Effects of miR-204-5p modulation on PAX6 regulation and corneal inflammation.

Congenital aniridia is a rare eye disease characterized by loss of PAX6 protein leading to aniridia-associated keratopathy that significantly reduces vision. The miR-204-5p is a possible regulator of PAX6 function and here we evaluate its effect in multiple in vitro and in vivo models. In vitro, miR-204-5p overexpression suppressed vascular factor ANGPT1 in human limbal stem cells (T-LSC) and Pax6-knockdown LSC (mut-LSC), and in primary human limbal epithelial cells (LEC) at the gene and protein levels and following LPS stimulation. However, miR-204-5p inhibited VEGFA expression only in mut-LSCs and LPS-stimulated LEC. Also, miR-204-5p increased PAX6 expression in mut-LSC and differentiated corneal epithelial cells, but not in LEC. Topical miR-204-5p after LPS-induced keratitis in mice failed to suppress Vegfa, Angpt1, Il-1ß, and Tnf-α or rescue Pax6 levels in contrast to in vitro results, although it significantly reduced the inflammatory infiltrate in the cornea. In Pax6Sey/+ aniridia mice, miR-204-5p did not rescue PAX6 levels or suppress Vegfa, Angpt1, or inhibit the ERK1/2 pathway. While short-term miR-204-5p treatment effectively suppresses VEGFA and ANGPT1 and enhances PAX6 expression in multiple corneal epithelia, effects are variable across primary and immortalized cells. Effects of longer-term in vivo treatment, however, require further study.

Impact of Previous Cataract Surgery in Corneal Donors on the Outcome of Descemet Membrane Endothelial Keratoplasty.

PURPOSE: The aim of this study was to investigate differences between phakic, pseudophakic, and scarred stromal donor tissue for their influence on complication rates during preparation or implantation and on the postoperative outcome of Descemet membrane endothelial keratoplasty (DMEK). METHODS: We retrospectively compared 484 eyes undergoing DMEK, divided into 3 subgroups of donor tissue (1: phakic, 2: pseudophakic, and 3: scarred stromal). Visual acuity, central corneal thickness (CCT), and endothelial cell count were monitored preoperatively and postoperatively at 6 weeks and 3, 6, 12, and 24 months. The incidence of intraoperative and postoperative complications was analyzed. RESULTS: The risk of adherence and tearing during preparation was significantly higher in group 2 than in the other groups (p's < 0.001). No significant difference was found for visual acuity (p's ≥ 0.368) and long-term CCT, but CCT recovery took longer in group 2 (P = 0.003), normalizing after 3 months (p's ≥ 0.096). The overall mean endothelial cell count was lower in group 2 compared with the other groups (P = 0.011). No difference in the rebubbling rate was detected (P = 0.890). However, the risk of repeat keratoplasty for phakic grafts was lower compared with group 2 (P = 0.008). CONCLUSIONS: Pseudophakic donor grafts are more difficult to prepare and implant, resulting in longer recovery times and a higher risk of graft failure. However, when the preparation is uneventful and no graft failure occurs, pseudophakic grafts show a comparable outcome. Given the shortage of corneal donors and the high prevalence of pseudophakic corneal donors, they should not generally be excluded from corneal donation for DMEK.

Retinal oxygen metabolic function in choroideremia and retinitis pigmentosa.

