Hyalocytes-guardians of the vitreoretinal interface.

Originally discovered in the nineteenth century, hyalocytes are the resident macrophage cell population in the vitreous body. Despite this, a comprehensive understanding of their precise function and immunological significance has only recently emerged. In this article, we summarize recent in-depth investigations deciphering the critical role of hyalocytes in various aspects of vitreous physiology, such as the molecular biology and functions of hyalocytes during development, adult homeostasis, and disease. Hyalocytes are involved in fetal vitreous development, hyaloid vasculature regression, surveillance and metabolism of the vitreoretinal interface, synthesis and breakdown of vitreous components, and maintenance of vitreous transparency. While sharing certain resemblances with other myeloid cell populations such as retinal microglia, hyalocytes possess a distinct molecular signature and exhibit a gene expression profile tailored to the specific needs of their host tissue. In addition to inflammatory eye diseases such as uveitis, hyalocytes play important roles in conditions characterized by anomalous posterior vitreous detachment (PVD) and vitreoschisis. These can be hypercellular tractional vitreo-retinopathies, such as macular pucker, proliferative vitreo-retinopathy (PVR), and proliferative diabetic vitreo-retinopathy (PDVR), as well as paucicellular disorders such as vitreo-macular traction syndrome and macular holes. Notably, hyalocytes assume a significant role in the early pathophysiology of these disorders by promoting cell migration and proliferation, as well as subsequent membrane contraction, and vitreoretinal traction. Thus, early intervention targeting hyalocytes could potentially mitigate disease progression and prevent the development of proliferative vitreoretinal disorders altogether, by eliminating the involvement of vitreous and hyalocytes.

Transcriptional Comparison of Human and Murine Retinal Neovascularization.

Purpose: Retinal neovascularization (RNV) is the leading cause of vision loss in diseases like proliferative diabetic retinopathy (PDR). A significant failure rate of current treatments indicates the need for novel treatment targets. Animal models are crucial in this process, but current diabetic retinopathy models do not develop RNV. Although the nondiabetic oxygen-induced retinopathy (OIR) mouse model is used to study RNV development, it is largely unknown how closely it resembles human PDR. Methods: We therefore performed RNA sequencing on murine (C57BL/6J) OIR retinas (n = 14) and human PDR RNV membranes (n = 7) extracted during vitrectomy, each with reference to control tissue (n=13/10). Differentially expressed genes (DEG) and associated biological processes were analyzed and compared between human and murine RNV to assess molecular overlap and identify phylogenetically conserved factors. Results: In total, 213 murine- and 1223 human-specific factors were upregulated with a small overlap of 94 DEG (7% of human DEG), although similar biological processes such as angiogenesis, regulation of immune response, and extracellular matrix organization were activated in both species. Phylogenetically conserved mediators included ANGPT2, S100A8, MCAM, EDNRA, and CCR7. Conclusions: Even though few individual genes were upregulated simultaneously in both species, similar biological processes appeared to be activated. These findings demonstrate the potential and limitations of the OIR model to study human PDR and identify phylogenetically conserved potential treatment targets for PDR.

[Role of vitreous in the pathogenesis of neovascular age-related macular degeneration]. / Die Rolle des Glaskörpers in der Pathogenese der exsudativen altersabhängigen Makuladegeneration.

Age-related changes in vitreous molecular and anatomic morphology begin early in life and involve two major processes: vitreous liquefaction and weakening of vitreo-retinal adhesion. An imbalance in these two processes results in anomalous posterior vitreous detachment (PVD), which comprises, among other conditions, vitreo-macular adhesion (VMA) and traction (VMT). VMA is more common in patients with neovascular age-related macular degeneration (nAMD) than age-matched control patients, with the site of posterior vitreous adherence to the inner retina correlating with location of neovascular complexes. The pernicious effects of an attached posterior vitreous on age-related macular degeneration (AMD) progression involve mechanical forces, enhanced fluid influx and inflammation in and between the retinal layers, hypoxia leading to an accumulation of vascular endothelial growth factor (VEGF) and other stimulatory cytokines, and probably an infiltration of hyalocytes. It has been shown that vitrectomy not only mitigates progression to end-stage AMD, but existing choroidal neovascularization regresses after surgery. Thus, surgical PVD induction during vitrectomy or by pharmacologic vitreolysis may be considered in non-responders to anti-VEGF treatment with concomitant VMA.

