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Background: Imaging mass cytometry (IMC) combines the principles of flow cytometry and mass spectrometry (MS) with laser scanning spatial resolution and offers unique advantages for the analysis of tissue samples in unprecedented detail. In contrast to conventional immunohistochemistry, which is limited in its application by the number of possible fluorochrome combinations, IMC uses isoptope-coupled antibodies that allow multiplex analysis of up to 40 markers in the same tissue section simultaneously. Methods: In this report we use IMC to analyze formalin-fixed, paraffin-embedded conjunctival tissue. We performed a 18-biomarkers IMC analysis of conjunctival tissue to determine and summarize the possibilities, relevance and limitations of IMC for deciphering the biology and pathology of ocular diseases. Results: Without modifying the manufacturer’s protocol, we observed positive and plausible staining for 12 of 18 biomarkers. Subsequent bioinformatical single-cell analysis and phenograph clustering identified 24 different cellular clusters with distinct expression profiles with respect to the markers used. Conclusions: IMC enables highly multiplexed imaging of ocular samples at subcellular resolution. IMC is an innovative and feasible method, providing new insights into ocular disease pathogenesis that will be valuable for basic research, drug discovery and clinical diagnostics. [ABSTRACT FROM AUTHOR]

A correction to the article "LRG1 promotes angiogenesis by modulating endothelial TGF-β signalling" that was published in the 2013 issue of the periodical is presented.

Central serous chorioretinopathy (CSC) is a relatively common disease that causes vision loss due to macular subretinal fluid leakage and it is often associated with reduced vision-related quality of life. In CSC, the leakage of subretinal fluid through defects in the retinal pigment epithelial layer's outer blood-retina barrier appears to occur secondary to choroidal abnormalities and dysfunction. The treatment of CSC is currently the subject of controversy, although recent data obtained from several large randomized controlled trials provide a wealth of new information that can be used to establish a treatment algorithm. Here, we provide a comprehensive overview of our current understanding regarding the pathogenesis of CSC, current therapeutic strategies, and an evidence-based treatment guideline for CSC. In acute CSC, treatment can often be deferred for up to 3-4 months after diagnosis; however, early treatment with either half-dose or half-fluence photodynamic therapy (PDT) with the photosensitive dye verteporfin may be beneficial in selected cases. In chronic CSC, half-dose or half-fluence PDT, which targets the abnormal choroid, should be considered the preferred treatment. If PDT is unavailable, chronic CSC with focal, non-central leakage on angiography may be treated using conventional laser photocoagulation. CSC with concurrent macular neovascularization should be treated with half-dose/half-fluence PDT and/or intravitreal injections of an anti-vascular endothelial growth factor compound. Given the current shortage of verteporfin and the paucity of evidence supporting the efficacy of other treatment options, future studies-ideally, well-designed randomized controlled trials-are needed in order to evaluate new treatment options for CSC., Competing Interests: Declaration of competing interest Bailey Freund is an consultant for Bayer, Genentech, Heidelberg Engineering, Nidek, Novartis, Regeneron, and Zeiss. The author authors declare not to have conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Recent studies deciphering the transcriptional profile of choroidal neovascularization (CNV) in body donor eyes with neovascular age-related macular degeneration are limited by the time span from death to preservation and the associated 5'-RNA degradation. This study therefore used CNV and control specimens that were formalin-fixed and paraffin-embedded immediately after surgical extraction and analyzed them by a 3'-RNA sequencing approach. Transcriptome profiles were analyzed to estimate content of immune and stromal cells and to define disease-associated gene signatures by using statistical and bioinformatics methods. This study identified 158 differentially expressed genes (DEGs) that were significantly increased in CNV compared with control tissue. Cell type enrichment analysis revealed a diverse cellular landscape with an enrichment of endothelial cells, macrophages, T cells, and natural killer T cells in the CNV. Gene ontology enrichment analysis found that DEGs contributed to blood vessel development, extracellular structure organization, response to wounding, and several immune-related terms. The S100 calcium-binding proteins A8 (S100A8) and A9 (S100A9) emerged among the top DEGs, as confirmed by immunohistochemistry on CNV tissue and protein analysis of vitreous samples. This study provides a high-resolution RNA-sequencing-based transcriptional signature of human CNV, characterizes its compositional pattern of immune and stromal cells, and reveals S100A8/A9 to be a novel biomarker and promising target for therapeutics and diagnostics directed at age-related macular degeneration., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The molecular chaperone Hsp90 is important for the functional maturation of many client proteins, and inhibitors are in clinical trials for multiple indications in cancer. Hsp90 inhibition activates the heat shock response and can improve viability in a cell model of the P23H misfolding mutation in rhodopsin that causes autosomal dominant retinitis pigmentosa (adRP). Here, we show that a single low dose of the Hsp90 inhibitor HSP990 enhanced visual function and delayed photoreceptor degeneration in a P23H transgenic rat model. This was associated with the induction of heat shock protein expression and reduced rhodopsin aggregation. We then investigated the effect of Hsp90 inhibition on a different type of rod opsin mutant, R135L, which is hyperphosphorylated, binds arrestin and disrupts vesicular traffic. Hsp90 inhibition with 17-AAG reduced the intracellular accumulation of R135L and abolished arrestin binding in cells. Hsf-1(-/-) cells revealed that the effect of 17-AAG on P23H aggregation was dependent on HSF-1, whereas the effect on R135L was HSF-1 independent. Instead, the effect on R135L was mediated by a requirement of Hsp90 for rhodopsin kinase (GRK1) maturation and function. Importantly, Hsp90 inhibition restored R135L rod opsin localization to wild-type (WT) phenotype in vivo in rat retina. Prolonged Hsp90 inhibition with HSP990 in vivo led to a posttranslational reduction in GRK1 and phosphodiesterase (PDE6) protein levels, identifying them as Hsp90 clients. These data suggest that Hsp90 represents a potential therapeutic target for different types of rhodopsin adRP through distinct mechanisms, but also indicate that sustained Hsp90 inhibition might adversely affect visual function.

