In vivo longitudinal 920†nm two-photon intravital kidney imaging of a dynamic 2,8-DHA crystal formation and tubular deterioration in the adenine-induced chronic kidney disease mouse model.

Chronic kidney disease (CKD) is one of the most common renal diseases manifested by gradual loss of kidney function with no symptoms in the early stage. The underlying mechanism in the pathogenesis of CKD with various causes such as high blood pressure, diabetes, high cholesterol, and kidney infection is not well understood. In vivo longitudinal repetitive cellular-level observation of the kidney of the CKD animal model can provide novel insights to diagnose and treat the CKD by visualizing the dynamically changing pathophysiology of CKD with its progression over time. In this study, using two-photon intravital microscopy with a single 920 nm fixed-wavelength fs-pulsed laser, we longitudinally and repetitively observed the kidney of an adenine diet-induced CKD mouse model for 30 days. Interestingly, we could successfully visualize the 2,8-dihydroxyadenine (2,8-DHA) crystal formation with a second-harmonics generation (SHG) signal and the morphological deterioration of renal tubules with autofluorescence using a single 920 nm two-photon excitation. The longitudinal in vivo two-photon imaging results of increasing 2,8-DHA crystals and decreasing tubular area ratio visualized by SHG and autofluorescence signal, respectively, were highly correlated with the CKD progression monitored by a blood test showing increased cystatin C and blood urea nitrogen (BUN) levels over time. This result suggests the potential of label-free second-harmonics generation crystal imaging as a novel optical technique for in vivo CKD progression monitoring.

In vivo observation of multi-phase spatiotemporal cellular dynamics of transplanted HSPCs during early engraftment.

Hematopoietic stem cell transplantation (HSCT) is commonly used to treat patients with various blood disorders, genetic and immunological diseases, and solid tumors. Several systemic complications following HSCT are critical limiting factors for achieving a successful outcome. These systemic complications are mainly due to the lack of initial engraftment after transplantation. However, the detailed underlying cellular dynamics of early engraftment have not been fully characterized yet. We performed in vivo longitudinal visualization of early engraftment characteristics of transplanted hematopoietic stem and progenitor cells (HSPCs) in the mouse calvarial bone marrow (BM). To achieve this, we utilized an in vivo laser-scanning confocal microscopy imaging system with a cranial BM imaging window and stereotaxic device. We observed two distinct cellular behaviors of HSPCs in vivo, cluster formation and cluster dissociation, early after transplantation. Furthermore, we successfully identified three cellular phases of engraftment with distinct cellular distances which are coordinated with cell proliferation and cell migration dynamics during initial engraftment.

Intravital longitudinal cellular visualization of oral mucosa in a murine model based on rotatory side-view confocal endomicroscopy.

Oral mucosa is a soft tissue lining the inside of the mouth, protecting the oral cavity from microbiological insults. The mucosal immune system is composed of diverse types of cells that defend against a wide range of pathogens. The pathophysiology of various oral mucosal diseases has been studied mostly by ex vivo histological analysis of harvested specimens. However, to analyze dynamic cellular processes in the oral mucosa, longitudinal in vivo observation of the oral mucosa in a single mouse during pathogenesis is a highly desirable and efficient approach. Herein, by utilizing micro GRIN lens-based rotatory side-view confocal endomicroscopy, we demonstrated non-invasive longitudinal cellular-level in vivo imaging of the oral mucosa, visualizing fluorescently labeled cells including various immune cells, pericytes, nerve cells, and lymphatic and vascular endothelial cells. With rotational and sliding movement of the side-view endomicroscope on the oral mucosa, we successfully achieved a multi-color wide-area cellular-level visualization in a noninvasive manner. By using a transgenic mouse expressing photoconvertible protein, Kaede, we achieved longitudinal repetitive imaging of the same microscopic area in the buccal mucosa of a single mouse for up to 10 days. Finally, we performed longitudinal intravital visualization of the oral mucosa in a DNFB-derived oral contact allergy mouse model, which revealed highly dynamic spatiotemporal changes of CSF1R or LysM expressing immune cells such as monocytes, macrophages, and granulocytes in response to allergic challenge for one week. This technique can be a useful tool to investigate the complex pathophysiology of oral mucosal diseases.

Characterization of junctional structures in the gingival epithelium as barriers against bacterial invasion.