[Vitreous body in the treatment of exudative age-related macular degeneration : The medium is the message]. / Glaskörper bei der Therapie der exsudativen altersabhängigen Makuladegeneration : Das Medium ist die Message.

BACKGROUND: Intravitreal anti-vascular endothelial growth factor (VEGF) is the standard treatment for exudative age-related macular degeneration (AMD). The constitution of the vitreomacular interface varies greatly in cases of attached (with or without traction) or detached vitreous body, which can impact the effectiveness of the anti-VEGF treatment. OBJECTIVE: Based on the current literature this article displays the current state of the science on whether the constitution of the vitreous body has an effect on the anti-VEGF treatment. MATERIAL AND METHODS: The published data extracted from current trials and post hoc analyses concerning this topic are presented and put into the clinical context. RESULTS: The presence of a vitreomacular adhesion reduces the efficacy of anti-VEGF treatment of exudative AMD. Posterior vitreous body detachment represents a positive prognostic factor concerning the efficacy of anti-VEGF treatment but not necessarily the prognosis for visual acuity. CONCLUSION: Patients with attached vitreous body need a more intensive treatment monitoring compared to patients with detached vitreous body. Therefore, in eyes with initial posterior vitreous body detachment receiving a treat and extend regimen, the interval between anti-VEGF injections can be extended to 4 instead of 2 weeks without endangering the success of treatment.

[Vitreoretinal surgery in age-related macular degeneration]. / Vitreoretinale Chirurgie bei altersabhängiger Makuladegeneration.

The structure of the vitreous body, its interaction with the retinal surface and tractive alterations of the vitreoretinal interface may play a role in the pathogenesis and the development of age-related macular degeneration (AMD). From clinical trials it can be concluded that posterior vitreous detachment (PVD) or vitreous removal may protect against the development of neovascular AMD. Vitreomacular adhesions may promote neovascular AMD and may have an impact on the efficacy and frequency of intravitreal vascular endothelial growth factor (VEGF) inhibition. Therefore, vitreomacular surgery may be considered as a treatment option in selected cases. Peeling of epimacular membranes and the internal limiting membrane (ILM) may contribute to stabilizing visual acuity and reducing the treatment burden of current intravitreal pharmacotherapy. At present, surgical interventions in AMD are mainly performed in cases of submacular hemorrhage involving the fovea. The treatment is not standardized and consists of liquefaction of the blood using recombinant tissue plasminogen activator (rTPA) and its pneumatic displacement, potentially combined with VEGF inhibition. Other submacular surgical strategies, such as choroidal neovascularization (CNV) extraction, macular translocation or transplantation of retinal pigment epithelium (RPE) are currently limited to selected cases as a salvage treatment; however, the development of these submacular surgical interventions has formed the basis for new approaches in the treatment of dry and neovascular AMD including submacular or intravitreal gene and stem cell therapy.

RECURRENT FLOATERS AFTER LIMITED VITRECTOMY FOR VISION DEGRADING MYODESOPSIA.

