Differences in Guttae Ultramorphology in Relation to Visual Function in Fuchs Endothelial Corneal Dystrophy.

PURPOSE: The purpose of this study was to investigate the differences in guttae ultramorphology and their relation to visual function in eyes with Fuchs endothelial corneal dystrophy (FECD). METHODS: Thirty FECD eyes without ocular comorbidities were included. Visual functional parameters (best-corrected visual acuity with high-contrast and low-contrast letters and contrast sensitivity/LogCS) and corneal morphology measured with Scheimpflug tomography (Pentacam) were assessed. The surgically removed Descemet membranes were examined by light and transmission electron microscopy. RESULTS: Preoperative mean best-corrected visual acuity (logarithm of the minimum angle of resolution) was 0.52 ± 0.18, LogCS 0.96 ± 0.21 and central corneal thickness 640 ± 55 µm. All eyes had signs of subclinical corneal edema in Scheimpflug tomography; clinically visible corneal edema was present in 40% of eyes. Histological findings included a posterior fibrillar zone (PFZ) in 10 specimens (33%) and abnormal collagen depositions in Descemet membranes in 14 specimens (47%). Guttae buried within the PFZ were present only in eyes with clinically visible edema (n = 4, 13%). There was no difference in visual function results and tomography parameters between eyes with and without PFZ or between protruding guttae and guttae embedded in a PFZ, respectively. CONCLUSIONS: Guttae morphology and density were not correlated with visual functional parameters. Guttae buried in a PFZ occurred only in eyes with clinically manifest edema, and thereby, they are an ultramorphological sign for advanced FECD. Subclinical edema was present in all eyes and might be more relevant for quality of vision than guttae ultramorphology.

Long-Term Outcomes of Keratoprosthesis With Biological Haptic: A Review of 25 Years of Single-Center Surgeries.

PURPOSE: To study the outcome of eyes that underwent surgery for keratoprosthesis with a biological haptic, osteo-odonto-keratoprosthesis (OOKP) or tibia keratoprosthesis, by a single surgeon over a time span of more than 25 years. METHODS: One hundred thirty eyes that had received a keratoprosthesis with a biological haptic between 1994 and 2022 by a single surgeon were included in this retrospective analysis. Main outcome parameters were postoperative best corrected visual acuity, postoperative refractive error, postoperative complications, anatomical and functional survival of the prosthesis as well as comparison of subgroups of the 2 different types (OOKP n = 78; tibia keratoprosthesis n = 52) of keratoprostheses, and subgroup analysis of different indications for surgery. Patients were examined every 6 months. RESULTS: The longest follow-up was 25.8 years. Reasons for implantation were graft-vs-host disease (6.9%), vascularized corneas and dry eye (22.9%), physical or chemical burns (29.8%), Stevens-Johnson syndrome (9.9%), and ocular cicatricial pemphigoid (30.5%). The functional success rate with postoperative visual acuity of better than 0.7 log MAR was achieved by 56.9%. The OOKP subgroup showed a better mean visual outcome. 14 keratoprostheses (10.7%) had to be explanted over the whole time span. In the time leading to explantation, refraction showed a statistically significant myopic shift when compared with the non-explanted prosthesis. Anatomical survival rates were better for the OOKP in the first 12 years after implantation. CONCLUSIONS: The study shows that keratoprosthesis with a biological haptic has favorable long-term outcomes. The retention rate stayed very high with excellent functional outcomes.

Pattern electroretinogram, blue-yellow visual evoked potentials and the risk of developing visual field defects in glaucoma suspects: a longitudinal "survival" analysis with a very long follow-up.

