCAM staining of these interlocking peripheral cell extensions reveals an elaborate system of lateral membrane folds that, when viewed in optical sections, increase in complexity from the apical to the basal pole. This not only produces a substantial increase in the basolateral, relative to the apical, membrane but also greatly extends the paracellular pathway as a highly convoluted space. CONCLUSIONS: Our analysis indicates that, far from being simple polygonal prisms, endothelial cells possess an elaborate multipolar shape. Their unusual geometry may be essential for the endothelium to carry out its role as the principal regulator of corneal extracellular fluid flux, and thus ultimately of tissue clarity.

Optimization of immunostaining on flat-mounted human corneas.

PURPOSE: In the literature, immunohistochemistry on cross sections is the main technique used to study protein expression in corneal endothelial cells (ECs), even though this method allows visualization of few ECs, without clear subcellular localization, and is subject to the staining artifacts frequently encountered at tissue borders. We previously proposed several protocols, using fixation in 0.5% paraformaldehyde (PFA) or in methanol, allowing immunostaining on flatmounted corneas for proteins of different cell compartments. In the present study, we further refined the technique by systematically assessing the effect of fixative temperature. Last, we used optimized protocols to further demonstrate the considerable advantages of immunostaining on flatmounted intact corneas: detection of rare cells in large fields of thousands of ECs and epithelial cells, and accurate subcellular localization of given proteins. METHODS: The staining of four ubiquitous proteins, ZO-1, hnRNP L, actin, and histone H3, with clearly different subcellular localizations, was analyzed in ECs of organ-cultured corneas. Whole intact human corneas were fixed for 30 min in 0.5% paraformaldehyde or pure methanol at four temperatures (4 °C for PFA, -20 °C for methanol, and 23, 37, and 50 °C for both). Experiments were performed in duplicate and repeated on three corneas. Standardized pictures were analyzed independently by two experts. Second, optimized immunostaining protocols were applied to fresh corneas for three applications: identification of rare cells that express KI67 in the endothelium of specimens with Fuch's endothelial corneal dystrophy (FECD), the precise localization of neural cell adhesion molecules (NCAMs) in normal ECs and of the cytokeratin pair K3/12 and CD44 in normal epithelial cells, and the identification of cells that express S100b in the normal epithelium. RESULTS: Temperature strongly influenced immunostaining quality. There was no ubiquitous protocol, but nevertheless, room temperature may be recommended as first-line temperature during fixation, instead of the conventional -20 °C for methanol and 4 °C for PFA. Further optimization may be required for certain target proteins. Optimized protocols allowed description of two previously unknown findings: the presence of a few proliferating ECs in FECD specimens, suggesting ineffective compensatory mechanisms against premature EC death, and the localization of NCAMs exclusively in the lateral membranes of ECs, showing hexagonal organization at the apical pole and an irregular shape with increasing complexity toward the basal pole. Optimized protocols were also effective for the epithelium, allowing clear localization of cytokeratin 3/12 and CD44 in superficial and basal epithelial cells, respectively. Finally, S100b allowed identification of clusters of epithelial Langerhans cells near the limbus and more centrally. CONCLUSIONS: Fixative temperature is a crucial parameter in optimizing immunostaining on flatmounted intact corneas. Whole-tissue overview and precise subcellular staining are significant advantages over conventional immunohistochemistry (IHC) on cross sections. This technique, initially developed for the corneal endothelium, proved equally suitable for the corneal epithelium and could be used for other superficial mono- and multilayered epithelia.

Assessing microstructures of the cornea with Gabor-domain optical coherence microscopy: pathway for corneal physiology and diseases.

Gabor-domain optical coherence microscopy (GD-OCM) was applied ex vivo in the investigation of corneal cells and their surrounding microstructures with particular attention to the corneal endothelium. Experiments using fresh pig eyeballs, excised human corneal buttons from patients with Fuchs' endothelial dystrophy (FED), and healthy donor corneas were conducted. Results show in a large field of view (1 mm×1 mm) high definition images of the different cell types and their surrounding microstructures through the full corneal thickness at both the central and peripheral locations of porcine corneas. Particularly, an image of the endothelial cells lining the bottom of the cornea is highlighted. As compared to healthy human corneas, the corneas of individuals with FED show characteristic microstructural alterations of the Descemet's membrane and increased size and number of keratocytes. The GD-OCM-based imaging system developed may constitute a novel tool for corneal imaging and disease diagnosis. Also, importantly, it may provide insights into the mechanism of corneal physiology and pathology, particularly in diseases of the corneal endothelium.

Anterior segment biometry using spectral-domain optical coherence tomography.

