Whole-body imaging with single-cell resolution by tissue decolorization.

The development of whole-body imaging at single-cell resolution enables system-level approaches to studying cellular circuits in organisms. Previous clearing methods focused on homogenizing mismatched refractive indices of individual tissues, enabling reductions in opacity but falling short of achieving transparency. Here, we show that an aminoalcohol decolorizes blood by efficiently eluting the heme chromophore from hemoglobin. Direct transcardial perfusion of an aminoalcohol-containing cocktail that we previously termed CUBIC coupled with a 10 day to 2 week clearing protocol decolorized and rendered nearly transparent almost all organs of adult mice as well as the entire body of infant and adult mice. This CUBIC-perfusion protocol enables rapid whole-body and whole-organ imaging at single-cell resolution by using light-sheet fluorescent microscopy. The CUBIC protocol is also applicable to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest that whole-body imaging of colorless tissues at high resolution will contribute to organism-level systems biology.

Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.

Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.

Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains.

Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.

A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues.

Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.

Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and Quantity of CRY1.

To conduct comprehensive characterization of molecular properties in organisms, we established an efficient method to produce knockout (KO)-rescue mice within a single generation. We applied this method to produce 20 strains of almost completely embryonic stem cell (ESC)-derived mice ("ES mice") rescued with wild-type and mutant Cry1 gene under a Cry1-/-:Cry2-/- background. A series of both phosphorylation-mimetic and non-phosphorylation-mimetic CRY1 mutants revealed that multisite phosphorylation of CRY1 can serve as a cumulative timer in the mammalian circadian clock. KO-rescue ES mice also revealed that CRY1-PER2 interaction confers a robust circadian rhythmicity in mice. Surprisingly, in contrast to theoretical predictions from canonical transcription/translation feedback loops, the residues surrounding the flexible P loop and C-lid domains of CRY1 determine circadian period without changing the degradation rate of CRY1. These results suggest that CRY1 determines circadian period through both its degradation-dependent and -independent pathways.

Blueprints from plane to space: outlook of next-generation three-dimensional histopathology.

Here, we summarize the literature relevant to recent advances in three-dimensional (3D) histopathology in relation to clinical oncology, highlighting serial sectioning, tissue clearing, light-sheet microscopy, and digital image analysis with artificial intelligence. We look forward to a future where 3D histopathology expands our understanding of human pathophysiology and improves patient care through cross-disciplinary collaboration and innovation.

Color-Changing Fluorescent Barcode Based on Strand Displacement Reaction Enables Simple Multiplexed Labeling.

Fluorescence imaging techniques have contributed to our understanding of various biological phenomenon; however, fluorescence spectral overlap significantly restricts multiplexing capability. Several strategies have been reported to overcome this limitation by utilizing the superior programmability of DNA technologies and nanostructures, but in practice, it remains challenging to achieve broad adoption of these multiplexed detection methods due to the complexities of these DNA designs. Here we report a color-changing fluorescent barcode (CCFB) approach that enables multiple labeling with simple and small nucleic acid structure design based on sequential toehold-mediated strand displacement reaction. The emission color of CCFB can vary in the predetermined sequence so that multiple targets can be detected simultaneously. The CCFB complex is composed of several oligonucleotides, and its color sequence can be easily expanded further. The CCFB approach is easy and time-saving to operate since the irreversible color-changing reaction occurs by simply adding complementary oligonucleotide. We herein developed 27 different CCFB labels, which required only 14 oligonucleotides. We demonstrated that the CCFB system can be used to label multiple targets by attaching CCFB label to polystyrene beads. Moreover, the CCFB can be used to detect intracellular proteins simultaneously when it is attached to antibodies. We expect that this practical platform will be adopted for comprehensive biomolecular imaging in cells.

Activation of Sympathetic Signaling in Macrophages Blocks Systemic Inflammation and Protects against Renal Ischemia-Reperfusion Injury.