PURPOSE: Estimating glaucoma suspects' risk for visual field defects helps to avoid under- and over-treatment. In this retrospective, longitudinal cohort study with a very long follow-up, we studied whether pattern electroretinograms (PERG) amplitudes and blue-on-yellow visual evoked potential (BY-VEP) latencies can predict visual field defects. METHODS: Participants of the Erlangen Glaucoma Study were examined with PERG and BY-VEP between 9/1991 and 8/2001. Stimuli were created using an optical bench with Maxwellian view and consisted of vertical gratings (0,88 cpd) in a 32° field for both PERG and BY-VEP. Patients were treated according to clinical standards and performed standard automated perimetry (SAP) annually. Retrospectively, patients with normal SAP at baseline were selected. Primary endpoint was conversion to perimetric glaucoma. Predictive value was modeled using Kaplan-Meier analyses and a multivariate cox proportional hazards model with the continuous variables PERG amplitude, BY-VEP peak time and SAP square-root of loss variance (sLV) after stratification for Jonas classification of the optic discs. RESULTS: Of 412 patients (288: Jonas 0, 103: I, and 21: II; baseline age: 20-60 years), 65 converted to perimetric glaucoma during follow-up (0.5-23.3 years; median 5.5 years). Optic disc classification was a strong risk factor for conversion (log rank p < 0.0001), and patients with more advanced changes progressed earlier. In the multivariate analysis (log rank p = 0.005), only PERG amplitude remained an independent risk factor after stratification for optic disc morphology (p = 0.021), with a ~ 30% higher risk per µV amplitude decrease. CONCLUSIONS: PERG helps to estimate glaucoma suspects' risk for visual field defects.

Characterisation of ectasia after penetrating keratoplasty in keratoconus eyes using anterior segment optical coherence tomography.

BACKGROUND/AIMS: Ectasia of the cornea can occur decades after penetrating keratoplasty (PK), especially in keratoconus eyes. The purpose of this study was to characterise ectasia after PK by morphological findings in anterior segment optical coherence tomography (AS-OCT). METHODS: In this retrospective, single-centre case series, 50 eyes of 32 patients with a history of PK at an average of 25±10 years earlier were included. The eyes were classified either as ectatic (n=35) or as non-ectatic (n=15). The main parameters included central corneal thickness (CCT), lowest corneal thickness at the interface (LCTI), anterior chamber depth, graft-host interface angle at the thinnest point and host cornea-iris angle. Furthermore, steep and flat keratometry readings obtained by AS-OCT (CASIA-2, Tomey) and Scheimpflug tomography (Pentacam, Oculus) were assessed. OCT findings were correlated with clinical grading of ectasia. RESULTS: There was a highly significant difference in LCTI, graft-host interface angle and anterior chamber depth (in pseudophakic eyes) between the groups. The ratio calculated by the quotient of LCTI divided by CCT was significantly lower in ectatic than non-ectatic eyes (p<0.001). In eyes with an LCTI/CCT ratio of ≤0.7, the OR for the occurrence of a clinical detectable ectasia was 2.4 (CI 1.5 to 3.7). Steep keratometry values were significantly higher in ectatic eyes. CONCLUSION: AS-OCT is a helpful tool to recognise and quantify ectasia in post-PK eyes objectively.

Risk factors for ocular hypotony after XEN Gel Stent implantation.

PURPOSE: To investigate the incidence of postoperative hypotony, and risk factors for the development of hypotony in eyes who had undergone XEN Gel Stent implantation. METHODS: In this retrospective, single-centre case series, medical records of 170 consecutive eyes who had undergone XEN Gel Stent implantation with or without simultaneous phacoemulsification for primary or secondary open angle glaucoma were analysed. Primary outcome parameters were the incidence of postoperative hypotony and potential risk factors for its development, and secondary parameters were pre- and postoperative visual acuity, intraocular pressure (IOP), and number of IOP-lowering eye drops. RESULTS: Postoperative hypotony ≤ 6 mmHg occurred in 57% of eyes. Hypotony was without complications in 70.1%, 13.4% had transient complications with spontaneous resolution, and 16.5% had complications requiring treatment. Mean visual acuity logMAR before surgery accounted for 0.47 ± 0.46 in all eyes and 0.47 ± 0.48 at the 4-week visit. There was no significant difference of BCVA in the group of eyes with and without postoperative hypotony before and after surgery. The mean IOP before surgery was 24.6 ± 8.4 mmHg and decreased significantly to 18.4 ± 10.2 after 4 weeks. Eyes with an axial length over 24.3 mm had a threefold increased risk for postoperative hypotony (OR 3.226, 95% confidence interval 1.121-9.279). This risk was decreased in eyes with simultaneous cataract surgery (OR 0.483, 95% confidence interval 0.258-0.903). CONCLUSION: In our sample, postoperative hypotony was a common complication after XEN Gel Stent implantation, but serious, persistent complications were rare. A longer axial length predisposes the eye for the development of hypotony.

Post-COVID-19 Syndrome: Retinal Microcirculation as a Potential Marker for Chronic Fatigue.