BACKGROUND AND OBJECTIVE: Adherens junctions (AJs) and tight junctions (TJs) are known to play a crucial role in maintaining the physical barrier function of the epithelium. Here, we aimed to characterize the distribution of AJs and TJs throughout the gingival epithelium and to obtain insights into the physiological importance of these junctional structures. METHODS: Sections of mouse gingival tissue were examined using transmission electron microscopy (TEM) and bio-high voltage electron microscopy tomography. The gingival sections were stained for E-cadherin and JAM-A as markers of AJs and TJs, respectively, and examined using confocal microscopy and lattice structured illumination microscopy. Bacteria within the gingival epithelium were examined using in situ hybridization. RESULTS: Junctional structures, including desmosomes, AJs, and TJs, were observed throughout the gingival epithelium. The expression levels of E-cadherin were particularly low in the granular/keratinized layers of the oral epithelium (OE), while extremely low JAM-A levels were detected in the granular/keratinized layers of the sulcular epithelium (SE). The three-dimensional rendering of the junctional structures revealed that both AJs and TJs in the gingival epithelium formed discontinuous short bands or patches. Interestingly, strong bacterial signals were observed at the granular/keratinized layers of both SE and OE, but a few bacteria were detected within the junctional epithelium (JE) and the basal/spinous layers of the SE and OE. CONCLUSIONS: AJs and TJs form a discontinuous barrier throughout paracellular passage in the gingival epithelium; nevertheless, they seem to play an important role in defending against invading bacteria.

Estrogen-Related Receptor γ Maintains Pancreatic Acinar Cell Function and Identity by Regulating Cellular Metabolism.

BACKGROUND & AIMS: Mitochondrial dysfunction disrupts the synthesis and secretion of digestive enzymes in pancreatic acinar cells and plays a primary role in the etiology of exocrine pancreas disorders. However, the transcriptional mechanisms that regulate mitochondrial function to support acinar cell physiology are poorly understood. Here, we aim to elucidate the function of estrogen-related receptor γ (ERRγ) in pancreatic acinar cell mitochondrial homeostasis and energy production. METHODS: Two models of ERRγ inhibition, GSK5182-treated wild-type mice and ERRγ conditional knock-out (cKO) mice, were established to investigate ERRγ function in the exocrine pancreas. To identify the functional role of ERRγ in pancreatic acinar cells, we performed histologic and transcriptome analysis with the pancreas isolated from ERRγ cKO mice. To determine the relevance of these findings for human disease, we analyzed transcriptome data from multiple independent human cohorts and conducted genetic association studies for ESRRG variants in 2 distinct human pancreatitis cohorts. RESULTS: Blocking ERRγ function in mice by genetic deletion or inverse agonist treatment results in striking pancreatitis-like phenotypes accompanied by inflammation, fibrosis, and cell death. Mechanistically, loss of ERRγ in primary acini abrogates messenger RNA expression and protein levels of mitochondrial oxidative phosphorylation complex genes, resulting in defective acinar cell energetics. Mitochondrial dysfunction due to ERRγ deletion further triggers autophagy dysfunction, endoplasmic reticulum stress, and production of reactive oxygen species, ultimately leading to cell death. Interestingly, ERRγ-deficient acinar cells that escape cell death acquire ductal cell characteristics, indicating a role for ERRγ in acinar-to-ductal metaplasia. Consistent with our findings in ERRγ cKO mice, ERRγ expression was significantly reduced in patients with chronic pancreatitis compared with normal subjects. Furthermore, candidate locus region genetic association studies revealed multiple single nucleotide variants for ERRγ that are associated with chronic pancreatitis. CONCLUSIONS: Collectively, our findings highlight an essential role for ERRγ in maintaining the transcriptional program that supports acinar cell mitochondrial function and organellar homeostasis and provide a novel molecular link between ERRγ and exocrine pancreas disorders.

Longitudinal intravital imaging of cerebral microinfarction reveals a dynamic astrocyte reaction leading to glial scar formation.

Cerebral microinfarct increases the risk of dementia. But how microscopic cerebrovascular disruption affects the brain tissue in cellular-level are mostly unknown. Herein, with a longitudinal intravital imaging, we serially visualized in vivo dynamic cellular-level changes in astrocyte, pericyte and neuron as well as microvascular integrity after the induction of cerebral microinfarction for 1 month in mice. At day 2-3, it revealed a localized edema with acute astrocyte loss, neuronal death, impaired pericyte-vessel coverage and extravascular leakage of 3 kDa dextran (but not 2 MDa dextran) indicating microinfarction-related blood-brain barrier (BBB) dysfunction for small molecules. At day 5, the local edema disappeared with the partial restoration of microcirculation and recovery of pericyte-vessel coverage and BBB integrity. But brain tissue continued to shrink with persisted loss of astrocyte and neuron in microinfarct until 30 days, resulting in a collagen-rich fibrous scar surrounding the microinfarct. Notably, reactive astrocytes expressing glial fibrillary acidic protein (GFAP) appeared at the peri-infarct area early at day 2 and thereafter accumulated in the peri-infarct until 30 days, inducing glial scar formation in cerebral cortex. Our longitudinal intravital imaging of serial microscopic neurovascular pathophysiology in cerebral microinfarction newly revealed that astrocytes are critically susceptible to the acute microinfarction and their reactive response leads to the fibrous glial scar formation.