PURPOSE: Limited vitrectomy improves vision degrading myodesopsia, but the incidence of recurrent floaters postoperatively is not known. We studied patients with recurrent central floaters using ultrasonography and contrast sensitivity (CS) testing to characterize this subgroup and identify the clinical profile of patients at risk of recurrent floaters. METHODS: A total of 286 eyes (203 patients, 60.6 ± 12.9 years) undergoing limited vitrectomy for vision degrading myodesopsia were studied retrospectively. Sutureless 25G vitrectomy was performed without intentional surgical posterior vitreous detachment (PVD) induction. CS (Freiburg Acuity Contrast test: Weber index, %W) and vitreous echodensity (quantitative ultrasonography) were assessed prospectively. RESULTS: No eyes (0/179) with preoperative PVD experienced new floaters. Recurrent central floaters occurred in 14/99 eyes (14.1%) without complete preoperative PVD (mean follow-up = 39 months vs. 31 months in 85 eyes without recurrent floaters). Ultrasonography identified new-onset PVD in all 14 (100%) recurrent cases. Young (younger than 52 years; 71.4%), myopic (≥-3D; 85.7%), phakic (100%) men (92.9%) predominated. Reoperation was elected by 11 patients, who had partial PVD preoperatively in 5/11 (45.5%). At study entry, CS was degraded (3.55 ± 1.79 %W) but improved postoperatively by 45.6% (1.93 ± 0.86 %W, P = 0.033), while vitreous echodensity reduced by 86.6% ( P = 0.016). New-onset PVD postoperatively degraded CS anew, by 49.4% (3.28 ± 0.96 %W; P = 0.009) in patients electing reoperation. Repeat vitrectomy normalized CS to 2.00 ± 0.74%W ( P = 0.018). CONCLUSION: Recurrent floaters after limited vitrectomy for vision degrading myodesopsia are caused by new-onset PVD, with younger age, male sex, myopia, and phakic status as risk factors. Inducing surgical PVD at the primary operation should be considered in these select patients to mitigate recurrent floaters.

Hyalocytes in proliferative vitreo-retinal diseases.

Introduction: Hyalocytes are sentinel macrophages residing within the posterior vitreous cortex anterior to the retinal inner limiting membrane (ILM). Following anomalous PVD and vitreoschisis, hyalocytes contribute to paucicellular (vitreo-macular traction syndrome, macular holes) and hypercellular (macular pucker, proliferative vitreo-retinopathy, proliferative diabetic vitreo-retinopathy) diseases. Areas covered: Studies of human tissues employing dark-field, phase, and electron microscopy; immunohistochemistry; and in vivo imaging of human hyalocytes. Expert opinion: Hyalocytes are important in early pathophysiology, stimulating cell migration and proliferation, as well as subsequent membrane contraction and vitreo-retinal traction. Targeting hyalocytes early could mitigate advanced disease. Ultimately, eliminating the role of vitreous and hyalocytes may prevent proliferative vitreo-retinal diseases entirely.

Single-Cell Protein and Transcriptional Characterization of Epiretinal Membranes From Patients With Proliferative Vitreoretinopathy.

Purpose: Proliferative vitreoretinopathy (PVR) remains an unresolved clinical challenge and can lead to frequent revision surgery and blindness vision loss. The aim of this study was to characterize the microenvironment of epiretinal PVR tissue, in order to shed more light on the complex pathophysiology and to unravel new treatment options. Methods: A total of 44 tissue samples were analyzed in this study, including 19 epiretinal PVRs, 13 epiretinal membranes (ERMs) from patients with macular pucker, as well as 12 internal limiting membranes (ILMs). The cellular and molecular microenvironment was assessed by cell type deconvolution analysis (xCell), RNA sequencing data and single-cell imaging mass cytometry. Candidate drugs for PVR treatment were identified in silico via a transcriptome-based drug-repurposing approach. Results: RNA sequencing of tissue samples demonstrated distinct transcriptional profiles of PVR, ERM, and ILM samples. Differential gene expression analysis revealed 3194 upregulated genes in PVR compared with ILM, including FN1 and SPARC, which contribute to biological processes, such as extracellular matrix (ECM) organization. The xCell and IMC analyses showed that PVR membranes were composed of macrophages, retinal pigment epithelium, and α-SMA-positive myofibroblasts, the latter predominantly characterized by the co-expression of immune cell signature markers. Finally, 13 drugs were identified as potential therapeutics for PVR, including aminocaproic acid and various topoisomerase-2A inhibitors. Conclusions: Epiretinal PVR membranes exhibit a unique and complex transcriptional and cellular profile dominated by immune cells and myofibroblasts, as well as a variety of ECM components. Our findings provide new insights into the pathophysiology of PVR and suggest potential targeted therapeutic options.

Transcriptional and Distributional Profiling of Microglia in Retinal Angiomatous Proliferation.