Post-COVID-19 syndrome (PCS) is characterized by persisting sequelae after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). PCS can affect patients with all COVID-19 disease severities. As previous studies have revealed impaired blood flow as a provoking factor triggering PCS, it was the aim of the present study to investigate the potential association between self-reported chronic fatigue and retinal microcirculation in patients with PCS, potentially indicating an objective biomarker. A prospective study was performed, including 201 subjects: 173 patients with PCS and 28 controls. Retinal microcirculation was visualized by OCT angiography (OCT-A) and quantified using the Erlangen-Angio-Tool as macula and peripapillary vessel density (VD). Chronic fatigue (CF) was assessed according to the variables of Bell's score, age and gender. VDs in the superficial vascular plexus (SVP), intermediate capillary plexus (ICP) and deep capillary plexus (DCP) were analyzed, considering the repetitions (12 times). Seropositivity for autoantibodies targeting G protein-coupled receptors (GPCR-AAbs) was determined by an established cardiomyocyte bioassay. Taking account of the repetitions, a mixed model was performed to detect possible differences in the least square means between the different groups included in the analysis. An age effect in relation to VD was observed between patients and controls (p < 0.0001). Gender analysis showed that women with PCS showed lower VD levels in the SVP compared to male patients (p = 0.0015). The PCS patients showed significantly lower VDs in the ICP as compared to the controls (p = 0.0001 (CI: 0.32; 1)). Moreover, considering PCS patients, the mixed model revealed a significant difference between those with chronic fatigue (CF) and those without CF with respect to VDs in the SVP (p = 0.0033 (CI: −4.5; −0.92)). The model included variables of age, gender and Bell's score, representing a subjective marker for CF. Consequently, retinal microcirculation might serve as an objective biomarker in subjectively reported chronic fatigue in patients with PCS.

Long COVID: Association of Functional Autoantibodies against G-Protein-Coupled Receptors with an Impaired Retinal Microcirculation.

Long COVID (LC) describes the clinical phenotype of symptoms after infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnostic and therapeutic options are limited, as the pathomechanism of LC is elusive. As the number of acute SARS-CoV-2 infections was and is large, LC will be a challenge for the healthcare system. Previous studies revealed an impaired blood flow, the formation of microclots, and autoimmune mechanisms as potential factors in this complex interplay. Since functionally active autoantibodies against G-protein-coupled receptors (GPCR-AAbs) were observed in patients after SARS-CoV-2 infection, this study aimed to correlate the appearance of GPCR-AAbs with capillary microcirculation. The seropositivity of GPCR-AAbs was measured by an established cardiomyocyte bioassay in 42 patients with LC and 6 controls. Retinal microcirculation was measured by OCT-angiography and quantified as macula and peripapillary vessel density (VD) by the Erlangen-Angio Tool. A statistical analysis yielded impaired VD in patients with LC compared to the controls, which was accentuated in female persons. A significant decrease in macula and peripapillary VD for AAbs targeting adrenergic ß2-receptor, MAS-receptor angiotensin-II-type-1 receptor, and adrenergic α1-receptor were observed. The present study might suggest that a seropositivity of GPCR-AAbs can be linked to an impaired retinal capillary microcirculation, potentially mirroring the systemic microcirculation with consecutive clinical symptoms.

Dysregulated Retinoic Acid Signaling in the Pathogenesis of Pseudoexfoliation Syndrome.

Pseudoexfoliation (PEX) syndrome, a stress-induced fibrotic matrix process, is the most common recognizable cause of open-angle glaucoma worldwide. The recent identification of PEX-associated gene variants uncovered the vitamin A metabolic pathway as a factor influencing the risk of disease. In this study, we analyzed the role of the retinoic acid (RA) signaling pathway in the PEX-associated matrix metabolism and evaluated its targeting as a potential candidate for an anti-fibrotic intervention. We provided evidence that decreased expression levels of RA pathway components and diminished RA signaling activity occur in an antagonistic crosstalk with TGF-ß1/Smad signaling in ocular tissues and cells from PEX patients when compared with age-matched controls. Genetic and pharmacologic modes of RA pathway inhibition induced the expression and production of PEX-associated matrix components by disease-relevant cell culture models in vitro. Conversely, RA signaling pathway activation by natural and synthetic retinoids was able to suppress PEX-associated matrix production and formation of microfibrillar networks via antagonization of Smad-dependent TGF-ß1 signaling. The findings indicate that deficient RA signaling in conjunction with hyperactivated TGF-ß1/Smad signaling is a driver of PEX-associated fibrosis, and that restoration of RA signaling may be a promising strategy for anti-fibrotic intervention in patients with PEX syndrome and glaucoma.