BACKGROUND: The sympathetic nervous system regulates immune cell dynamics. However, the detailed role of sympathetic signaling in inflammatory diseases is still unclear because it varies according to the disease situation and responsible cell types. This study focused on identifying the functions of sympathetic signaling in macrophages in LPS-induced sepsis and renal ischemia-reperfusion injury (IRI). METHODS: We performed RNA sequencing of mouse macrophage cell lines to identify the critical gene that mediates the anti-inflammatory effect of ß2-adrenergic receptor (Adrb2) signaling. We also examined the effects of salbutamol (a selective Adrb2 agonist) in LPS-induced systemic inflammation and renal IRI. Macrophage-specific Adrb2 conditional knockout (cKO) mice and the adoptive transfer of salbutamol-treated macrophages were used to assess the involvement of macrophage Adrb2 signaling. RESULTS: In vitro, activation of Adrb2 signaling in macrophages induced the expression of T cell Ig and mucin domain 3 (Tim3), which contributes to anti-inflammatory phenotypic alterations. In vivo, salbutamol administration blocked LPS-induced systemic inflammation and protected against renal IRI; this protection was mitigated in macrophage-specific Adrb2 cKO mice. The adoptive transfer of salbutamol-treated macrophages also protected against renal IRI. Single-cell RNA sequencing revealed that this protection was associated with the accumulation of Tim3-expressing macrophages in the renal tissue. CONCLUSIONS: The activation of Adrb2 signaling in macrophages induces anti-inflammatory phenotypic alterations partially via the induction of Tim3 expression, which blocks LPS-induced systemic inflammation and protects against renal IRI.

Lipopolysaccharides and Cellular Senescence: Involvement in Atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of the vascular walls related to aging. Thus far, the roles of cellular senescence and bacterial infection in the pathogenesis of atherosclerosis have been speculated to be independent of each other. Some types of macrophages, vascular endothelial cells, and vascular smooth muscle cells are in a senescent state at the sites of atherosclerotic lesions. Likewise, bacterial infections and accumulations of lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria, have also been observed in the atherosclerotic lesions of patients. This review introduces the integration of these two potential pathways in atherosclerosis. Previous studies have suggested that LPS directly induces cellular senescence in cultured monocytes/macrophages and vascular cells. In addition, LPS enhances the inflammatory properties (senescence-associated secretory phenotype [SASP]) of senescent endothelial cells. Thus, LPS derived from Gram-negative bacteria could exaggerate the pathogenesis of atherosclerosis by inducing and enhancing cellular senescence and the SASP-associated inflammatory properties of specific vascular cells in atherosclerotic lesions. This proposed mechanism can provide novel approaches to preventing and treating this common age-related disease.

The oral hypoxia-inducible factor prolyl hydroxylase inhibitor enarodustat counteracts alterations in renal energy metabolism in the early stages of diabetic kidney disease.

Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors, also known as HIF stabilizers, increase endogenous erythropoietin production and serve as novel therapeutic agents against anemia in chronic kidney disease. HIF induces the expression of various genes related to energy metabolism as an adaptive response to hypoxia. However, it remains obscure how the metabolic reprogramming in renal tissue by HIF stabilization affects the pathophysiology of kidney diseases. Previous studies suggest that systemic metabolic disorders such as hyperglycemia and dyslipidemia cause alterations of renal metabolism, leading to renal dysfunction including diabetic kidney disease. Here, we analyze the effects of enarodustat (JTZ-951), an oral HIF stabilizer, on renal energy metabolism in the early stages of diabetic kidney disease, using streptozotocin-induced diabetic rats and alloxan-induced diabetic mice. Transcriptome analysis revealed that enarodustat counteracts the alterations in diabetic renal metabolism. Transcriptome analysis showed that fatty acid and amino acid metabolisms were upregulated in diabetic renal tissue and downregulated by enarodustat, whereas glucose metabolism was upregulated. These symmetric changes were confirmed by metabolome analysis. Whereas glycolysis and tricarboxylic acid cycle metabolites were accumulated and amino acids reduced in renal tissue of diabetic animals, these metabolic disturbances were mitigated by enarodustat. Furthermore, enarodustat increased the glutathione to glutathione disulfide ratio and relieved oxidative stress in renal tissue of diabetic animals. Thus, HIF stabilization counteracts alterations in renal energy metabolism occurring in incipient diabetic kidney disease.

Comparison of the 3-D patterns of the parasympathetic nervous system in the lung at late developmental stages between mouse and chicken.

