Cerebral Organoid Arrays for Batch Phenotypic Analysis in Sections and Three Dimensions.

Organoids can recapitulate human-specific phenotypes and functions in vivo and have great potential for research in development, disease modeling, and drug screening. Due to the inherent variability among organoids, experiments often require a large sample size. Embedding, staining, and imaging each organoid individually require a lot of reagents and time. Hence, there is an urgent need for fast and efficient methods for analyzing the phenotypic changes in organoids in batches. Here, we provide a comprehensive strategy for array embedding, staining, and imaging of cerebral organoids in both agarose sections and in 3D to analyze the spatial distribution of biomarkers in organoids in situ. We constructed several disease models, particularly an aging model, as examples to demonstrate our strategy for the investigation of the phenotypic analysis of organoids. We fabricated an array mold to produce agarose support with microwells, which hold organoids in place for live/dead imaging. We performed staining and imaging of sectioned organoids embedded in agarose and 3D imaging to examine phenotypic changes in organoids using fluorescence micro-optical sectioning tomography (fMOST) and whole-mount immunostaining. Parallel studies of organoids in arrays using the same staining and imaging parameters enabled easy and reliable comparison among different groups. We were able to track all the data points obtained from every organoid in an embedded array. This strategy could help us study the phenotypic changes in organoids in disease models and drug screening.

Three-dimensional mapping in multi-samples with large-scale imaging and multiplexed post staining.

Dissection of the anatomical information at the single-cell level is crucial for understanding the organization rule and pathological mechanism of biological tissues. Mapping the whole organ in numerous groups with multiple conditions brings the challenges in imaging and analysis. Here, we describe an approach, named array fluorescent micro-optical sectioning tomography (array-fMOST), to identify the three-dimensional information at single-cell resolution from multi-samples. The pipeline contains array embedding, large-scale imaging, post-imaging staining and data analysis, which could image over 24 mouse brains simultaneously and collect the slices for further analysis. With transgenic mice, we acquired the distribution information of neuropeptide somatostatin neurons during natural aging and compared the changes in the microenvironments by multi-component labeling of serial sections with precise co-registration of serial datasets quantitatively. With viral labeling, we also analyzed the input circuits of the medial prefrontal cortex in the whole brain of Alzheimer's disease and autism model mice. This pipeline is highly scalable to be applied to anatomical alterations screening and identification. It provides new opportunities for combining multi-sample whole-organ imaging and molecular phenotypes identification analysis together. Such integrated high-dimensional information acquisition method may accelerate our understanding of pathogenesis and progression of disease in situ at multiple levels.

Multiscale Analysis of Cellular Composition and Morphology in Intact Cerebral Organoids.

Cerebral organoids recapitulate in vivo phenotypes and physiological functions of the brain and have great potential in studying brain development, modeling diseases, and conducting neural network research. It is essential to obtain whole-mount three-dimensional (3D) images of cerebral organoids at cellular levels to explore their characteristics and applications. Existing histological strategies sacrifice inherent spatial characteristics of organoids, and the strategy for volume imaging and 3D analysis of entire organoids is urgently needed. Here, we proposed a high-resolution imaging pipeline based on fluorescent labeling by viral transduction and 3D immunostaining with fluorescence micro-optical sectioning tomography (fMOST). We were able to image intact organoids using our pipeline, revealing cytoarchitecture information of organoids and the spatial localization of neurons and glial fibrillary acidic protein positive cells (GFAP+ cells). We performed single-cell reconstruction to analyze the morphology of neurons and GFAP+ cells. Localization and quantitative analysis of cortical layer markers revealed heterogeneity of organoids. This pipeline enabled acquisition of high-resolution spatial information of millimeter-scale organoids for analyzing their cell composition and morphology.

Extension of Endocardium-Derived Vessels Generate Coronary Arteries in Neonates.