PURPOSE: To measure the retinal oxygen metabolic function with retinal oximetry (RO) in patients with choroideremia (CHM) and compare these findings with retinitis pigmentosa (RP) patients and controls. METHODS: Prospective observational study including 18 eyes of 9 molecularly confirmed CHM patients (9♂; 40.2 ± 21.2 years (mean ± SD), 77 eyes from 39 patients with RP (15♀ 24♂; 45.6 ± 14.7 years) and 100 eyes from 53 controls (31♀ 22♂; 40.2 ± 13.4 years). Main outcome parameters were the mean arterial (A-SO2; %), venular (V-SO2; %) oxygen saturation, and their difference (A-V SO2; %) recorded with the oxygen saturation tool of the Retinal Vessel Analyzer (IMEDOS Systems UG, Germany). Statistical analyses were performed with linear mixed-effects models. RESULTS: Eyes suffering from CHM differed significantly from both RP and control eyes, when the retinal oxygen metabolic parameters were taken into account. While RP showed significantly higher A-SO2 and V-SO2 values when compared to controls, CHM showed opposite findings with significantly lower values when compared to both RP and controls (P < 0.001). The A-V SO2, which represents the retinal oxygen metabolic consumption, showed significantly lower values in CHM compared to controls. CONCLUSION: The retina in CHM is a relatively hypoxic environment. The decrease in oxygen levels may be due to the profound choroidal degeneration, leading to decreased oxygen flux to the retina. RO measurements may help understand the pathogenesis of CHM and RP. These findings may provide useful details to inform the planning of clinical trials of emerging therapies for CHM. KEY MESSAGES: What was known before? Retinal oxygen metabolic function measured with retinal oximetry (RO) shows significant alterations in patients with retinitis pigmentosa. WHAT THIS STUDY ADDS: RO function in choroideremia is significantly altered when compared to controls. Furthermore, RO in choroideremia shows opposing findings within different oxygen metabolic parameters to those that were so far known for retinitis pigmentosa. By providing insights into the retinal oxygen metabolic mechanisms, RO can help understand the underlying pathophysiology in choroideremia.

Effect of Ritanserin and Duloxetine on the Gene Expression of Primary Aniridia and Healthy Human Limbal Stromal Cells, In Vitro.

INTRODUCTION: In congenital aniridia caused by mutations in paired box 6 (PAX6), PAX6 influences the migration and differentiation of limbal epithelial cells (LECs), thereby playing a pivotal role in aniridia-associated keratopathy. The antidepressants ritanserin and duloxetine affect PAX6 expression in LECs. Limbal stromal cells, which support limbal epithelial stem cells, are crucial in the limbal stem cell niche. This study explores how ritanserin and duloxetine influence gene expression in primary human limbal stromal cells from subjects with congenital aniridia and from healthy subjects, in vitro. METHODS: Primary human limbal stromal cells from corneas affected by aniridia (AN-LSCs) (n = 8) and from healthy corneas (LSCs) (n = 8) were isolated and cultured in either low-glucose serum-free (LGSF) or normal-glucose serum-containing (NGSC) media. Cells were treated with 4 µM ritanserin or duloxetine for 24 h. Quantitative PCR (qPCR) and western blot were used to assess the expression of PAX6, FOSL2, TGF-ß1, ACTA2A1, LUM, COL1A1, COL5A1, DSG1, FABP5 and ADH7. RESULTS: In AN-LSCs with LGSF-medium, ritanserin increased PAX6 messenger RNA (mRNA) (p = 0.007) and decreased TGF-ß1 and FOSL2 mRNA levels (P = 0.005, P = 0.038). In addition, TGF-ß1 protein levels decreased with both treatments (P = 0.02, P = 0.007), and FABP5 protein level increased, using ritanserin (P = 0.019). In LSCs with LGSF-medium, ACTA2A1 mRNA levels decreased using ritanserin and duloxetine (P = 0.028; P = 0.031), while FABP5 mRNA levels increased with ritanserin treatment (P = 0.003). Also, duloxetine use reduced α-SMA protein (P = 0.013) and increased FABP5 protein levels (P = 0.029). In LSCs with NGSC-medium, ritanserin elevated LUM, FABP5 and ADH7 mRNA and protein levels (P = 0.025, P = 0.003, P = 0.047, P = 0.024, P = 0.013, P = 0.039). CONCLUSIONS: The results of our study confirmed that the antipsychotropic drugs ritanserin and duloxetine alter PAX6 and TGF-ß1 gene expression in AN-LSCs cultured in LGSF-medium. These drugs were found to have an impact on retinoic acid signaling pathways and keratocyte characteristic markers both in LSCs and AN-LSCs, using different culture media.

Early REperfusion therapy with intravenous alteplase for recovery of VISION in acute central retinal artery occlusion (REVISION): Study protocol of a phase III trial.