Macular neovascularization type 3, formerly known as retinal angiomatous proliferation (RAP), is a hallmark of age-related macular degeneration and is associated with an accumulation of myeloid cells, such as microglia (MG) and infiltrating blood-derived macrophages (MAC). However, the contribution of MG and MAC to the myeloid cell pool at RAP sites and their exact functions remain unknown. In this study, we combined a microglia-specific reporter mouse line with a mouse model for RAP to identify the contribution of MG and MAC to myeloid cell accumulation at RAP and determined the transcriptional profile of MG using RNA sequencing. We found that MG are the most abundant myeloid cell population around RAP, whereas MAC are rarely, if ever, associated with late stages of RAP. RNA sequencing of RAP-associated MG showed that differentially expressed genes mainly contribute to immune-associated processes, including chemotaxis and migration in early RAP and proliferative capacity in late RAP, which was confirmed by immunohistochemistry. Interestingly, MG upregulated only a few angiomodulatory factors, suggesting a rather low angiogenic potential. In summary, we showed that MG are the dominant myeloid cell population at RAP sites. Moreover, MG significantly altered their transcriptional profile during RAP formation, activating immune-associated processes and exhibiting enhanced proliferation, however, without showing substantial upregulation of angiomodulatory factors.

In-Depth Molecular Profiling Specifies Human Retinal Microglia Identity.

Microglia are the tissue-resident macrophages of the retina and brain, being critically involved in organ development, tissue homeostasis, and response to cellular damage. Until now, little is known about the molecular signature of human retinal microglia and how it differs from the one of brain microglia and peripheral monocytes. In addition, it is not yet clear to what extent murine retinal microglia resemble those of humans, which represents an important prerequisite for translational research. The present study applies fluorescence-activated cell sorting to isolate human retinal microglia from enucleated eyes and compares their transcriptional profile with the one of whole retinal tissue, human brain microglia as well as classical, intermediate and non-classical monocytes. Finally, human retinal microglia are compared to murine retinal microglia, isolated from Cx3cr1GFP/+ mice. Whereas human retinal microglia exhibited a high grade of similarity in comparison to their counterparts in the brain, several enriched genes were identified in retinal microglia when compared to whole retinal tissue, as well as classical, intermediate, and non-classical monocytes. In relation to whole retina sequencing, several risk genes associated with age-related macular degeneration (AMD) and diabetic retinopathy (DR) were preferentially expressed in retinal microglia, indicating their potential pathophysiological involvement. Although a high degree of similarity was observed between human and murine retinal microglia, several species-specific genes were identified, which should be kept in mind when employing mouse models to investigate retinal microglia biology. In summary, this study provides detailed insights into the molecular profile of human retinal microglia, identifies a plethora of tissue-specific and species-specific genes in comparison to human brain microglia and murine retinal microglia, and thus highlights the significance of retinal microglia in human retinal diseases and for translational research approaches.

Deciphering the Molecular Signature of Human Hyalocytes in Relation to Other Innate Immune Cell Populations.

Purpose: Hyalocytes are the tissue-resident innate immune cell population of the vitreous body with important functions in health and vitreoretinal disease. The purpose of this study is to gain new insights into the biology and function of human hyalocytes in comparison to other innate immune cells. Methods: The present study applies fluorescence-activated cell sorting and RNA sequencing to compare the transcriptional profiles of human hyalocytes, retinal microglia (rMG) and classical, intermediate, and non-classical monocytes isolated from the same patients. Immunohistochemistry was applied for morphological characterization of human hyalocytes. Results: Pairwise analysis indicates distinct differences between hyalocytes and monocytes, whereas a high degree of similarity to rMG is apparent, with comparable expression levels of established microglia markers, such as TREM2, P2RY12, and TMEM119. Among the top expressed genes in hyalocytes, SPP1, CD74, and C3, were significantly upregulated when compared with monocytes. Despite the high level of similarity of hyalocytes and rMG, ten highly expressed genes in hyalocytes compared to microglia were identified, among them FOS, DUSP1, and EGR2. Conclusions: This study reveals a high degree of similarity between hyalocytes and retinal microglia. Nevertheless, hyalocytes exhibit some expression differences that may adapt them to the specific needs of the vitreous and provide the basis for deciphering the multiple roles of this fascinating cell population in health and vitreoretinal diseases.