Management of Late Descemet's Membrane Detachment After Penetrating Keratoplasty in Keratoconus.

PURPOSE: The purpose of this study was to describe the feasibility of Descemet membrane endothelial keratoplasty (DMEK) as a treatment modality for spontaneous detachment of DM (DMD) decades after penetrating keratoplasty (PK) for keratoconus. METHODS: We describe the clinical characteristics and therapeutic surgical approach in 6 eyes of 5 patients with DMD. Clinical images, anterior segment optical coherence tomography scans, and histological findings are presented. RESULTS: Mean age of patients at time of diagnosis was 60 years (range 56-66 years). Mean interval between PK and occurrence of DM detachment was 36 years (range 29-45 years). In 4 of 6 eyes, air injections into the anterior chamber were initially attempted to reattach DM to the stroma but without long-lasting effect. Two eyes underwent repeat PK because of pronounced ectasia after long-standing DMD and stromal scars. DMEK was performed successfully in 4 eyes leading to an increase in visual acuity and a reduction in central corneal thickness. Electron microscopy showed abnormal vacuolar inclusions and collagenous material in the posterior nonbanded layer and a separation of the anterior banded layer from the posterior nonbanded layer. CONCLUSIONS: This case series provides evidence that DMEK is a viable option in eyes with spontaneous DM detachment after PK. Visual outcome is limited by the persisting high astigmatism in the ectatic cornea. Illustrated by a small series of patients, the results of DMEK in this condition are presented and new findings about the pathophysiology are given.

Identification, Isolation, and Characterization of Melanocyte Precursor Cells in the Human Limbal Stroma.

Given their vital role in the homeostasis of the limbal stem cell niche, limbal melanocytes have emerged as promising candidates for tissue engineering applications. This study aimed to isolate and characterize a population of melanocyte precursors in the limbal stroma, compared with melanocytes originating from the limbal epithelium, using magnetic-activated cell sorting (MACS) with positive (CD117/c-Kit microbeads) or negative (CD326/EpCAM or anti-fibroblast microbeads) selection approaches. Both approaches enabled fast and easy isolation and cultivation of pure limbal epithelial and stromal melanocyte populations, which differed in phenotype and gene expression, but exhibited similar functional properties regarding proliferative potential, pigmentation, and support of clonal growth of limbal epithelial stem/progenitor cells (LEPCs). In both melanocyte populations, limbus-specific matrix (laminin 511-E8) and soluble factors (LEPC-derived conditioned medium) stimulated melanocyte adhesion, dendrite formation, melanogenesis, and expression of genes involved in UV protection and immune regulation. The findings provided not only a novel protocol for the enrichment of pure melanocyte populations from limbal tissue applying easy-to-use MACS technology, but also identified a population of stromal melanocyte precursors, which may serve as a reservoir for the replacement of damaged epithelial melanocytes and an alternative resource for tissue engineering applications.

Descemet membrane endothelial keratoplasty: analysis of clinical outcomes of patients with 8-10 years follow-up.

PURPOSE: This study aimed to evaluate the clinical outcomes up to 10 years after Descemet membrane endothelial keratoplasty (DMEK). METHODS: In this retrospective, consecutive, single-center case series the medical files of eyes which have received DMEK between 2009 and 2012 for the treatment of endothelial dysfunction was evaluated regarding follow-up time and clinical outcomes. Annual examinations of best-corrected visual acuity (BCVA), endothelial cell density (ECD), central corneal thickness (CCT) of 66 eyes which fulfilled the criterion of a minimum of 8 years follow-up were analyzed. RESULTS: BCVA improved from 0.55 ± 0.37 logMAR (n = 54) to 0.15 ± 0.11 (n = 47) in eyes without ocular comorbidities one year after DMEK (p < 0.001), and remained stable up to 10 years after DMEK. Mean ECD decreased to 744 ± 207 cells/mm2 (n = 39) after 9 years, and to 729 ± 167 cells/mm2 (n = 21) after 10 years, respectively. CCT decreased from 650 ± 67 µm before DMEK to 525 ± 40 µm (n = 56) after 1 year, increasing slowly to 563 ± 40 µm (n = 39) after 9 years, and to 570 ± 42 µm (n = 21) after 10 years, respectively. Graft failure occurred in 4 of 66 eyes after year 8. These 4 eyes required repeat DMEK after 101-127 months. CONCLUSION: This study shows the long-term outcomes in a small subset of DMEK grafts. Visual acuity remained stable in spite of slowly increasing corneal thickness and diminishing endothelial cell density during the 10-year period after DMEK.

