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To discuss and evaluate new technologies for a better diagnosis of corneal diseases and limbal stem cell deficiency, the outcomes of a consensus process within the European Vision Institute (and of a workshop at the University of Cologne) are outlined. Various technologies are presented and analyzed for their potential clinical use also in defining new end points in clinical trials. The disease areas which are discussed comprise dry eye and ocular surface inflammation, imaging, and corneal neovascularization and corneal grafting/stem cell and cell transplantation. The unmet needs in the abovementioned disease areas are discussed, and realistically achievable new technologies for better diagnosis and use in clinical trials are outlined. To sum up, it can be said that there are several new technologies that can improve current diagnostics in the field of ophthalmology in the near future and will have impact on clinical trial end point design.

PURPOSE: We sought to assess the correlation of corneal endothelial cell (CEC) density to alterations of collagen composition of Descemet membrane (DM) in advanced Fuchs endothelial corneal dystrophy (FECD) and to image such changes by slit-lamp biomicroscopy in vivo. DESIGN: Prospective, observational consecutive case series. METHODS: Fifty eyes (50 subjects) with advanced FECD were enrolled. After slit-lamp biomicroscopy and corneal Scheimpflug imaging, the Descemet endothelium complex (DEC) was retrieved during DM endothelial keratoplasty (DMEK) surgery. The expression of collagens I, III, and IV (COL I, COL III, and COL IV) and corresponding CEC density were analyzed by immunofluorescence flat mount-staining. Presence, diameter and surface area of collagen expression, and CEC density served as the main outcome measures. RESULTS: Immunofluorescence staining revealed central coherent collagen positive areas (mean surface area = 10 mm(2) +/- 6 mm(2)) corresponding to a fibrillar layer burying the guttae of DM in 84% (42/50) of DECs. CEC density overlying the fibrillar layer compared with the periphery was significantly reduced (- 54.8%, P < .0001) with a steep decline of CEC density at its borders. Subgroup analysis revealed that the fibrillar layer may be imaged by slit-lamp biomicroscopy in vivo with significant positive correlation of mean maximum diameter detected by slit-lamp biomicroscopy (d(SL max) = 4.1 mm +/- 0.9 mm) and by immunofluorescence staining (d(IF max) = 4.7 mm +/- 1.1 mm; r = 0.76; P = .001). CONCLUSION: A fibrillar layer with a clear geographic pattern marks areas of pronounced loss of CEC density in advanced FECD eyes and may be imaged by slit-lamp biomicroscopy in vivo. (C) 2020 Elsevier Inc. All rights reserved.

Eugene Appenteng Osae,1,2 Reynolds Kwame Ablordeppey,1 Jens Horstmann,2 David Ben Kumah,1 Philipp Steven2,3 1Department of Optometry and Visual Science, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana; 2Department of Ophthalmology, Division for Dry-Eye and Ocular GvHD, Medical Faculty, University of Cologne, Cologne, Germany; 3Cluster of Excellence: Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, GermanyCorrespondence: Eugene Appenteng OsaeDepartment of Optometry and Visual Science, Kwame Nkrumah University of Science and Technology, Private Mail Bag, Kumasi, GhanaTel +233 200 466 637Email osaeappen@gmail.comAim: To compare differences in clinical dry eye features and meibomian gland health status between dry eye patients from rural and urban populations in Ghana.Methods: We examined 211 (rural=109, urban=102) participants with subjective dry eye symptoms. Tear film break -up time (TBUT), Schirmer’s test and ocular surface staining (OSS) were assessed. Symptoms were evaluated using the SPEED II questionnaire. Meibomian glands (MG) in the right eye upper (UL) and lower lids (LL) were imaged using a custom meibographer. MG area was determined by intensity threshold segmentation using Image J software. MG loss (MGL) was also graded based on Pult’s grading scheme. Mann–Whitney, Spearman correlation, chi-square and odds analyses were performed; p< 0.05 was considered significant.Results: Rural participants showed greater SPEED scores, reduced TBUT, and lower Schirmer scores, p < 0.05. The proportion of rural participants with MGL were significantly more (82.3%) than urban participants (63.3%), p < 0.05. They also showed greater MGL than urban participants, p < 0.05. Chi-square test revealed significantly different meiboscale distributions (UL: χ2=13.58, LL: χ2=15.29) between the groups, p < 0.05. Overall significant relationsh