3D Visualization of Dynamic Cellular Reaction of Pulpal CD11c+ Dendritic Cells against Pulpitis in Whole Murine Tooth.

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.

Longitudinal Intravital Imaging of Tumor-Infiltrating Lymphocyte Motility in Breast Cancer Models.

Immunoreactive dynamics of tumor-infiltrating lymphocytes (TILs) within the tumor microenvironment in breast cancer are not well understood. This study aimed to investigate the spatiotemporal cellular dynamics of TILs in breast cancer models. Breast cancer cells were implanted into the dorsal skinfold chamber of BALB/c nude mice, and T lymphocytes were adoptively transferred. Longitudinal intravital imaging was performed, and the spatiotemporal dynamics of TILs were assessed. In the 4T1 model, TILs progressively exhibited increased motility, and their motility inside the tumor was significantly higher than that outside the tumor. In the MDA-MB-231 model, the motility of TILs progressively decreased after an initial increase. TIL motility in the MDA-MB-231 and MCF-7 models differed significantly, suggesting an association between programmed death-ligand 1 expression levels and TIL motility, which warrants further investigation. Furthermore, intravital imaging of TILs can be a useful method for addressing dynamic interactions between TILs and breast cancer cells.

Intravital Imaging of Circulating Red Blood Cells in the Retinal Vasculature of Growing Mice.

Purpose: To establish a custom-built, high-speed 90 frame-per-second laser-scanning confocal microscope for real-time in vivo retinal imaging of individual flowing red blood cells (RBCs) in retinal vasculature of live mouse model. Methods: Fluorescently labeled RBCs were injected into mice of different ages (3 to 62 weeks old). Anti-CD31 antibody conjugated with Alexa Fluor 647 was injected to visualize retinal endothelial cells (ECs). Longitudinal and cross-sectional intravital retinal imaging of flowing RBCs and ECs was performed in two strains (C57BL/6 and Balb/c) by using the custom-built confocal microscope. Results: Simultaneous tracking of the routes of many fluorescently labeled individual RBCs flowing from a large artery and vein to a single capillary in the retina of live mice was achieved, which enabled in vivo measurement of retinal RBC flow velocities in each vessel type in growing mice from 3 to 62 weeks after birth. Average RBC flow velocities were gradually increased during growing from 3 to 14 weeks by more than two times. Then the average RBC flow velocity was maintained at about 20 mm/s in artery and 16 mm/s in vein until 62 weeks. Conclusions: Our study successfully established a custom-built high-speed 90-Hz retinal confocal microscope for measuring RBC flow velocity at the single cell level. It could be a useful tool to investigate the pathophysiology of various retinal diseases associated with blood flow impairment. Translational Relevance: This technological method could be a valuable assessment tool to help the development of novel therapeutics for retinal diseases.

Development and evaluation of an ultrasound-triggered microbubble combined transarterial chemoembolization (TACE) formulation on rabbit VX2 liver cancer model.

Transarterial chemoembolization (TACE) is an image-guided locoregional therapy used for the treatment of patients with primary or secondary liver cancer. However, conventional TACE formulations are rapidly dissociated due to the instability of the emulsion, resulting in insufficient local drug concentrations in the target tumor. Methods: To overcome these limitations, a doxorubicin-loaded albumin nanoparticle-conjugated microbubble complex in an iodized oil emulsion (DOX-NPs-MB complex in Lipiodol) has been developed as a new ultrasound-triggered TACE formulation. Results: (1) Microbubbles enhanced therapeutic efficacy by effectively delivering doxorubicin- loaded nanoparticles into liver tumors via sonoporation under ultrasound irradiation (US+). (2) Microbubbles constituting the complex retained their function as an ultrasound contrast agent in Lipiodol. In a rabbit VX2 liver cancer model, the in vivo study of DOX-NPs-MB complex in Lipiodol (US+) decreased the viability of tumor more than the conventional TACE formulation, and in particular, effectively killed cancer cells in the tumor periphery. Conclusion: Incorporation of doxorubicin-loaded microbubble in the TACE formulation facilitated drug delivery to the tumor with real-time monitoring and enhanced the therapeutic efficacy of TACE. Thus, the enhanced TACE formulation may represent a new treatment strategy against liver cancer.