RATIONALE: Meta-analyses of case series of non-arteritic central retinal artery occlusion (CRAO) indicate beneficial effects of intravenous thrombolysis when initiated early after symptom onset. Randomized data are lacking to address this question. AIMS: The REperfusion therapy with intravenous alteplase for recovery of VISION in acute central retinal artery occlusion (REVISION) investigates intravenous alteplase within 4.5 h of monocular vision loss due to acute CRAO. METHODS: This study is the randomized (1:1), double-blind, placebo-controlled, multicenter adaptive phase III trial. STUDY OUTCOMES: Primary outcome is functional recovery to normal or mildly impaired vision in the affected eye defined as best-corrected visual acuity of the Logarithm of the Minimum Angle of Resolution of 0.5 or less at 30 days (intention-to-treat analysis). Secondary efficacy outcomes include modified Rankin Score at 90 days and quality of life. Safety outcomes include symptomatic intracranial hemorrhage, major bleeding (International Society on Thrombosis and Haemostasis definition) and mortality. Exploratory analyses of optical coherence tomography/angiography, ultrasound and magnetic resonance imaging (MRI) biomarkers will be conducted. SAMPLE SIZE: Using an adaptive design with interim analysis at 120 patients, up to 422 participants (211 per arm) would be needed for 80% power (one-sided alpha = 0.025) to detect a difference of 15%, assuming functional recovery rates of 10% in the placebo arm and 25% in the alteplase arm. DISCUSSION: By enrolling patients within 4.5 h of CRAO onset, REVISION uses insights from meta-analyses of CRAO case series and randomized thrombolysis trials in acute ischemic stroke. Increased rates of early reperfusion and good neurological outcomes in stroke may translate to CRAO with its similar pathophysiology. TRIAL REGISTRATION: ClinicalTrials.gov: NCT04965038; EU Trial Number: 2023-507388-21-00.

Acute bilateral vision loss after endovascular treatment of an intracranial aneurysm.

Acute bilateral vision loss (ABVL) is a rare and challenging diagnostic issue that is most often caused by a neurological disorder. Since it can be the presenting symptom of potentially life-threatening diseases, priority should be given to excluding such diagnoses. Special caution is required if ABVL symptoms result after an intracranial intervention. This article reports on a diagnostic approach for a patient suffering from ABVL due to vitreous hemorrhage related to a subarachnoid hemorrhage (SAH) after endovascular intracranial aneurysm treatment. This case study highlights the importance of imaging interpretation and its consequences.

Protein profiling of conjunctival impression cytology samples of aniridia subjects.

PURPOSE: Congenital aniridia is a rare disease, which is in most cases related to PAX6 haploinsufficiency. Aniridia associated keratopathy (AAK) also belongs to ocular signs of congenital aniridia. In AAK, there is corneal epithelial thinning, corneal inflammation, vascularization and scarring. In advanced stage AAK, typically, conjunctival epithelial cells slowly replace the corneal epithelium. Based on previous results we hypothesize that alterations of the conjunctival cells in congenital aniridia may also support the corneal conjunctivalization process. The aim of this study was to identify deregulated proteins in conjunctival impression cytology samples of congenital aniridia subjects. METHODS: Conjunctival impression cytology samples of eight patients with congenital aniridia [age 34.5 ± 9.9 (17-51) years, 50% female] and eight healthy subjects [age 34.1 ± 11.9 (15-54) years, 50% female] were collected and analysed using mass spectrometry. Proteomic profiles were analysed in terms of molecular functions, biological processes, cellular components and pathway enrichment using the protein annotation of the evolutionary relationship (PANTHER) classification system. RESULTS: In total, 3323 proteins could be verified and there were 127 deregulated proteins (p < 0.01) in congenital aniridia. From the 127 deregulated proteins (DEPs), 82 altered biological processes, 63 deregulated cellular components, 27 significantly altered molecular functions and 31 enriched signalling pathways were identified. Pathological alteration of the biological processes and molecular functions of retinol binding and retinoic acid biosynthesis, as well as lipid metabolism and apoptosis related pathways could be demonstrated. CONCLUSIONS: Protein profile of conjunctival impression cytology samples of aniridia subjects identifies alterations of retinol binding, retinoic acid biosynthesis, lipid metabolism and apoptosis related pathways. Whether these changes are directly related to PAX6 haploinsufficiency, must be investigated in further studies. These new findings offer the possibility to identify potential new drug targets.

Secondary Data Analysis of Inflammation-Related mRNAs in Conjunctival Impression Cytology Samples of Aniridia Patients.