PURPOSE: To compare the anterior chamber and anterior chamber angle measurements obtained with spectral-domain anterior segment optical coherence tomography (AS-OCT) and time-domain AS-OCT. METHODS: The anterior chamber of healthy participants was imaged with spectral-domain AS-OCT (Casia SS-1000; Tomey, Nagoya, Japan) and time-domain AS-OCT (Visante; Carl Zeiss Meditec, Dublin, CA). Central corneal thickness (CCT), anterior chamber depth (ACD), angle opening distance at 500 and 750 µm (AOD 500 and AOD 750), trabecular iris space area at 500 and 750 µm (TISA 500 and TISA 750), and scleral spur angle were measured. The intraclass correlation coefficients (ICCs) were calculated. The Pearson correlation test was used to evaluate the correlation between the measurements and Bland-Altman plots to evaluate the agreement. RESULTS: One hundred one eyes of 101 healthy participants were analyzed. Excellent repeatability was found with both devices for CCT, AOD 500, AOD 750, TISA 750, and scleral spur angle (ICC = 0.90 to 0.98 and 0.89 to 0.97, respectively) and excellent to moderate repeatability was found for TISA 500 (ICC = 0.68 to 0.97 and 0.70 to 0.93, respectively). For all parameters, Casia and Visante measurements were significantly correlated (r = 0.76 to 0.98; P < .02). ACD measured with the Casia was significantly larger (mean difference = 0.12 ± 0.08 mm; P < .0001), and the scleral spur angle measured with the Casia was significantly lower (mean difference = 4.85° ± 5.30°; P < .01). There were nonsignificant differences between the devices for the other parameters (P > .06). CONCLUSIONS: Both Casia and Visante AS-OCT demonstrate high repeatability. Except for the ACD and scleral spur angles, the two devices show good agreement.

The role of in vivo confocal microscopy in the diagnosis of eyelid margin tumors: 47 cases.

BACKGROUND: Handheld in vivo reflectance confocal microscopy (IVCM) is a new imaging method that allows noninvasive diagnosis of cutaneous tumors but to date it has not been used in the study of eyelid tumors. OBJECTIVE: We sought to evaluate the suitability of IVCM for eyelid margin tumors. METHODS: We prospectively evaluated the IVCM features of 47 eyelid margin lesions, clinically suspicious of malignancy; 35 of these were excised whereas the other 12, with no IVCM malignant features, were followed up for at least 1 year. Clinical, IVCM, and histologic diagnoses were compared. RESULTS: IVCM showed sensitivity and specificity of 100% and 69.2%, respectively, for malignancy (basal cell carcinoma, squamous cell carcinoma, and melanoma). The follow-up of the 12 nonexcised lesions did not show any clinical progression. LIMITATIONS: The lesions showing neither clinical nor IVCM features for malignancies were not biopsied in view of the potential functional and aesthetic consequences of eyelid margin surgery. CONCLUSION: Used with a handheld dermatology-specific microscope, IVCM can play a role in the noninvasive diagnosis of eyelid margin lesions. Further studies are needed to better define diagnostic criteria of eyelid tumors and improve the specificity of this technique.

Non-invasive measurement of transparency, arcus senilis, and scleral rim diameter of corneas during eye banking.

We developed a non-invasive device to quantify transparency (T), clear corneal diameter (CCD) excluding arcus senilis, and scleral rim diameter (SRD) of stored corneas. The T value (expressed in % on a relative scale), based on the modulation transfer function principle, referred to the ratio of local contrasts of a special LED backlit chart measured with and without cornea. CCD and SRD (in mm) were automatically calculated by morphologic operations. Firstly, we assessed measurement reproducibility. We then determined the agreement of T and CCD values with 3-level scores given independently by three experts on 179 scientific corneas. Thirdly, an eye bank was equipped with the device, and 358 consecutive organ-cultured (OC) corneas were tested for donor- and storage- related factors possibly influencing T and CCD. Reproducibility of T, CCD and SRD measurements was high, with intraclass correlation coefficients of 0.982, 0.886, and 0.999 respectively. Capacity to discriminate the three levels of transparency and arcus senilis was good, with T of 20.0 (10.0-33.6), 38.3 (24.3-75.4) and 57.9 (33.9-90.0) % respectively for T deemed poor, average, and good (P < 0.001), and CCD of 9.8 (7.3-10.6), 10.5 (8.2-11.5), and 11.1 (9.9-12.0) mm respectively for arcus senilis deemed prominent, moderate or absent (P < 0.001). T was correlated with neither donor age nor endothelial cell density nor storage time, but slightly worsened during OC for corneas assessed twice. In conclusion, the device, which can be easily integrated in the facilities of an eye bank, provides reliable objective measurement of T, CCD, and SRD. This could be a useful tool for standardizing quality assessment of stored corneas and consequently optimizing their selection for penetrating, endothelial or anterior lamellar keratoplasty.