Blindness from age-related macular degeneration (AMD) is an escalating problem, yet AMD pathogenesis is incompletely understood and treatments are limited. The intestinal microbiota is highly influential in ocular and extraocular diseases with inflammatory components, such as AMD. This article reviews data supporting the role of the intestinal microbiota in AMD pathogenesis. Multiple groups have found an intestinal dysbiosis in advanced AMD. There is growing evidence that environmental factors associated with AMD progression potentially work through the intestinal microbiota. A high-fat diet in apo-E-/- mice exacerbated wet and dry AMD features, presumably through changes in the intestinal microbiome, though other independent mechanisms related to lipid metabolism are also likely at play. AREDS supplementation reversed some adverse intestinal microbial changes in AMD patients. Part of the mechanism of intestinal microbial effects on retinal disease progression is via microbiota-induced microglial activation. The microbiota are at the intersection of genetics and AMD. Higher genetic risk was associated with lower intestinal bacterial diversity in AMD. Microbiota-induced metabolite production and gene expression occur in pathways important in AMD pathogenesis. These studies suggest a crucial link between the intestinal microbiota and AMD pathogenesis, thus providing a novel potential therapeutic target. Thus, the need for large longitudinal studies in patients and germ-free or gnotobiotic animal models has never been more pressing.

Background and aimsConnections between the gut microbiome and retinal diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), retinopathy of prematurity (ROP), and primary open-angle glaucoma (POAG) are recently being established. Communication between the gut microbiome and retina, referred to as the gut-retina axis, has been proposed; however, the biologic pathways and mediators involved in the interactions have not yet been elucidated. Using high-throughput RNA sequencing (RNA-seq) of whole retinas, we compare the retinal transcriptome from germ-free (GF) and specific pathogen-free (SPF) mice to investigate effects of the gut-microbiome on both retinal gene expression and biologic pathways.MethodsRNA was extracted from whole retinas of GF and SPF mice (four animals per group) and cDNA libraries were created. RNA-seq was performed on NovaSEQ6000 using the paired-end method. After preprocessing the RNA-seq data, gene expression value was calculated by count per million (CPM). The differentially expressed genes (DEGs) were identified with edgeR Bioconductor analysis of expression data. Functional enrichment and protein-protein interaction (PPI) network analyses were created for the differentially expressed genes (DEGs).ResultsRNA-sequencing reveals a cohort of 396 DEGs, of which, 173 are upregulated and 223 are downregulated in GF mouse retina. Enrichment analysis reveals that the DEGs are involved in glucocorticoid effects, transcription factor binding, cytoskeletal stability, lipid metabolism, and mitogen-activated protein kinase (MAPK). Multiple biologic pathways, including obesity/metabolic syndrome, longevity, and 5’ AMP-activated protein kinase (AMPK) signaling pathway are affected in the GF retinas. PPARG1a (PGC1a) gene is involved in 12 of the 16 significantly modulated pathways. Proteins with the most number of interactions in the PPI are E1A binding protein P300(EP300), forkhead box O3(FOXO3) and PGC1a.ConclusionsTo our knowledge, this is the first study demonstrating the involvement of the gut microbiome in driving the retinal transcriptome, providing evidence for the presence of a gut-retina axis. Future studies are needed to define the precise role of the gut-retina axis in the pathogenesis of retinal diseases.

The relationship between retinal disease, diet, and the gut microbiome has shown increasing importance over recent years. In particular, high-fat diets (HFDs) are associated with development and progression of several retinal diseases, including age-related macular degeneration (AMD) and diabetic retinopathy. However, the complex, overlapping interactions between diet, gut microbiome, and retinal homeostasis are poorly understood. Using high-throughput RNA-sequencing (RNA-seq) of whole retinas, we compare the retinal transcriptome from germ-free (GF) mice on a regular diet (ND) and HFD to investigate transcriptomic changes without influence of gut microbiome. After correction of raw data, 53 differentially expressed genes (DEGs) were identified, of which 19 were upregulated and 34 were downregulated in GF-HFD mice. Key genes involved in retinal inflammation, angiogenesis, and RPE function were identified. Enrichment analysis revealed that the top 3 biological processes affected were regulation of blood vessel diameter, inflammatory response, and negative regulation of endopeptidase. Molecular functions altered include endopeptidase inhibitor activity, protease binding, and cysteine-type endopeptidase inhibitor activity. Human and mouse pathway analysis revealed that the complement and coagulation cascades are significantly affected by HFD. This study demonstrates novel data that diet can directly modulate the retinal transcriptome independently of the gut microbiome.