BACKGROUND: Unraveling how new coronary arteries develop may provide critical information for establishing novel therapeutic approaches to treating ischemic cardiac diseases. There are 2 distinct coronary vascular populations derived from different origins in the developing heart. Understanding the formation of coronary arteries may provide insights into new ways of promoting coronary artery formation after myocardial infarction. METHODS: To understand how intramyocardial coronary arteries are generated to connect these 2 coronary vascular populations, we combined genetic lineage tracing, light sheet microscopy, fluorescence micro-optical sectioning tomography, and tissue-specific gene knockout approaches to understand their cellular and molecular mechanisms. RESULTS: We show that a subset of intramyocardial coronary arteries form by angiogenic extension of endocardium-derived vascular tunnels in the neonatal heart. Three-dimensional whole-mount fluorescence imaging showed that these endocardium-derived vascular tunnels or tubes adopt an arterial fate in neonates. Mechanistically, we implicate Mettl3 (methyltransferase-like protein 3) and Notch signaling in regulating endocardium-derived intramyocardial coronary artery formation. Functionally, these intramyocardial arteries persist into adulthood and play a protective role after myocardial infarction. CONCLUSIONS: A subset of intramyocardial coronary arteries form by extension of endocardium-derived vascular tunnels in the neonatal heart.

A spatial and cellular distribution of rabies virus infection in the mouse brain revealed by fMOST and single-cell RNA sequencing.

BACKGROUND: Neurotropic virus infection can cause serious damage to the central nervous system (CNS) in both humans and animals. The complexity of the CNS poses unique challenges to investigate the infection of these viruses in the brain using traditional techniques. METHODS: In this study, we explore the use of fluorescence micro-optical sectioning tomography (fMOST) and single-cell RNA sequencing (scRNA-seq) to map the spatial and cellular distribution of a representative neurotropic virus, rabies virus (RABV), in the whole brain. Mice were inoculated with a lethal dose of a recombinant RABV encoding enhanced green fluorescent protein (EGFP) under different infection routes, and a three-dimensional (3D) view of RABV distribution in the whole mouse brain was obtained using fMOST. Meanwhile, we pinpointed the cellular distribution of RABV by utilizing scRNA-seq. RESULTS: Our fMOST data provided the 3D view of a neurotropic virus in the whole mouse brain, which indicated that the spatial distribution of RABV in the brain was influenced by the infection route. Interestingly, we provided evidence that RABV could infect multiple nuclei related to fear independent of different infection routes. More surprisingly, our scRNA-seq data revealed that besides neurons RABV could infect macrophages and the infiltrating macrophages played at least three different antiviral roles during RABV infection. CONCLUSION: This study draws a comprehensively spatial and cellular map of typical neurotropic virus infection in the mouse brain, providing a novel and insightful strategy to investigate the pathogenesis of RABV and other neurotropic viruses.

Diltiazem-Induced Reversible Cardiogenic Shock in Thyroid Storm.

Early recognition of underlying thyroid disease in patients presenting with new-onset tachyarrhythmia is central to management, as usual rate-control strategies can result in significant mortality and morbidity. Hyperthyroidism-induced cardiomyopathy complicated by cardiogenic shock is a life-threatening condition. Thyroid storm can lead to irreversible cardiovascular collapse and death if proper treatment is not initiated as soon as possible. In this case, we report a 44-year-old female who presented to the emergency department (ED) with the chief complaints of anxiety and palpitations. Her past medical history was significant for anxiety, but she was otherwise healthy. An electrocardiogram (ECG) in the ED demonstrated atrial fibrillation (A. fib) with rapid ventricular response (RVR) felt to be secondary to thyroid storm based on the Burch-Wartofsky point scale (BWPS), which is a quantitative diagnostic tool that uses clinical manifestation in diagnosing thyroid storm. The patient was initially managed with a continuous infusion of diltiazem to achieve rate control with a goal heart rate <115 beats per minute (bpm). Shortly after initiating the infusion, the patient developed signs and symptoms consistent with cardiogenic shock. Bedside echocardiogram revealed an estimated ejection fraction (EF) <10% with concomitant pulmonary edema. Repeat echocardiogram within 72 hours after stopping diltiazem and starting appropriate treatment for thyroid storm showed improvement of EF to 35%.

High-Throughput Strategy for Profiling Sequential Section With Multiplex Staining of Mouse Brain.