A Close Look at the Clinical Efficacy of Rho-Associated Protein Kinase Inhibitor Eye Drops for Fuchs Endothelial Corneal Dystrophy.

ABSTRACT: The current understanding on the clinical efficacy of Rho-associated protein kinase (ROCK) inhibitor for treating Fuchs endothelial corneal dystrophy is summarized to clarify whether the "off-label" ROCK-inhibitor eye-drop application are appropriate. ROCK-inhibitor eye drops may eventually be deemed a cutting-edge therapy for Fuchs endothelial corneal dystrophy patients with acute corneal endothelial defect.

Melanocytes as emerging key players in niche regulation of limbal epithelial stem cells.

PURPOSE: Limbal melanocytes (LMel) represent essential components of the corneal epithelial stem cell niche and are known to protect limbal epithelial stem/progenitor cells (LEPCs) from UV damage by transfer of melanosomes. Here, we explored additional functional roles for LMel in niche homeostasis, immune regulation and angiostasis. METHODS: Human corneoscleral tissues were morphologically analyzed in normal, inflammatory and wound healing conditions. The effects of LMel on LEPCs were analyzed in direct and indirect co-culture models using electron microscopy, immunocytochemistry, qRT-PCR, Western blotting and functional assays; limbal mesenchymal stromal cells and murine embryonic 3T3 fibroblasts served as controls. The immunophenotype of LMel was assessed by flow cytometry before and after interferon-γ stimulation, and their immunomodulatory properties were analyzed by mixed lymphocytes reaction, monocyte adhesion assays and cytometric bead arrays. Their angiostatic effects on human umbilical cord endothelial cells (HUVECs) were evaluated by proliferation, migration, and tube formation assays. RESULTS: LMel and LEPCs formed structural units in the human limbal stem cell niche in situ, which could be functionally replicated, including melanosome transfer, by co-cultivation in vitro. LMel supported LEPCs during clonal expansion and during epithelial wound healing by stimulating proliferation and migration, and suppressed their differentiation through direct contact and paracrine effects. Under inflammatory conditions, LMel were increased in numbers and upregulated expression of ICAM-1 and MHC II molecules (HLA-DR), but lacked expression of HLA-G, -DP, -DQ and costimulatory molecules CD80 and CD86. They were also found to be potent suppressors of alloreactive T- cell proliferation and cytokine secretion, which largely depended on direct cell-cell interaction. Moreover, the LMel secretome exerted angiostatic activity by inhibiting vascular endothelial cell proliferation and capillary network formation. CONCLUSION: These findings suggest that LMel are not only professional melanin-producing cells, but exert various non-canonical functions in limbal niche homeostasis by regulating LEPC maintenance, immune responses, and angiostasis. Their potent regulatory, immunomodulatory and anti-angiogenic properties may have important implications for future regenerative cell therapies.

Dysfunction of the limbal epithelial stem cell niche in aniridia-associated keratopathy.

PURPOSE: Abnormalities in the limbal niche microenvironment have been suggested to be causally involved in aniridia-associated keratopathy (AAK), but histological analyses on the limbal structure and composition in AAK are lacking. Here, we investigated morphologic and molecular alterations of the limbal epithelial stem cell niche in human congenital aniridia. METHODS: The blind, buphthalmic and painful left eye of a 16-year old girl with congenital aniridia and juvenile glaucoma had to be enucleated because of uncontrolled intraocular pressure. The diagnosis of AAK was based on classical clinical features and partial limbal stem cell deficiency in the superior half. Genetic analysis identified a large heterozygous PAX6 gene deletion encompassing exons 11-15 as well as exon 9 of the neighboring ELP4 gene. Three limbal biopsies were taken from the superior, nasal and temporal regions to isolate and cultivate limbal epithelial progenitor cells and subject them to mRNA expression analyses. The globe was vertically bisected and processed for light and transmission electron microscopy and immunohistochemistry. RESULTS: Comparative analysis of the superior and inferior limbal zones showed a gradual degradation of palisade structures associated with the transition from a hyperplastic to an attenuated corneal epithelium, inflammatory cell infiltrations and basement membrane irregularities. The clinically unaffected inferior part revealed no distinct stem cell clusters in the preserved palisade region, but a uniform population of hyperproliferative, undifferentiated progenitor cells in the basal/suprabasal layers of limbal and corneal epithelia, which gave rise to maldifferentiated epithelial cells exhibiting a conjunctival/epidermal phenotype and nuclear-to-cytoplasmic translocation of Pax6. The structure of the limbal niche was fundamentally perturbed, showing marked alterations in extracellular matrix composition, dislocation of atypical melanocytes lacking melanosomes and melanin, aberrant Wnt/ß-catenin and retinoic acid signaling, and massive immune cell infiltration. CONCLUSIONS: Considering the limitations of a single Case study, the findings suggest that ocular surface alterations in AAK are caused by a primary dysfunction and gradual breakdown of the limbal stem cell niche through Pax6-related effects on both melanogenesis and epithelial differentiation.