Deficiency in Retinal TGFß Signaling Aggravates Neurodegeneration by Modulating Pro-Apoptotic and MAP Kinase Pathways.

Transforming growth factor ß (TGFß) signaling has manifold functions such as regulation of cell growth, differentiation, migration, and apoptosis. Moreover, there is increasing evidence that it also acts in a neuroprotective manner. We recently showed that TGFß receptor type 2 (Tgfbr2) is upregulated in retinal neurons and Müller cells during retinal degeneration. In this study we investigated if this upregulation of TGFß signaling would have functional consequences in protecting retinal neurons. To this end, we analyzed the impact of TGFß signaling on photoreceptor viability using mice with cell type-specific deletion of Tgfbr2 in retinal neurons and Müller cells (Tgfbr2ΔOC) in combination with a genetic model of photoreceptor degeneration (VPP). We examined retinal morphology and the degree of photoreceptor degeneration, as well as alterations of the retinal transcriptome. In summary, retinal morphology was not altered due to TGFß signaling deficiency. In contrast, VPP-induced photoreceptor degeneration was drastically exacerbated in double mutant mice (Tgfbr2ΔOC; VPP) by induction of pro-apoptotic genes and dysregulation of the MAP kinase pathway. Therefore, TGFß signaling in retinal neurons and Müller cells exhibits a neuroprotective effect and might pose promising therapeutic options to attenuate photoreceptor degeneration in humans.

The Human Eye Transcriptome Atlas: A searchable comparative transcriptome database for healthy and diseased human eye tissue.

The applications of deep sequencing technologies in life science research and clinical diagnostics have increased rapidly over the last decade. Although fast algorithms for data processing exist, intuitive, portable solutions for data analysis are still rare. For this purpose, we developed a web-based transcriptome database, which provides a platform-independent, intuitive solution to easily explore and compare ocular gene expression of 100 diseased and healthy human tissue samples from 15 different tissue types collected at the Eye Center of the University of Freiburg. To ensure comparability of expression between different tissues, reads were normalized across all 100 samples. Differentially expressed genes were calculated between each tissue type to determine tissue-specific genes. Unsupervised analysis of all 100 samples revealed an accurate clustering according to different tissue types and a high tissue specificity by analyzing known tissue-specific marker genes. Bioinformatic cell type deconvolution using xCell provided detailed insights into the cellular profiles of each tissue type. Several new tissue-specific marker genes were identified. These genes were involved in tissue- or disease-specific processes, such as myelination for the optic nerve, visual perception for retina, keratinocyte differentiation for conjunctival carcinoma, as well as endothelial cell migration for choroidal neovascularization membranes. The results are accessible at the Human Eye Transcriptome Atlas website at https://www.eye-transcriptome.com. In summary, this searchable transcriptome database enables easy exploration of ocular gene expression in healthy and diseased human ocular tissues without bioinformatics expertise. Thus, it provides rapid access to detailed insights into the molecular mechanisms of various ocular tissues and diseases, as well as the rapid retrieval of potential new diagnostic and therapeutic targets.

Comparative transcriptome analysis of human and murine choroidal neovascularization identifies fibroblast growth factor inducible-14 as phylogenetically conserved mediator of neovascular age-related macular degeneration.

BACKGROUND: Visual outcome of patients with neovascular age-related macular degeneration has significantly improved during the last years following the introduction of anti-vascular endothelial growth factor (VEGF) therapy. However, about one third of patients show persistent exudation and decreasing visual acuity despite recurrent anti-VEGF treatment, which implies a role of other, still unknown proangiogenic mediators. METHODS: The present study applied transcriptional profiling of human and mouse (C57BL/6J wildtype) choroidal neovascularization (CNV) membranes each with reference to healthy control tissue to identify yet unrecognized mediators of CNV formation. Key factors were further investigated by immunohistochemistry as well as by intravitreal inhibition experiments and multiplex protein assays in the laser-induced CNV mouse model. FINDINGS: Transcriptional profiles of CNV membranes were characterized by enhanced activation of blood vessel development, cytoskeletal organization, and cytokine production, with angiogenesis and wound healing processes predominating in humans and activation of immune processes in mice. Besides several species-specific factors, 95 phylogenetically conserved CNV-associated genes were detected, among which fibroblast growth factor inducible-14 (FN14), a member of the tumor necrosis factor (TNF) receptor family, was identified as a key player of CNV formation. Blocking the pathway by intravitreal injection of a FN14 decoy receptor modulated the cytokine profile - most notably IL-6 - and led to a significant reduction of CNV size in vivo. INTERPRETATION: This study characterizes the transcriptome of human and mouse CNV membranes in an unprejudiced manner and identifies FN14 as a phylogenetically conserved mediator of CNV formation and a promising new therapeutic target for neovascular AMD. FUNDING: This study was funded by the Helmut Ecker Foundation and the Volker Homann Foundation.