In the past decade, novel lamellar keratoplasty techniques such as Deep Anterior Lamellar Keratoplasty (DALK) for anterior keratoplasty and Descemet stripping automated endothelial keratoplasty (DSAEK)/Descemet membrane endothelial keratoplasty (DMEK) for posterior keratoplasty have been developed. DALK eliminates the possibility of endothelial allograft rejection, which is the main reason for graft failure after penetrating keratoplasty (PK). Compared to PK, the risk of endothelial graft rejection is significantly reduced after DSAEK/ DMEK. Thus, with modern lamellar techniques, the clinical problem of endothelial graft rejection seems to be nearly solved in the low-risk situation. However, even with lamellar grafts there are epithelial, subepithelial and stromal immune reactions in DALK and endothelial immune reactions in DSAEK/DMEK, and not all keratoplasties can be performed in a lamellar fashion. Therefore, endothelial graft rejection in PK is still highly relevant, especially in the high-risk setting, where the cornea's (lymph)angiogenic and immune privilege is lost due to severe inflammation and pathological neovascularization. For these eyes, currently available treatment options are still unsatisfactory. In this review, we will describe currently used keratoplasty techniques, namely PK, DALK, DSAEK, and DMEK. We will summarize their indications, provide surgical descriptions, and comment on their complications and outcomes. Furthermore, we will give an overview on corneal transplant immunology. A specific focus will be placed on endothelial graft rejection and we will report on its incidence, clinical presentation, and current/future treatment and prevention options. Finally, we will speculate how the field of keratoplasty and prevention of corneal allograft rejection will develop in the future.

Excimer laser phototherapeutic keratectomy (PTK) is a safe treatment for superficial corneal opacities, e.g., in corneal dystrophies or degenerations. Nevertheless, no standardized treatment protocols are available and intraoperative monitoring was not possible, so far. Here we evaluate the potential benefits of the intraoperative assessment by microscope-integrated intraoperative optical coherence tomography (MI-OCT) of corneal optical properties during PTK. Retrospective study of eight patients (one male, seven females; age range, 43-80 years, mean = 66.1 years) using an 840-nm microscope-integrated spectral-domain OCT (iOCT; OptoMedical Technologies, Luebeck, Germany adapted to HS Hi-R Neo 900A, Haag Streit Surgical, Wedel, Germany). Images were acquired before and after corneal abrasion and after PTK. For PTK, a SCHWIND Amaris 750S excimer laser (SCHWIND eye-tech-solutions GmbH und KO. KG) was used. Parameters assessed were the central corneal thickness (CCT), changes in central depth-dependent corneal tissue intensity (TI), and corneal surface roughness (SR) in cross-sectional images of the cornea. Intraoperative monitoring using microscope-integrated OCT was possible in all patients at all time points. TI of the anterior corneal stroma decreased significantly (p = 0.037) after PTK (T1 = 15.1 +/- 3.6, T2 = 15.0 +/- 3.84, T3 = 13.7 +/- 3.38), but not after corneal abrasion alone, indicating increased transparency caused by excimer laser PTK. CCT was significantly lower after corneal abrasion (p = 0.017), but not after PTK (T1 = 630.4 +/- 70 mu m, T2 = 544.1 +/- 59.4 mu m, T3 = 558.3 +/- 52.5 mu m. SR significantly decreased (p = 0.043) after PTK (T1 = 614.4 +/- 37.5 pixels, T2 = 634.4 +/- 35.6 pixels, T3 = 611.0 +/- 40.3 pixels). Intraoperative OCT allows real-time imaging during PTK and the assessment of corneal optical transparency and its surface roughness. It has to be clarified in larger studies if these parameters correlate with later postoperative visual

Purpose: Corneal lymphatic vessels are clinically invisible because of their thin walls and clear lymph fluid. There is no easy and established method for in vivo imaging of corneal lymphatic vessels so far. In this study, we present a novel approach to visualize corneal lymphatic vessels in vivo by injecting intrastromal fluorescein sodium. Methods: Six-to eight-week-old female BALB/c mice were used in the mouse model of suture-induced corneal neovascularization. Two weeks after the suture placement, fluorescein sodium was injected intrastromally. The fluorescein, taken up by the presumed lymphatic vessels, was then tracked using a clinically used Spectralis HRA + OCT device. Immunohistochemistry staining with specific lymphatic marker LYVE-1 and pan-endothelial marker CD31 was used to confirm the indirect lymphangiography findings. Results: By injecting fluorescein intrastromally, both corneal blood and lymphatic vessels were detected. While the lymphatic vessels were visible as bright vessel-like structures using HRA, the blood vessels appeared as dark networks. Fluorescein-labeled lymphatic vessels were colocalized with LYVE-1 in immunohistochemically stained sections of the same specimen. Conclusions: Corneal lymphatic vessels can be easily imaged in vivo in the murine model using intrastromal fluorescein injection.