PURPOSE: Aniridia is a rare corneal disease that is often associated with aniridia-associated keratopathy (AAK). In AAK, the conjunctival tissue crosses the limbal border, forming a corneal pannus that extends into the corneal center. With increasing AAK severity, corneal pannus formation, vascularization, and ocular surface inflammation increase. The purpose of this study was to investigate inflammation-related mRNA expression in conjunctival epithelial cells in AAK and its relationship with AAK severity. METHODS: Using impression cytology, bulbar conjunctival cells were sampled from 20 subjects with congenital aniridia and 20 age-matched and sex-matched healthy control subjects. RNA was extracted, and mRNA analyses were performed using microarray, which was evaluated for inflammatory markers. RESULTS: In the analyzed aniridia subjects, 70 deregulated mRNAs encoding proinflammatory or antiinflammatory cytokines or factors associated with chronic inflammation, including increased IL-1, IL-8, and MIP3A/CCL20 mRNA. The most downregulated mRNA was TIMP3, and the most upregulated mRNA was Protein c-Fos.Of the 70 mRNAs, 14 inflammation-related genes were altered only in the mild AAK forms, whereas only 2 mRNAs were altered only in the severe AAK forms (TLR4 and PPARG). CONCLUSIONS: The expression of numerous proinflammatory and antiinflammatory cytokines is deregulated at the ocular surface of aniridia subjects with mild AAK. Thus, early antiinflammatory treatment may prevent or slow down corneal scarring and pannus formation in aniridia subjects.

Dry eye disease in astronauts: a narrative review.

Long-duration spaceflight can have adverse effects on human health. One of the most common ocular conditions experienced by astronauts is dry eye disease (DED). Symptoms of DED include feelings of eye irritation, eye strain, foreign body sensation and blurred vision. Over 30% of International Space Station expedition crew members reported irritation and foreign body sensation. We reviewed the current literature on the prevalence and mechanisms of DED in astronauts and its potential implications for long-duration spaceflight, including the influence of environmental factors, such as microgravity and fluid shift on tear film physiology in space. DED has negative effects on astronaut performance, which is why there is a need for further research into the pathophysiology and countermeasures. As an in-flight countermeasure, neurostimulation seems to be among the most promising options.

Early ocular surface and tear film status in congenital aniridia indicates a supportive treatment window.

AIM: To evaluate changes in the ocular surface and tear film with age and mutational status in congenital aniridia. METHODS: 45 participants with congenital aniridia (89 eyes) in a prospective, cross-sectional study. Whole-exome sequencing identified the causative mutation. Examinations included slit-lamp biomicroscopy, in vivo confocal microscopy, Ocular Surface Disease Index (OSDI) score, blink rate, Schirmer I test, Oxford Staining Score (OSS), tear film break-up time (TFBUT) and Ocular Protection Index (OPI). RESULTS: There were age-dependent increases in OSDI (ß=0.34, 95% CI 0.03 to 0.66; p=0.030), blink rate (ß=0.18, 95% CI 0.08 to 0.27; p<0.001) and OSS (ß=0.05, 95% CI 0.03 to 0.07; p<0.001) and age-dependent reductions in tear production (ß=-0.23, 95% CI -0.43 to 0.02; p=0.029) and TFBUT (ß=-0.10, 95% CI -0.17 to -0.04; p<0.001). Perturbed OSDI, OSS, blink rate, tear production and TFBUT were noted after the age of ten and OSDI, OSS, blink rate and TFBUT correlated with deficient corneal nerves and limbal stem cell function. OSDI, blink rate, Schirmer, OSS, TFBUT and OPI were not associated with type of PAX6 mutation, but OSDI, OSS and blink rate associated with grade of aniridia-associated keratopathy. CONCLUSIONS: Ocular surface damage and dry eye signs appear in congenital aniridia regardless of mutation, appearing after 10 years of age and progressing thereafter. An early treatment window may exist for therapies to protect the ocular surface homoeostasis and limbal function, to possibly delay keratopathy development and progression.

Decreased FABP5 and DSG1 protein expression following PAX6 knockdown of differentiated human limbal epithelial cells.

