Deciphering perivascular macrophages and microglia in the retinal ganglion cell layers.

Introduction: The classically defined two retinal microglia layers are distributed in inner and outer plexiform layers. Although there are some reports that retinal microglia are also superficially located around the ganglion cell layer (GCL) in contact with the vitreous, there has been a lack of detailed descriptions and not fully understood yet. Methods: We visualized the microglial layers by using CX3CR1-GFP (C57BL6) transgenic mice with both healthy and disease conditions including NaIO3-induced retinal degeneration models and IRBP-induced auto-immune uveitis models. Result: We found the GCL microglia has two subsets; peripheral (pph) microglia located on the retinal parenchyma and BAM (CNS Border Associated Macrophage) which have a special stretched phenotype only located on the surface of large retinal veins. First, in the pph microglia subset, but not in BAM, Galectin-3 and LYVE1 are focally expressed. However, LYVE1 is specifically expressed in the amoeboid or transition forms, except the typical dendritic morphology in the pph microglia. Second, BAM is tightly attached to the surface of the retinal veins and has similar morphology patterns in both the healthy and disease conditions. CD86+ BAM has a longer process which vertically passes the proximal retinal veins. Our data helps decipher the basic anatomy and pathophysiology of the retinal microglia in the GCL. Discussion: Our data helps decipher the basic anatomy and pathophysiology of the retinal microglia in the GCL.

Dynamic and stable hippocampal representations of social identity and reward expectation support associative social memory in male mice.

Recognizing an individual and retrieving and updating the value information assigned to the individual are fundamental abilities for establishing social relationships. To understand the neural mechanisms underlying the association between social identity and reward value, we developed Go-NoGo social discrimination paradigms that required male subject mice to distinguish between familiar mice based on their individually unique characteristics and associate them with reward availability. We found that mice could discriminate individual conspecifics through a brief nose-to-nose investigation, and this ability depended on the dorsal hippocampus. Two-photon calcium imaging revealed that dorsal CA1 hippocampal neurons represented reward expectation during social, but not non-social tasks, and these activities were maintained over days regardless of the identity of the associated mouse. Furthermore, a dynamically changing subset of hippocampal CA1 neurons discriminated between individual mice with high accuracy. Our findings suggest that the neuronal activities in CA1 provide possible neural substrates for associative social memory.

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.

An agonistic anti-Tie2 antibody suppresses the normal-to-tumor vascular transition in the glioblastoma invasion zone.

Tumor progression is intimately associated with the vasculature, as tumor proliferation induces angiogenesis and tumor cells metastasize to distant organs via blood vessels. However, whether tumor invasion is associated with blood vessels remains unknown. As glioblastoma (GBM) is featured by aggressive invasion and vascular abnormalities, we characterized the onset of vascular remodeling in the diffuse tumor infiltrating zone by establishing new spontaneous GBM models with robust invasion capacity. Normal brain vessels underwent a gradual transition to severely impaired tumor vessels at the GBM periphery over several days. Increasing vasodilation from the tumor periphery to the tumor core was also found in human GBM. The levels of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) showed a spatial correlation with the extent of vascular abnormalities spanning the tumor-invading zone. Blockade of VEGFR2 suppressed vascular remodeling at the tumor periphery, confirming the role of VEGF-VEGFR2 signaling in the invasion-associated vascular transition. As angiopoietin-2 (ANGPT2) was expressed in only a portion of the central tumor vessels, we developed a ligand-independent tunica interna endothelial cell kinase 2 (Tie2)-activating antibody that can result in Tie2 phosphorylation in vivo. This agonistic anti-Tie2 antibody effectively normalized the vasculature in both the tumor periphery and tumor center, similar to the effects of VEGFR2 blockade. Mechanistically, this antibody-based Tie2 activation induced VE-PTP-mediated VEGFR2 dephosphorylation in vivo. Thus, our study reveals that the normal-to-tumor vascular transition is spatiotemporally associated with GBM invasion and may be controlled by Tie2 activation via a novel mechanism of action.

Establishment of the reproducible branch retinal artery occlusion mouse model and intravital longitudinal imaging of the retinal CX3CR1-GFP+ cells after spontaneous arterial recanalization.