Immunologic graft rejection is the main complication after corneal transplant into pathologically prevascularized so-called high-risk eyes. The aim of this study was to evaluate whether ultraviolet (UV) light crosslinking can regress pathologic corneal blood and lymphatic vessels and thereby improve subsequent graft survival. Using the murine model of suture-induced corneal neovascularization, we found that corneal crosslinking with UVA light and riboflavin regressed both preexisting blood and lymphatic vessels significantly via induction of apoptosis in vascular endothelial cells. In addition, macrophages and CD45(+) cell counts were significantly reduced. Consistently, corneal crosslinking reduced keratocyte density and corneal thickness without affecting corneal nonvascular endothelial cells, iris, and lens depending on the crosslinking duration. Furthermore, using the murine model of corneal transplant, long-term graft survival was significantly promoted (P < .05) and CD4(+)CD25(+)FoxP3(+) T regulatory cells were upregulated (P < .01) in high-risk eyes preoperatively treated with crosslinking. Our results suggest UV light crosslinking as a novel method to regress both pathologic corneal blood and lymphatic vessels and to reduce CD45(+) inflammatory cells. Furthermore, this study demonstrates for the first time that preoperative corneal crosslinking in prevascularized high-risk eyes can significantly improve subsequent graft survival and may become a promising novel therapy in the clinic.

Lymphangiogenesis is essential for fluid homeostasis in vascularized tissues. In the normally avascular cornea, however, pathological lymphangiogenesis mediates diseases like corneal transplant rejection, dry eye disease, and allergy. So far, a physiological role for lymphangiogenesis in a primarily avascular site such as the cornea has not been described. Using a mouse model of perforating corneal injury that causes acute and severe fluid accumulation in the cornea, we show that lymphatics transiently and selectively invade the cornea and regulate the resolution of corneal edema. Pharmacological blockade of lymphangiogenesis via VEGFR-3 inhibition results in increased corneal thickness due to delayed drainage of corneal edema and a trend towards prolonged corneal opacification. Notably, lymphatics are also detectable in the cornea of a patient with acute edema due to spontaneous Descemet's (basement) membrane rupture in keratoconus, mimicking this animal model and highlighting the clinical relevance of lymphangiogenesis in corneal fluid homeostasis. Together, our findings provide evidence that lymphangiogenesis plays an unexpectedly beneficial role in the regulation of corneal edema and transparency. This might open new treatment options in blinding diseases associated with corneal edema and transparency loss. Furthermore, we demonstrate for the first time that physiological lymphangiogenesis also occurs in primarily avascular sites.

A holographic method for high-speed, noncontact photoacoustic tomography is introduced and evaluated. Relative changes of the object's topography, induced by the impact of thermoelastic pressure waves, were determined at nanometer sensitivity without physical contact. The object's surface was illuminated with nanosecond laser pulses and imaged with a high-speed CMOS camera. From two interferograms measured before and after excitation of the acoustic wave, surface displacement was calculated and then used as the basis for a tomographic reconstruction of the initial pressure caused by optical absorption. The holographic detection scheme enables variable sampling rates of the photoacoustic signal of up to 50 MHz. The total acquisition times for complete volumes with 230 MVoxel is far below 1 s. Measurements of silicone and porcine skin tissue phantoms with embedded artificial absorbers, which served as a model for human subcutaneous vascular networks, were possible. Three-dimensional reconstructions of the absorbing structures show details with a diameter of 310 aem up to a depth of 2.5 mm. Theoretical limitations and the experimental sensitivity, as well as the potential for in vivo imaging depending on the detection repetition rate, are analyzed and discussed. (C) 2017 Society of Photo-Optical Instrumentation Engineers (SPIE).

PURPOSE. Corneal neovascularization, in particular lymphangiogenesis, is a limiting factor in corneal transplant survival. Novel treatment approaches focus on (selective) inhibition and regression of lymphatic vessels. Imaging clinically invisible corneal lymphatic vessels is a prerequisite for these strategies. Using a murine model, this study investigates whether corneal lymphatic vessels can be imaged using microscopic optical coherence tomography (mOCT). METHODS. Corneal neovascularization was induced by intrastromal placement of 11.0 nylon sutures in one eye of BALB/c mice. After 2 weeks, cross-sectional images and volumes of the corneas with a 0.5 mm lateral and axial field of view were acquired using a custom-built mOCT system enabling a resolution of 1 mu m at a B-scan rate of 165/s. Three of the six animals received an additional intrastromal injection of India ink 24 hours before the measurement to stain the corneal lymphatic system in vivo. Immunohistochemistry using CD31 and LYVE-1 was used to validate the mOCT findings. RESULTS. Using mOCT, lymphatic vessels were visible as dark vessel-like structures with the lumen lacking a hyperreflective wall and mostly lacking cells. However, individual, slowly moving particles, which most likely are immune cells, occasionally could be observed inside the lumen. In lymphatic vessels of ink-stained corneas, hyperreflection and shadowing underneath was observed. Ink-filled lymphatic vessels were colocalized in consecutive corneal flat mounts of the same specimen. CONCLUSIONS. Corneal lymphatic vessels can be imaged using mOCT This novel approach opens new options for noninvasive clinical imaging of corneal lymphatic vessels for diagnostic and therapeutic indications.