Although the basic schema of the body plan is similar among different species of amniotes (mammals, birds, and reptiles), the lung is an exception. Here, anatomy and physiology are considerably different, particularly between mammals and birds. In mammals, inhaled and exhaled airs mix in the airways, whereas in birds the inspired air flows unidirectionally without mixing with the expired air. This bird-specific respiration system is enabled by the complex tubular structures called parabronchi where gas exchange takes place, and also by the bellow-like air sacs appended to the main part of the lung. That the lung is predominantly governed by the parasympathetic nervous system has been shown mostly by physiological studies in mammals. However, how the parasympathetic nervous system in the lung is established during late development has largely been unexplored both in mammals and birds. In this study, by combining immunocytochemistry, the tissue-clearing CUBIC method, and ink-injection to airways, we have visualized the 3-D distribution patterns of parasympathetic nerves and ganglia in the lung at late developmental stages of mice and chickens. These patterns were further compared between these species, and three prominent similarities emerged: (1) parasympathetic postganglionic fibers and ganglia are widely distributed in the lung covering the proximal and distal portions, (2) the gas exchange units, alveoli in mice and parabronchi in chickens, are devoid of parasympathetic nerves, (3) parasympathetic nerves are in close association with smooth muscle cells, particularly at the base of the gas exchange units. These observations suggest that despite gross differences in anatomy, the basic mechanisms underlying parasympathetic control of smooth muscles and gas exchange might be conserved between mammals and birds.

Comprehensive three-dimensional analysis (CUBIC-kidney) visualizes abnormal renal sympathetic nerves after ischemia/reperfusion injury.

The sympathetic nervous system is critical in maintaining the homeostasis of renal functions. However, its three-dimensional (3D) structures in the kidney have not been elucidated due to limitation of conventional imaging methods. CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) is a newly developed tissue-clearing technique, which enables whole-organ 3D imaging without thin-sectioning. Comprehensive 3D imaging by CUBIC found that sympathetic nerves are primarily distributed around arteries in the mouse kidney. Notably, the sympathetic innervation density was significantly decreased 10 days after ischemia-reperfusion injury (voluminal ratio of innervation area to kidney) by about 70%. Moreover, norepinephrine levels in kidney tissue (output of sympathetic nerves) were significantly reduced in injured kidneys by 77%, confirming sympathetic denervation after ischemia-reperfusion injury. Time-course imaging indicated that innervation partially recovered although overall denervation persisted 28 days after injury, indicating a continuous sympathetic nervous abnormality during the progression of chronic kidney disease. Thus, CUBIC-kidney, the 3D imaging analysis, can be a strong imaging tool, providing comprehensive, macroscopic perspectives for kidney research.

Novel 3D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis.

Recent developments in optical tissue clearing have been difficult to apply for the morphometric analysis of organs with high cellular content and small functional structures, such as the kidney. Here, we establish combinations of genetic and immuno-labelling for single cell identification, tissue clearing and subsequent de-clarification for histoimmunopathology and transmission electron microscopy. Using advanced light microscopy and computational analyses, we investigated a murine model of crescentic nephritis, an inflammatory kidney disease typified by immune-mediated damage to glomeruli leading to the formation of hypercellular lesions and the rapid loss of kidney function induced by nephrotoxic serum. Results show a graded susceptibility of the glomeruli, significant podocyte loss and capillary injury. These effects are associated with activation of parietal epithelial cells and formation of glomerular lesions that may evolve and obstruct the kidney tubule, thereby explaining the loss of kidney function. Thus, our work provides new high-throughput endpoints for the analysis of complex tissues with single-cell resolution.

Establishment of TSH ß real-time monitoring system in mammalian photoperiodism.

Organisms have seasonal physiological changes in response to day length. Long-day stimulation induces thyroid-stimulating hormone beta subunit (TSHß) in the pars tuberalis (PT), which mediates photoperiodic reactions like day-length measurement and physiological adaptation. However, the mechanism of TSHß induction for day-length measurement is largely unknown. To screen candidate upstream molecules of TSHß, which convey light information to the PT, we generated Luciferase knock-in mice, which quantitatively report the dynamics of TSHß expression. We cultured brain slices containing the PT region from adult and neonatal mice and measured the bioluminescence activities from each slice over several days. A decrease in the bioluminescence activities was observed after melatonin treatment in adult and neonatal slices. These observations indicate that the experimental system possesses responsiveness of the TSHß expression to melatonin. Thus, we concluded that our experimental system monitors TSHß expression dynamics in response to external stimuli.

Unlocking the potential of large-scale 3D imaging with tissue clearing techniques.

The three-dimensional (3D) anatomical structure of living organisms is intrinsically linked to their functions, yet modern life sciences have not fully explored this aspect. Recently, the combination of efficient tissue clearing techniques and light-sheet fluorescence microscopy (LSFM) for rapid 3D imaging has improved access to 3D spatial information in biological systems. This technology has found applications in various fields, including neuroscience, cancer research, and clinical histopathology, leading to significant insights. It allows imaging of entire organs or even whole bodies of animals and humans at multiple scales. Moreover, it enables a form of spatial omics by capturing and analyzing cellome information, which represents the complete spatial organization of cells. While current 3D imaging of cleared tissues has limitations in obtaining sufficient molecular information, emerging technologies such as multi-round tissue staining and super-multicolor imaging are expected to address these constraints. 3D imaging using tissue clearing and light-sheet microscopy thus offers a valuable research tool in the current and future life sciences for acquiring and analyzing large-scale biological spatial information.