Animal models of retinal artery occlusion (RAO) have been widely used in many studies. However, most of these studies prefer using a central retinal artery occlusion (CRAO) which is a typical global ischemia model of the retina, due to the technical limitation of producing single vessel targeted modeling with real-time imaging. A focal ischemia model, such as branch retinal artery occlusion (BRAO), is also needed for explaining interactions, including the immunological reaction between the ischemic retina and adjacent healthy retina. Accordingly, a relevant model for clinical RAO patients has been demanded to understand the pathophysiology of the RAO disease. Herein, we establish a convenient BRAO mouse model to research the focal reaction of the retina. As a photo-thrombotic agent, Rose bengal was intravenously injected into 7 week-old transgenic mice (CX3CR1-GFP) for making embolism occlusion, which causes pathology similarly to clinical cases. In an optimized condition, a 561 nm laser (13.1 mw) was projected to a targeted vessel to induce photo-thrombosis for 27 s by custom-built retinal confocal microscopy. Compared to previous BRAO models, the procedures of thrombosis generation were naturally and minimal invasively generated with real-time retinal imaging. In addition, by utilizing the self-remission characteristics of Rose bengal thrombus, a reflow of the BRAO with immunological reactions of the CX3CR1-GFP+ inflammatory cells such as the retinal microglia and monocytes was monitored and analyzed. In this models, reperfusion began on day 3 after modeling. Simultaneously, the activation of CX3CR1-GFP+ inflammatory cells, including the increase of activation marker and morphologic change, was confirmed by immunohistochemical (IHC) staining and quantitative real-time PCR. CD86 and Nox2 were prominently expressed on day 3 after the modeling. At day 7, blood flow was almost restored in the large vessels. CX3CR1-GFP+ populations in both superficial and deep layers of the retina also increased around even in the BRAO peri-ischemic area. In summary, this study successfully establishes a reproducible BRAO modeling method with convenient capabilities of easily controllable time points and selection of a specific single vessel. It can be a useful tool to analyze the behavior of inflammatory cell after spontaneous arterial recanalization in BRAO and further investigate the pathophysiology of BRAO.

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.

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.

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.

Intravital longitudinal imaging of hepatic lipid droplet accumulation in a murine model for nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a rapidly increasing chronic liver disorder worldwide accompanied by hepatic steatosis, inflammation, fibrosis, and severe liver failure. Unfortunately, an effective treatment strategy for NAFLD has not yet been established, which has been hampered by the limited understanding of the pathophysiological drivers for NAFLD. To examine the unknown cellular and molecular mechanisms in the pathogenesis of NAFLD, there is an increasing need for the direct in vivo observation of hepatic microenvironments over extended periods of time. In this work, using a custom-built intravital imaging system and a novel fluorescent lipid droplet labeling dye, Seoul-Fluor 44 (SF44), we established an intravital imaging method to visualize individual lipid droplets and microvasculature simultaneously in the liver of live mice in vivo. In addition, in the nonalcoholic steatosis and steatohepatitis mouse model induced by a methionine and choline-deficient diet, we longitudinally visualized and quantitatively analyzed the development of lipid droplets in hepatocytes and sinusoid at a subcellular resolution during the progression of NAFLD up to 21 days in vivo.

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.

In vivo longitudinal depth-wise visualization of tumorigenesis by needle-shaped side-view confocal endomicroscopy.

In vivo, longitudinal observation of tumorigenesis in a live mouse model over an extended time period has been actively pursued to obtain a better understanding of the cellular and molecular mechanism in a highly complex tumor microenvironment. However, common intravital imaging approaches based on a conventional laser scanning confocal or a two-photon microscope have been mostly limited to the observation of superficial parts of the solid tumor tissue. In this work, we implemented a small diameter needle-shaped side-view confocal endomicroscope that can be directly inserted into a solid tumor in a minimally-invasive manner in vivo. By inserting the side-view endomicroscope into the breast tumor from the surface, we achieved in vivo depth-wise cellular-level visualization of microvasculature and fluorescently labeled tumor cells located deeply inside the tumor. In addition, we successfully performed longitudinal depth-wise visualization of a growing breast tumor over three weeks in a live mouse model, which revealed dynamic changes in microvasculature such as a decreasing amount of intratumoral blood vessels over time.

Lissajous Scanning Two-photon Endomicroscope for In vivo Tissue Imaging.

An endomicroscope opens new frontiers of non-invasive biopsy for in vivo imaging applications. Here we report two-photon laser scanning endomicroscope for in vivo cellular and tissue imaging using a Lissajous fiber scanner. The fiber scanner consists of a piezoelectric (PZT) tube, a single double-clad fiber (DCF) with high fluorescence collection, and a micro-tethered-silicon-oscillator (MTSO) for the separation of biaxial resonant scanning frequencies. The endomicroscopic imaging exhibits 5 frames/s with 99% in scanning density by using the selection rule of scanning frequencies. The endomicroscopic scanner was compactly packaged within a stainless tube of 2.6 mm in diameter with a high NA gradient-index (GRIN) lens, which can be easily inserted into the working channel of a conventional laparoscope. The lateral and axial resolutions of the endomicroscope are 0.70 µm and 7.6 µm, respectively. Two-photon fluorescence images of a stained kidney section and miscellaneous ex vivo and in vivo organs from wild type and green fluorescent protein transgenic (GFP-TG) mice were successfully obtained by using the endomicroscope. The endomicroscope also obtained label free images including autofluorescence and second-harmonic generation of an ear tissue of Thy1-GCaMP6 (GP5.17) mouse. The Lissajous scanning two-photon endomicroscope can provide a compact handheld platform for in vivo tissue imaging or optical biopsy applications.