Subretinal fibrosis in neovascular age-related macular degeneration: current concepts, therapeutic avenues, and future perspectives.

Age-related macular degeneration (AMD) is a progressive, degenerative disease of the human retina which in its most aggressive form is associated with the formation of macular neovascularization (MNV) and subretinal fibrosis leading to irreversible blindness. MNVs contain blood vessels as well as infiltrating immune cells, myofibroblasts, and excessive amounts of extracellular matrix proteins such as collagens, fibronectin, and laminin which disrupts retinal function and triggers neurodegeneration. In the mammalian retina, damaged neurons cannot be replaced by tissue regeneration, and subretinal MNV and fibrosis persist and thus fuel degeneration and visual loss. This review provides an overview of subretinal fibrosis in neovascular AMD, by summarizing its clinical manifestations, exploring the current understanding of the underlying cellular and molecular mechanisms and discussing potential therapeutic approaches to inhibit subretinal fibrosis in the future.

Immunosenescence in Choroidal Neovascularization (CNV)-Transcriptional Profiling of Naïve and CNV-Associated Retinal Myeloid Cells during Aging.

Immunosenescence is considered a possible factor in the development of age-related macular degeneration and choroidal neovascularization (CNV). However, age-related changes of myeloid cells (MCs), such as microglia and macrophages, in the healthy retina or during CNV formation are ill-defined. In this study, Cx3cr1-positive MCs were isolated by fluorescence-activated cell sorting from six-week (young) and two-year-old (old) Cx3cr1GFP/+ mice, both during physiological aging and laser-induced CNV development. High-throughput RNA-sequencing was performed to define the age-dependent transcriptional differences in MCs during physiological aging and CNV development, complemented by immunohistochemical characterization and the quantification of MCs, as well as CNV size measurements. These analyses revealed that myeloid cells change their transcriptional profile during both aging and CNV development. In the steady state, senescent MCs demonstrated an upregulation of factors contributing to cell proliferation and chemotaxis, such as Cxcl13 and Cxcl14, as well as the downregulation of microglial signature genes. During CNV formation, aged myeloid cells revealed a significant upregulation of angiogenic factors such as Arg1 and Lrg1 concomitant with significantly enlarged CNV and an increased accumulation of MCs in aged mice in comparison to young mice. Future studies need to clarify whether this observation is an epiphenomenon or a causal relationship to determine the role of immunosenescence in CNV formation.

In-Depth Molecular Characterization of Neovascular Membranes Suggests a Role for Hyalocyte-to-Myofibroblast Transdifferentiation in Proliferative Diabetic Retinopathy.