No abstract

Objectives To present an index case and review the histologic and electron microscopic findings in chloroquine (CQ) and hydroxychloroquine (HCQ) myopathy, focusing primarily on cardiomyopathy. CQ and HCQ are antimalarial drugs with disease-modifying activity in rheumatic diseases (DMARD) and now are among the most widely used DMARDs. Although they are rare, severe adverse effects caused mainly by deposition of intracellular metabolites in both cardiac and skeletal muscle have been described. Currently, both CQ and HCQ have been proposed to have efficacy for patients with coronavirus disease 2019, and several large centers in the United States and other countries have started clinical trials. Methods A case of HCQ cardiotoxicity diagnosed on an endomyocardial biopsy is presented. A review of the pathology archives was performed to identify additional cases of CQ or HCQ myopathy, and histologic changes were recorded. A brief literature review with an emphasis on pathologic findings in myopathies was performed. Results Including the index case, 4 cases of CQ or HCQ myopathy were identified. Light microscopic findings included vacuolated myopathy, and electron microscopic findings included myeloid bodies and curvilinear inclusion bodies. Conclusion CQ and HCQ myopathy can present following long-term administration of the drug. The pathologic findings are nonspecific and overlap with other vacuolated myopathies, necessitating careful correlation of the histologic changes with the patient’s medical history.

Purpose Compelling new evidence reveals a close link between the gut microbiome and the pathogenesis of neovascular age-related macular degeneration (nAMD). Germ-free (GF) animal models are the current gold standard for studying host the microbe interactions in vivo; yet, no GF animal models of nAMD are available today. This protocol describes gnotobiotic operations and assembly for a laser-induced choroidal neovascularization (CNV) model in GF mice to study the gut microbiome in neovascular AMD. Methods We developed a step-wise approach to performing retinal laser photocoagulation in GF C57BL/6J mice that were bred and maintained at the gnotobiotic facility. Following a strict sterility protocol, we administered laser photocoagulation via an Argon 532-nm laser attached to a customized slit-lamp delivery system. Sterility was confirmed by weekly fecal cultures and reverse transcriptase–polymerase chain reaction. Results The experiment was repeated twice at different time points using seven mice (14 eyes). Stool cultures and RT-PCR remained negative for 14 days post-procedure in all mice. Lectin immunostaining performed on choroidal flatmounts confirmed the presence of CNV lesions 2 weeks after laser treatment. Conclusions We established a GF mouse model of nAMD with detailed guidelines to deliver retinal laser in GF mice maintaining sterility after the laser procedure. Translational Relevance To our knowledge, this is the first protocol that describes a GF murine model of laser-induced CNV. In addition to nAMD, this animal model can be used to investigate host–microbial interactions in other eye diseases with laser-induced mouse models such as glaucoma and retinal vein occlusion.

A 25-year-old African-American woman with end-stage renal disease presented with new-onset heart failure. Transthoracic echocardiography indicated a significantly hyperechoic myocardium, and computed tomography noted a circumferential hyperattenuated myocardium. Endomyocardial biopsy revealed focal interstitial and intramyocyte calcium deposition in the heart, confirming a rare diagnosis of massive myocardial calcium deposition. ( Level of Difficulty: Beginner. )

Collection of a blood sample defined by the term “blood liquid biopsy” is commonly used to detect diagnostic, prognostic, and therapeutic decision‐making markers of metastatic tumors including circulating tumor cells (CTCs). Many tumors also release CTCs and other markers into lymph fluid, but the utility of lymphatic markers largely remains unexplored. Here, we introduce lymph liquid biopsy through collection of peripheral (afferent) and central (thoracic duct [TD]) lymph samples and demonstrates its feasibility for detection of stem‐like CTCs potentially responsible for metastasis development and tumor relapse. Stemness of lymphatic CTCs (L‐CTCs) was determined by spheroid‐forming assay in vitro. Simultaneously, we tested blood CTCs by conventional blood liquid biopsy, and monitored the primary tumor size, early metastasis in a sentinel lymph node (SLN) and distant metastasis in lungs. Using a mouse model at early melanoma stage with no distant metastasis, we identified stem‐like L‐CTCs in lymph samples from afferent lymphatic vessels. Since these vessels transport cells from the primary tumor to SLN, our finding emphasizes the significance of the lymphatic pathway in development of SLN metastasis. Surprisingly, in pre‐metastatic disease, stem‐like L‐CTCs were detected in lymph samples from the TD, which directly empties lymph into blood circulation. This suggests a new contribution of the lymphatic system to initiation of distant metastasis. Integration of lymph and blood liquid biopsies demonstrated that all mice with early melanoma had stem‐like CTCs in at least one of three samples (afferent lymph, TD lymph, and blood). At the stage of distant metastasis, spheroid‐forming L‐CTCs were detected in TD lymph, but not in afferent lymph. Altogether, our results demonstrated that lymph liquid biopsy and testing L‐CTCs holds promise for diagnosis and prognosis of early metastasis. © 2020 International Society for Advancement of Cytometry