The retina has a uniquely high metabolic demand for oxygen that is normally met by a highly efficient vascular supply. Oxygen plays an essential role in oxidative phosphorylation as an electron acceptor in the mitochondrial respiratory chain in the synthesis of adenosine triphosphate required to support the metabolic demand, including that of the visual cycle. Maintenance of normal retinal function depends on a continuous supply of oxygen and on the capability to detect and respond rapidly to local oxygen deficiency (hypoxia). The functional reserve of oxygen is small and retinal hypoxia can cause neuroretinal dysfunction and degeneration that lead directly to vision loss. Local oxygen sensing mechanisms control adaptive responses that can help protect against ischaemic injury. In the retina, powerful oxygen sensing mechanisms rapidly detect alterations in intracellular oxygen tension and respond with adaptive changes that redress the balance between oxygen supply and demand. These responses include rapid changes in blood flow, protective metabolic adaptations and angiogenesis. In the eye, however, the angiogenic response to hypoxia is typically associated with oedema, haemorrhage and fibrosis that can exacerbate hypoxic neuroretinal injury, causing severe vision loss. This aberrant response is the target of novel therapies including inhibitors of vascular endothelial growth factor. However, non-specific angiostatic agents fail to consider appropriate beneficial adaptive responses to hypoxia, and risk compromising neuroprotective mechanisms. In this review, we discuss the current understanding of retinal oxygenation and oxygen sensing in health and disease, focussing on the central role of hypoxia-inducible transcription factors, and suggest that therapeutic strategies may be improved by considering more targeted interventions., (Copyright © 2011 S. Karger AG, Basel.)

Retinal pathologies are frequently accompanied by retinal vascular responses, including the formation of new vessels by angiogenesis (neovascularization). Pathological vascular changes may also include less well characterized traits of vascular remodeling that are non-neovascular, such as vessel pruning and the emergence of dilated and tortuous vessel phenotypes (telangiectasis). The molecular mechanisms underlying neovascular growth versus non-neovascular remodeling are poorly understood. We therefore undertook to identify novel regulators of non-neovascular remodeling in the retina by using the dystrophic Royal College of Surgeons (RCS) rat and the retinal dystrophy 1 (RD1) mouse, both of which display pronounced non-neovascular remodeling. Gene expression profiling of isolated retinal vessels from these mutant rodent models and wild-type controls revealed 60 differentially expressed genes. These included the genes for apelin (Apln) and for its receptor (Aplnr), both of which were strongly up-regulated in the mutants. Crossing RD1 mice into an Apln-null background substantially reduced vascular telangiectasia. In contrast, Apln gene deletion had no effect in two models of neovascular pathology [laser-induced choroidal neovascularization and the very low density lipoprotein receptor (Vldlr)-knockout mouse]. These findings suggest that in these models apelin has minimal effect on sprouting retinal angiogenesis, but contributes significantly to pathogenic non-neovascular remodeling., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: To determine the preretinal distribution of oxygen in advanced proliferative diabetic retinopathy, and to investigate the relationship between intraocular oxygen tensions and vitreous cytokine concentrations., Design: Comparative cross-sectional study., Methods: Oxygen levels were measured at sites in the vitreous and at the inner retinal surface using an optical oxygen sensor in 14 control subjects and in 14 subjects with advanced proliferative diabetic retinopathy who had developed tractional retinal detachments despite previous panretinal photocoagulation. The vitreous and plasma concentrations of 42 cytokines were measured using multiplex cytokine arrays and their correlation with intraocular oxygen tension was investigated., Results: The mean oxygen tension in the mid-vitreous in diabetic retinopathy was 46% lower than that in control subjects (P = .017). However, the mean preretinal oxygen tension at the posterior pole in diabetic retinopathy was 37% higher than in controls (P = .039). We measured significant alterations in the vitreous concentrations of 9 cytokines-eotaxin, Flt-3 ligand, growth-related oncogene (GRO), interleukin (IL)-6, IL-8, IL-9, IFN-inducible protein-10 (IP-10), macrophage-derived cytokine (MDC), and vascular endothelial growth factor (VEGF)-in advanced proliferative diabetic retinopathy, and found that oxygen tension at the posterior pole was directly correlated with vitreous VEGF concentration., Conclusion: We identified significant intraocular oxygen gradients in proliferative diabetic retinopathy. Our findings are consistent with the hypothesis that VEGF induces the development of neovascular complexes in the posterior retina that are richly perfused but nonetheless fail to redress hypoxia in the mid-vitreous. Upregulation of vitreous VEGF may be a consequence of retinal hypoxia at unidentified sites or of chronic inflammatory processes in advanced proliferative diabetic retinopathy., (Copyright © 2011 Elsevier Inc. All rights reserved.)