Background: Retinal neovascularization (RNV) membranes can lead to a tractional retinal detachment, the primary reason for severe vision loss in end-stage disease proliferative diabetic retinopathy (PDR). The aim of this study was to characterize the molecular, cellular and immunological features of RNV in order to unravel potential novel drug treatments for PDR. Methods: A total of 43 patients undergoing vitrectomy for PDR, macular pucker or macular hole (control patients) were included in this study. The surgically removed RNV and epiretinal membranes were analyzed by RNA sequencing, single-cell based Imaging Mass Cytometry and conventional immunohistochemistry. Immune cells of the vitreous body, also known as hyalocytes, were isolated from patients with PDR by flow cytometry, cultivated and characterized by immunohistochemistry. A bioinformatical drug repurposing approach was applied in order to identify novel potential drug options for end-stage diabetic retinopathy disease. Results: The in-depth transcriptional and single-cell protein analysis of diabetic RNV tissue samples revealed an accumulation of endothelial cells, macrophages and myofibroblasts as well as an abundance of secreted ECM proteins such as SPARC, FN1 and several types of collagen in RNV tissue. The immunohistochemical staining of cultivated vitreal hyalocytes from patients with PDR showed that hyalocytes express α-SMA (alpha-smooth muscle actin), a classic myofibroblast marker. According to our drug repurposing analysis, imatinib emerged as a potential immunomodulatory drug option for future treatment of PDR. Conclusion: This study delivers the first in-depth transcriptional and single-cell proteomic characterization of RNV tissue samples. Our data suggest an important role of hyalocyte-to-myofibroblast transdifferentiation in the pathogenesis of diabetic vitreoretinal disease and their modulation as a novel possible clinical approach.

Imaging mass cytometry for high-dimensional tissue profiling in the eye.

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.

The role of interferon regulatory factor 8 for retinal tissue homeostasis and development of choroidal neovascularisation.

BACKGROUND: Microglia cells represent the resident innate immune cells of the retina and are important for retinal development and tissue homeostasis. However, dysfunctional microglia can have a negative impact on the structural and functional integrity of the retina under native and pathological conditions. METHODS: In this study, we examined interferon-regulatory factor 8 (Irf8)-deficient mice to determine the transcriptional profile, morphology, and temporospatial distribution of microglia lacking Irf8 and to explore the effects on retinal development, tissue homeostasis, and formation of choroidal neovascularisation (CNV). RESULTS: Our study shows that Irf8-deficient MG exhibit a considerable loss of microglial signature genes accompanied by a severely altered MG morphology. An in-depth characterisation by fundus photography, fluorescein angiography, optical coherence tomography and electroretinography revealed no major retinal abnormalities during steady state. However, in the laser-induced CNV model, Irf8-deficient microglia showed an increased activity of biological processes critical for inflammation and cell adhesion and a reduced MG cell density near the lesions, which was associated with significantly increased CNV lesion size. CONCLUSIONS: Our results suggest that loss of Irf8 in microglia has negligible effects on retinal homeostasis in the steady state. However, under pathological conditions, Irf8 is crucial for the transformation of resident microglia into a reactive phenotype and thus for the suppression of retinal inflammation and CNV formation.

Transcriptional Profiling Identifies Upregulation of Neuroprotective Pathways in Retinitis Pigmentosa.

Hereditary retinal degenerations like retinitis pigmentosa (RP) are among the leading causes of blindness in younger patients. To enable in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death and to allow testing of therapeutic strategies that could prevent retinal degeneration, animal models have been created. In this study, we deeply characterized the transcriptional profile of mice carrying the transgene rhodopsin V20G/P23H/P27L (VPP), which is a model for autosomal dominant RP. We examined the degree of photoreceptor degeneration and studied the impact of the VPP transgene-induced retinal degeneration on the transcriptome level of the retina using next generation RNA sequencing (RNASeq) analyses followed by weighted correlation network analysis (WGCNA). We furthermore identified cellular subpopulations responsible for some of the observed dysregulations using in situ hybridizations, immunofluorescence staining, and 3D reconstruction. Using RNASeq analysis, we identified 9256 dysregulated genes and six significantly associated gene modules in the subsequently performed WGCNA. Gene ontology enrichment showed, among others, dysregulation of genes involved in TGF-ß regulated extracellular matrix organization, the (ocular) immune system/response, and cellular homeostasis. Moreover, heatmaps confirmed clustering of significantly dysregulated genes coding for components of the TGF-ß, G-protein activated, and VEGF signaling pathway. 3D reconstructions of immunostained/in situ hybridized sections revealed retinal neurons and Müller cells as the major cellular population expressing representative components of these signaling pathways. The predominant effect of VPP-induced photoreceptor degeneration pointed towards induction of neuroinflammation and the upregulation of neuroprotective pathways like TGF-ß, G-protein activated, and VEGF signaling. Thus, modulation of these processes and signaling pathways might represent new therapeutic options to delay the degeneration of photoreceptors in diseases like RP.