Influence of corneal guttae and nuclear cataract on contrast sensitivity.

AIMS: To evaluate the contrast sensitivity in patients with nuclear cataract and corneal guttae compared to patients with nuclear cataract without guttae. METHODS: In this retrospective, single-centre case series, 50 eyes of 50 patients fulfilling the inclusion criteria were enrolled. Patients with corneal guttae and nuclear cataract (n=25, study group) underwent triple Descemet membrane endothelial keratoplasty (DMEK). Patients with nuclear cataract and healthy corneas underwent cataract surgery (n=25, control group). Inclusion criteria were preoperative best-corrected visual acuity ≥20/40, no corneal oedema and similar lens opacity (nuclear opalescence 2.0-2.9). Outcome measures included MARS letter and OPTEC 6500P contrast sensitivity test, corneal volume, central corneal thickness and anterior and posterior corneal densitometry. RESULTS: Preoperative MARS letter and OPTEC 6500P contrast sensitivity was significantly worse in the study group (MARS: p<0.001; OPTEC 6500P: p<0.007 at low spatial frequencies in daylight with and without glare and nightlight without glare). After surgery, there was no significant difference in MARS letter contrast sensitivity between groups (p=0.225). OPTEC 6500P contrast sensitivity remained significantly lower in the study group in daylight and nightlight with and without glare at most spatial frequencies (p<0.01) postoperatively. Preoperative and postoperative corneal volume, central corneal thickness and anterior corneal densitometry were equal in both groups (p>0.05). Posterior densitometry was significantly higher in the study group than in the control group preoperatively (p<0.001) but turned into equal values postoperatively (p=0.07). CONCLUSIONS: Corneal guttae cause an additional significant decrease in contrast sensitivity in eyes with nuclear cataract. This is in favour of performing a triple DMEK even in eyes with a visual acuity of ≥20/40.

Refractive Outcomes After Descemet Membrane Endothelial Keratoplasty + Cataract/Intraocular Lens Triple Procedure: A Fellow Eye Comparison.

PURPOSE: Descemet membrane endothelial keratoplasty is often combined with phacoemulsification and intraocular lens implantation (DMEK + cataract/IOL triple procedure) in phakic patients. This procedure results in a refractive shift that is difficult to predict. The aim of this study was to evaluate the hypothesis that the refractive shift in the second eye follows the shift in the first eye. METHODS: In this retrospective, single-center, consecutive case series, the refractive outcomes of 254 eyes of 127 patients who underwent DMEK + cataract/IOL triple procedure in both eyes for Fuchs endothelial corneal dystrophy have been analyzed. Main outcome measures were spherical equivalent outcome (shift calculations), best spectacle-corrected visual acuity, central corneal thickness, and posterior simulated keratometry. RESULTS: The mean best spectacle-corrected visual acuity before surgery was 0.51 ± 0.24 and increased to 0.19 ± 0.15 (logMAR) after surgery (P < 0.001). After surgery, a mean hyperopic shift of 0.98 ± 0.89 D was observed. The refractive shift was 1.03 ± 0.93 D and 0.92 ± 1.02 D, in the first and second eyes, respectively (P = 0.435). In a paired analysis, the mean difference of the refractive shift between the first and second eyes was 0.49 ± 0.43 D. CONCLUSIONS: In our fellow eye comparison, the refractive shift after DMEK + cataract/IOL triple procedure in the second eye was comparable with the shift in the first eye. As a consequence, the refractive outcome of the first eye might serve as a reference for optimizing the refractive target in the second eye. Further studies investigating the influence of corneal parameters on refractive shift are needed for a more predictable lens power selection.