PAX6 haploinsufficiency related aniridia is characterized by disorder of limbal epithelial cells (LECs) and aniridia related keratopathy. In the limbal epithelial cells of aniridia patients, deregulated retinoic acid (RA) signaling components were identified. We aimed to visualize differentiation marker and RA signaling component expression in LECs, combining a differentiation triggering growth condition with a small interfering RNA (siRNA) based aniridia cell model (PAX6 knock down). Primary LECs were isolated from corneoscleral rims of healthy donors and cultured in serum free low Ca2+ medium (KSFM) and in KSFM supplemented with 0.9 mmol/L Ca2+. In addition, LECs were treated with siRNA against PAX6. DSG1, PAX6, KRT12, KRT 3, ADH7, RDH10, ALDH1A1, ALDH3A1, STRA6, CYP1B1, RBP1, CRABP2, FABP5, PPARG, VEGFA and ELOVL7 expression was determined using qPCR and western blot. DSG1, FABP5, ADH7, ALDH1A1, RBP1, CRABP2 and PAX6 mRNA and FABP5 protein expression increased (p ≤ 0.03), PPARG, CYP1B1 mRNA expression decreased (p ≤ 0.0003) and DSG1 protein expression was only visible after Ca2+ supplementation. After PAX6 knock down and Ca2+ supplementation, ADH7 and ALDH1A1 mRNA and DSG1 and FABP5 protein expression decreased (p ≤ 0.04), compared to Ca2+ supplementation alone. Using our cell model, with Ca2+ supplementation and PAX6 knockdown with siRNA treatment against PAX6, we provide evidence that haploinsufficiency of the master regulatory gene PAX6 contributes to differentiation defect in the corneal epithelium through alterations of RA signalling. Upon PAX6 knockdown, DSG1 differentiation marker and FABP5 RA signaling component mRNA expression decreases. A similar effect becomes apparent at protein level though differentiation triggering Ca2+ supplementation in the siRNA-based aniridia cell model. Expression data from this cell model and from our siRNA aniridia cell model strongly indicate that FABP5 expression is PAX6 dependent. These new findings may lead to a better understanding of differentiation processes in LECs and are able to explain the insufficient cell function in AAK.

[Employee survey after introduction of the FIDUS electronic patient file at the Saarland University Eye Hospital]. / Mitarbeiterbefragung nach Einführung der elektronischen Patientenakte FIDUS an der Universitätsaugenklinik des Saarlandes.

AIM: This study compared Saarland University Eye Hospital employees' satisfaction with the FIDUS electronic patient record between December 2016 and September 2020, after its introduction in January 2016. METHODS: For the retrospective study, the FIDUS team at the Saarland University Eye Hospital created a questionnaire which was distributed to all participants (physicians, nursing staff, and administrative staff) in December 2016 and September 2020. The questionnaire contained 19 questions, each scored from 0 to 10 (0: disagree; 5: partly agree; 10: completely agree). RESULTS: The number of participants increased significantly from 60/136 (47%) to 89/140 (64%) between 2016 and 2020. Of these participants, 23 (25.8%) were physicians, 27 (30.3%) were nurses, 12 (13.4%) were in administration, 16 (17.9%) were in other employment, and 11 (12.3%) did not disclose their employment. In 2020, 75.6% of employees agreed with a score of 10 that implementation of electronic medical records was the right step, compared to only 36.7% in 2016 (p < 0.001). The highest rating for "record entries are faster to write with a computer than handwritten" was obtained in 38% in 2020 vs. 25% in 2016 (p < 0.001). CONCLUSION: Satisfaction with the FIDUS electronic patient record has improved significantly in 2020 compared to 2016, particularly concerning file clarity and faster workflows. The proportion of respondents who see their initial expectations of electronic patient files as 100% fulfilled has tripled. However, there is still a need for optimization of details.

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.

[Uniform classification of the pachychoroid spectrum disorders]. / Einheitliche Klassifikation des pachychorioidalen Krankheitsspektrums.

Pachychoroid spectrum disorders are characterized by a thickening of the choroid. The spectrum includes pachychoroid pigment epitheliopathy (PPE), central serous chorioretinopathy (CCS), pachychoroid neovasculopathy (PNV), polypoidal choroidal vasculopathy (PCV)/aneurysmal type 1 choroidal neovascularization (ACNV-1), focal choroidal excavation (FCE) and peripapillary pachychoroid syndrome (PPS). If the choroid is thickened and there is a pathological alteration in the retinal pigment epithelium, the diagnosis is PPE; if the thickened choroid is accompanied by subretinal fluid, the diagnosis is CCS; if choroidal neovascularization is present, the diagnosis is PNV; if accompanied by aneurysms, the diagnosis is ACNV­1. The PPE, CCS, PNV and ACNV­1 were formerly regarded as independent disease entities but can be classified into four forms of a single disease family, pachychoroid macular disease.