A novel tauopathy model mimicking molecular and spatial aspects of human tau pathology.

Creating a mouse model that recapitulates human tau pathology is essential for developing strategies to intervene in tau-induced neurodegeneration. However, mimicking the pathological features seen in human pathology often involves a trade-off with artificial effects such as unexpected gene insertion and neurotoxicity from the expression system. To overcome these issues, we developed the rTKhomo mouse model by combining a transgenic CaMKII-tTA system with a P301L mutated 1N4R human tau knock-in at the Rosa26 locus with a C57BL/6J background. This model closely mimics human tau pathology, particularly in the hippocampal CA1 region, showing age-dependent tau accumulation, neuronal loss and neuroinflammation. Notably, whole-brain 3D staining and light-sheet microscopy revealed a spatial gradient of tau deposition from the entorhinal cortex to the hippocampus, similar to the spatial distribution of Braak neurofibrillary tangle staging. Furthermore, [18F]PM-PBB3 positron emission tomography imaging enabled the quantification and live monitoring of tau deposition. The rTKhomo mouse model shows potential as a promising next-generation preclinical tool for exploring the mechanisms of tauopathy and for developing interventions targeting the spatial progression of tau pathology.

Cortical parvalbumin neurons are responsible for homeostatic sleep rebound through CaMKII activation.

The homeostatic regulation of sleep is characterized by rebound sleep after prolonged wakefulness, but the molecular and cellular mechanisms underlying this regulation are still unknown. In this study, we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent activity control of parvalbumin (PV)-expressing cortical neurons is involved in homeostatic regulation of sleep in male mice. Prolonged wakefulness enhances cortical PV-neuron activity. Chemogenetic suppression or activation of cortical PV neurons inhibits or induces rebound sleep, implying that rebound sleep is dependent on increased activity of cortical PV neurons. Furthermore, we discovered that CaMKII kinase activity boosts the activity of cortical PV neurons, and that kinase activity is important for homeostatic sleep rebound. Here, we propose that CaMKII-dependent PV-neuron activity represents negative feedback inhibition of cortical neural excitability, which serves as the distributive cortical circuits for sleep homeostatic regulation.

descSPIM: an affordable and easy-to-build light-sheet microscope optimized for tissue clearing techniques.

Despite widespread adoption of tissue clearing techniques in recent years, poor access to suitable light-sheet fluorescence microscopes remains a major obstacle for biomedical end-users. Here, we present descSPIM (desktop-equipped SPIM for cleared specimens), a low-cost ($20,000-50,000), low-expertise (one-day installation by a non-expert), yet practical do-it-yourself light-sheet microscope as a solution for this bottleneck. Even the most fundamental configuration of descSPIM enables multi-color imaging of whole mouse brains and a cancer cell line-derived xenograft tumor mass for the visualization of neurocircuitry, assessment of drug distribution, and pathological examination by false-colored hematoxylin and eosin staining in a three-dimensional manner. Academically open-sourced ( https://github.com/dbsb-juntendo/descSPIM ), descSPIM allows routine three-dimensional imaging of cleared samples in minutes. Thus, the dissemination of descSPIM will accelerate biomedical discoveries driven by tissue clearing technologies.

Signal Improved ultra-Fast Light-sheet Microscope (SIFT) for large tissue imaging.

Light-sheet fluorescence microscopy (LSFM) in conjunction with tissue clearing techniques enables morphological investigation of large tissues faster and with excellent optical sectioning. Recently, cleared tissue axially swept light-sheet microscope (ctASLM) demonstrated three-dimensional isotropic resolution in millimeter-scaled tissues. But ASLM based microscopes suffer from low detection signal and slow imaging speed. Here we report a simple and efficient imaging platform that employs precise control of two fixed distant light-sheet foci to carry out ASLM. This allowed us to carry out full field of view (FOV) imaging at 40 frames per second (fps) which is a four-fold improvement compared to the current state-of-the-art. In addition, in a particular frame rate, our method doubles the signal compared to the current ASLM technique. To augment the overall imaging performance, we also developed a deep learning based tissue information classifier that enables faster determination of tissue boundary. We demonstrated the performance of our imaging platform on various cleared tissue samples and demonstrated its robustness over a wide range of clearing protocols.