Intravital two-photon imaging and quantification of hepatic steatosis and fibrosis in a live small animal model.

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.

In vivo longitudinal visualization of the brain neuroinflammatory response at the cellular level in LysM-GFP mice induced by 3-nitropropionic acid.

Blood-brain barrier (BBB) dysfunction is related to the development of neuroinflammation in the central nervous system (CNS). Neuroinflammation has been implicated as one of the key factors in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. Despite its importance, the impacts and underlying cellular mechanisms of chronic BBB impairment in neurodegenerative diseases are poorly understood. In this work, we performed a longitudinal intravital brain imaging of mouse model with neuroinflammation induced by 3-nitropropionic acid (3-NP). For this, we obtained a transgenic LysM-GFP mouse expressing the green fluorescence protein (GFP) in a subset of leukocytes. By using intravenously injected fluorescence blood tracers, we longitudinally observed in vivo dynamic cellular behaviors and the BBB integrity through a 30-day neuroinflammatory state. Vascular leakages in the cerebral cortex reflecting BBB impairment were observed at two weeks, which persisted to the third week, followed by a severe inflammatory response with massive leukocytes infiltration at day 30. These descriptions can help in the development of novel approaches to treat neurodegenerative conditions.

A Novel Pancreatic Imaging Window for Stabilized Longitudinal In Vivo Observation of Pancreatic Islets in Murine Model.

Longitudinal imaging of murine pancreas is technically challenging due to the mechanical softness of the tissue influenced by peristalsis. Here, we report a novel pancreatic imaging window for long-term stabilized cellular-level observation of the islets in the pancreas in vivo. By spatially separating the pancreas from the bowel movement and physiologic respiration with a metal plate integrated in the imaging window, we successfully tracked the pancreatic islets up to three weeks and visualized the dumbbell-shape transformation from the single islet. This window can be a useful tool for long-term cellular-level visualization of the microstructure in the pancreas.

3D cellular visualization of intact mouse tooth using optical clearing without decalcification.

Dental pulp is composed of nerves, blood vessels, and various types of cells and surrounded by a thick and hard enamel-dentin matrix. Due to its importance in the maintenance of tooth vitality, there have been intensive efforts to analyze the complex cellular-level organization of the dental pulp in teeth. Although conventional histologic analysis has provided microscopic images of the dental pulp, 3-dimensional (3D) cellular-level visualization of the whole dental pulp in an intact tooth has remained a technically challenging task. This is mainly due to the inevitable disruption and loss of microscopic structural features during the process of mechanical sectioning required for the preparation of the tooth sample for histological observation. To accomplish 3D microscopic observation of thick intact tissue, various optical clearing techniques have been developed mostly for soft tissue, and their application for hard tissues such as bone and teeth has only recently started to be investigated. In this work, we established a simple and rapid optical clearing technique for intact mouse teeth without the time-consuming process of decalcification. We achieved 3D cellular-level visualization of the microvasculature and various immune cell distributions in the whole dental pulp of mouse teeth under normal and pathologic conditions. This technique could be used to enable diverse research methods on tooth development and regeneration by providing 3D visualization of various pulpal cells in intact mouse teeth.

Effect of ginseng mixture on osteoporosis in ovariectomized rats.

In this study, the authors have characterized the effect of HER-S (red ginseng, Angelicae gigantis Radix, Phyllostachys folium, and soybean extracts) on osteoporosis-associated phenomena in ovariectomized (OVX) rats by measuring body weights and bone histomorphometries in control, sham, OVX, OVX(beta-estradiol-treated), and OVX(HER-S-treated) rats. Light microscopic analyses showed a porous or eroded appearance on the femoral trabecular bone surface in OVX rats, whereas the femoral trabecular bone surfaces of the other groups (control, sham, OVX(17beta-estradiol-treated), and OVX(HER-S-treated) rats) were composed of fine particles. The femoral trabecular bone area and number were decreased in OVX rats, but these reductions were significantly prevented by the administration of HER-S for 7 weeks, similar to estrogen. In the blood biochemistry results, serum phosphorus, calcium, T(3), and T(4) remained unchanged, but blood estrogen levels were significantly increased in HER-S-treated rats, which suggests that estrogen is related to the mechanism of the HER-S-induced antiosteoporosis function in OVX rats.