Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases closely associated with the metabolic system, including obesity and type 2 diabetes. The progression of NAFLD with advanced fibrosis is associated with an increased risk of liver cirrhosis and cancer as well as various extra-hepatic diseases. Yet, the underlying mechanism is not fully understood partly due to the absence of effective high-resolution in vivo imaging methods and the appropriate animal models recapitulating the pathology of NAFLD. To improve our understanding about complex pathophysiology of NAFLD, the need for an advanced imaging methodology to visualize and quantify subcellular-level features of NAFLD in vivo over time is ever-increasing. In this study, we established an advanced in vivo two-photon imaging technique to visualize and quantify subcellular-level pathological features of NAFLD in a live mouse animal developing hepatic steatosis, fibrosis, and disrupted microvasculature.

Glutamate, a crucial player in hepatic amino acid metabolism, has been relatively unexplored in immune cell activation. We show in a study with male mice that hepatic glutamate accumulates in vesicles of perivenous hepatocytes through vesicular glutamate transporter 3 (VGLUT3), regulated by the aryl hydrocarbon receptor upon chronic alcohol intake. Additional binge drinking triggers the exocytosis of glutamate by altering the intracellular Ca²⁺ level, stimulating metabotropic glutamate receptor 5 (mGluR5) and subsequent NADPH oxidase 2 (NOX2)-mediated ROS production in Kupffer cells (KCs). This interaction between hepatocytes and KCs is facilitated by pseudosynapse formation, arising from alcohol-induced ballooning of perivenous hepatocytes. Genetic or pharmacological interference of mGluR5 or NOX2 in KCs alleviates alcohol-related steatohepatitis (ASH). Analysis of patient samples confirmed some of the findings from mice, showing that plasma glutamate concentration and VGLUT3 levels correlate with ASH development. Conclusively, our findings highlight glutamate storage and release in mediating ASH, particularly through the pseudosynapse between hepatocytes and KCs.

The development of choroidal neovascularization (CNV) is a crucial factor in the pathophysiology and prognosis of exudative age-related macular degeneration (AMD). Therefore, the detection of CNV is essential for establishing an appropriate diagnosis and treatment plan. Current ophthalmic imaging techniques, such as fundus fluorescent angiography and optical coherence tomography, have limitations in accurately visualizing CNV lesions and expressing CNV activity, owing to issues such as excessive dye leakage with pooling and the inability to provide functional information. Here, using the arginine−glycine−aspartic acid (RGD) peptide’s affinity for integrin α_vβ₃, which is expressed in the neovascular endothelial cells in ocular tissues, we propose the use of fluorescein isothiocyanate (FITC)-labeled RGD peptide as a novel dye for effective molecular imaging of CNV. FITC-labeled RGD peptides (FITC-RGD₂), prepared by bioconjugation of one FITC molecule with two RGD peptides, demonstrated better visualization and precise localization of CNV lesions than conventional fluorescein dyes in laser-induced CNV rodent models, as assessed using various imaging techniques, including a commercially available clinical fundus camera (Optos). These results suggest that FITC-RGD₂ can serve as an effective novel dye for the diagnosis of neovascular retinal diseases, including AMD, by enabling early detection and treatment of disease occurrence and recurrence after treatment.

Background/aims Circumscribed choroidal haemangioma (CCH) can cause visual impairment, primarily managed with photodynamic therapy (PDT). This study aimed to identify predictive factors of therapeutic response in patients with CCH treated with PDT and to analyse serial anatomical outcomes, focusing on visual acuity, tumour characteristics, and inner retinal microstructures. Methods This retrospective study included patients diagnosed with CCH at Severance Eye Hospital, Yonsei University, from January 2005 to December 2022. We reviewed patient records and multimodal imaging, assessing demographics, including post-PDT visual acuity changes, age, laterality and sex, and multimodal imaging features such as tumour location and height, presence of subfoveal subretinal fluid (SRF), enhanced reflectivity of the retinal pigment epithelium and external limiting membrane (ELM) disruption. Results Among 114 patients with CCH (82 men, 65.6%; mean age 52.1±14.0 years), 45 (39.5%) had asymptomatic CCH, while 69 (60.5%) presented with symptomatic CCH. PDT was administered to 41 symptomatic patients (59.4%). The pretreatment LogMAR visual acuity was 0.50±0.63, which remained stable 1 year after PDT (0.53±0.64, p=0.666). However, it worsened 3 years after PDT (0.81±0.89, p=0.024), around the time tumour regrowth was observed. At 1-year post-PDT, tumour volume and height decreased by an average of 34.6% and 32.4%, respectively, accompanied by a 75.0% reduction in SRF. Patients with >75% vol reduction showed greater SRF persistence, while a higher initial SRF height was associated with complete SRF absorption following PDT. ELM disruption was identified as a significant predictor of poorer visual acuity, reduced tumour volume response and limited SRF absorption. Conclusions The presence of SRF predicts favourable tumour size reduction and SRF absorption post-PDT, while ELM disruption serves as an important prognostic factor for visual acuity and structural outcomes, aiding in pre-PDT treatment planning.

In dental pulp, diverse types of cells mediate the dental pulp immunity in a highly complex and dynamic manner. Yet, 3D spatiotemporal changes of various pulpal immune cells dynamically reacting against foreign pathogens during immune response have not been well characterized. It is partly due to the technical difficulty in detailed 3D comprehensive cellular-level observation of dental pulp in whole intact tooth beyond the conventional histological analysis using thin tooth slices. In this work, we validated the optical clearing technique based on modified Murray’s clear as a valuable tool for a comprehensive cellular-level analysis of dental pulp. Utilizing the optical clearing, we successfully achieved a 3D visualization of CD11c+ dendritic cells in the dentin-pulp complex of a whole intact murine tooth. Notably, a small population of unique CD11c+ dendritic cells extending long cytoplasmic processes into the dentinal tubule while located at the dentin-pulp interface like odontoblasts were clearly visualized. 3D visualization of whole murine tooth enabled a reliable observation of these rarely existing cells with a total number less than a couple of tens in one tooth. These CD11c+ dendritic cells with processes in the dentinal tubule were significantly increased in the dental pulpitis model induced by mechanical and chemical irritation. Additionally, the 3D visualization revealed a distinct spatial 3D arrangement of pulpal CD11c+ cells in the pulp into a front-line barrier-like formation in the pulp within 12 h after the irritation. Collectively, these observations demonstrated the unique capability of optical clearing-based comprehensive 3D cellular-level visualization of the whole tooth as an efficient method to analyze 3D spatiotemporal changes of various pulpal cells in normal and pathological conditions.