Potential Functional Restoration of Corneal Endothelial Cells in Fuchs Endothelial Corneal Dystrophy by ROCK Inhibitor (Ripasudil).

PURPOSE: Rho-associated kinase (ROCK) inhibitors have been successfully used as a rescue strategy in eyes that failed to clear after descemetorhexis without endothelial graft for treatment of Fuchs endothelial corneal dystrophy (FECD). The functional mechanisms by which ROCK inhibitors modulate corneal endothelial cell regeneration in FECD patients have, however, not been clarified. Here, we analyzed the effect of the ROCK inhibitor ripasudil on corneal endothelial cells of FECD patients and normal donors using ex vivo tissue and in vitro cellular models. DESIGN: Experimental study: laboratory investigation. METHODS: This institutional study used endothelial cell-Descemet membrane lamellae from FECD patients (n = 450) undergoing Descemet membrane endothelial keratoplasty (FECD ex vivo model), normal research-grade donor corneas (n = 30) after scraping off central endothelial cells (ex vivo wound healing model), normal donor corneas (n = 20) without endothelial injury, and immortalized cell lines (n = 3) generated from FECD patients (FECD in vitro model). Descemet membrane lamellae were dissected into halves and incubated for 24-72 hours in storage medium with or without a single dose of 30 µM ripasudil. The effects of ripasudil on expression of genes and proteins related to endothelial cell proliferation, migration, functionality, and endothelial-to-mesenchymal transition were analyzed and complemented by functional assays on FECD cell lines. RESULTS: A single dose of ripasudil induced significant upregulation of genes and proteins related to cell cycle progression, cell-matrix adhesion and migration, as well as endothelial barrier and pump function up to 72 hours, whereas classical markers of endothelial-to-mesenchymal transition were downregulated in both FECD and normal specimens compared to unstimulated controls ex vivo. In addition to stimulation of proliferation and migration, ripasudil-induced changes in expression of functional signature genes could be also verified in FECD cell lines in vitro. CONCLUSIONS: These data support the concept that inhibition of ROCK signaling represents a potent tool in regenerative therapies in FECD patients through reactivation of cell proliferation and migration as well as restoration of endothelial pump and barrier function without inducing adverse phenotypic changes.

Laminin-511-E8 promotes efficient in vitro expansion of human limbal melanocytes.

Limbal melanocytes, located in the basal epithelial layer of the corneoscleral limbus, represent essential components of the corneal epithelial stem cell niche, but, due to difficulties in their isolation and cultivation, their biological roles and potential for stem cell-based tissue engineering approaches have not been comprehensively studied. Here, we established a protocol for the efficient isolation and cultivation of pure populations of human limbal melanocytes, which could be expanded at high yield by using recombinant laminin (LN)-511-E8 as culture substrate. Co-cultivation of limbal melanocytes with limbal epithelial stem/progenitor cells on fibrin hydrogels pre-incubated with LN-511-E8 resulted in multilayered stratified epithelial constructs within ten days. By reproducing physiological cell-cell and cell-matrix interactions of the native niche environment, these biomimetic co-culture systems provide a promising experimental model for investigating the functional roles of melanocytes in the limbal stem cell niche and their suitability for developing advanced epithelial grafts for ocular surface surface reconstruction.

Isolation and ex vivo Expansion of Human Limbal Epithelial Progenitor Cells.

Limbal stem cell transplantation has been used successfully to treat patients with limbal stem cell deficiency all over the world. However, long term clinical results often proved less satisfactory due to the low quality of the graft or inadequate properties of transplanted cells. To enhance the ex vivo expansion of human limbal epithelial stem or progenitor cells (LEPC) by preserving stem cell phenotype and to improve subsequent transplantation efficiency, cell-matrix interactions ex vivo should mimic the condition in vivo. The laminin isoforms preferentially expressed in the limbal niche can be used as a culture matrix for epithelial tissue engineering. We recently published the expansion of LEPC on various laminin isoforms and observed that laminin alpha 5-derived matrices support the efficient expansion of LEPC compared to tissue culture plates and other laminin isoforms by preserving stem/progenitor cell phenotype. Here, we describe an optimized protocol for the isolation of LEPC from cadaveric corneal limbal tissue by collagenase digestion and efficient expansion of LEPC using recombinant human laminin-511 E8 fragment (LN-511E8) as culture substrate.