The brain modulates specific functions in its various regions. Understanding the organization of different cells in the whole brain is crucial for investigating brain functions. Previous studies have focused on several regions and have had difficulty analyzing serial tissue samples. In this study, we introduced a pipeline to acquire anatomical and histological information quickly and efficiently from serial sections. First, we developed a serial brain-slice-staining method to stain serial sections and obtained more than 98.5% of slices with high integrity. Subsequently, using the self-developed analysis software, we registered and quantified the signals of imaged sections to the Allen Mouse Brain Common Coordinate Framework, which is compatible with multimodal images and slant section planes. Finally, we validated the pipeline with immunostaining by analyzing the activity variance in the whole brain during acute stress in aging and young mice. By removing the problems resulting from repeated manual operations, this pipeline is widely applicable to serial brain slices from multiple samples in a rapid and convenient manner, which benefits to facilitate research in life sciences.

Whole-Brain Three-Dimensional Profiling Reveals Brain Region Specific Axon Vulnerability in 5xFAD Mouse Model.

Axonopathy is a pathological feature observed in both Alzheimer's disease (AD) patients and animal models. However, identifying the temporal and regional progression of axonopathy during AD development remains elusive. Using the fluorescence micro-optical sectioning tomography system, we acquired whole-brain datasets in the early stage of 5xFAD/Thy1-GFP-M mice. We reported that among GFP labeled axons, GFP-positive axonopathy first formed in the lateral septal nucleus, subiculum, and medial mammillary nucleus. The axonopathy further increased in most brain regions during aging. However, most of the axonopathic varicosities disappeared significantly in the medial mammillary nucleus after 8 weeks old. Continuous three-dimensional datasets showed that axonopathy in the medial mammillary nucleus was mainly located on axons from hippocampal GFP-positive neurons. Using the rabies viral tracer in combination with immunohistochemistry, we found that axons in the medial mammillary nucleus from the subiculum were susceptible to lesions that prior to the occurrence of behavioral disorders. In conclusion, we created an early-stage spatiotemporal map of axonopathy in 5xFAD/Thy1-GFP-M mice and identified specific neural circuits which are vulnerable to axon lesions in an AD mouse model. These findings underline the importance of early interventions for AD, and may contribute to the understanding of its progression and its early symptom treatment.

Mapping the Architecture of Ferret Brains at Single-Cell Resolution.

Mapping the cytoarchitecture of the whole brain can reveal the organizational logic of neural systems. However, this remains a significant challenge, especially for gyrencephalic brains with a large volume. Here we propose an integrated pipeline for generating a cytoarchitectonic atlas with single-cell resolution of the whole brain. To analyze a large-volume brain, we used a modified en-bloc Nissl staining protocol to achieve uniform staining of large-scale brain specimens from ferret (Mustela putorius furo). By combining whole-brain imaging and big data processing, we established strategies for parsing cytoarchitectural information at a voxel resolution of 0.33 µm × 0.33 µm × 1 µm and terabyte-scale data analysis. Using the cytoarchitectonic datasets for adult ferret brain, we identified giant pyramidal neurons in ferret brains and provide the first report of their morphological diversity, neurochemical phenotype, and distribution patterns in the whole brain in three dimensions. This pipeline will facilitate studies on the organization and development of the mammalian brains, from that of rodents to the gyrencephalic brains of ferret and even primates.

On-line optical clearing method for whole-brain imaging in mice.

The combination of optical clearing with light microscopy has a number of applications in the whole-brain imaging of mice. However, the initial processing time of optical clearing is time consuming, and the protocol is complicated. We propose a novel method based on on-line optical clearing. Agarose-embedded mouse brain was immersed in the optical clearing reagent, and clearing of the brain was achieved ~100 µm beneath the sample surface. After imaging, the cleared layer was removed, thereby allowing layer-by-layer clearing and imaging. No pre-immersion was required, and we demonstrated that on-line optical clearing can reduce the whole-brain imaging time by half.

A platform for efficient identification of molecular phenotypes of brain-wide neural circuits.

A neural circuit is a structural-functional unit of achieving particular information transmission and processing, and have various inputs, outputs and molecular phenotypes. Systematic acquisition and comparative analysis of the molecular features of neural circuits are crucial to elucidating the operating mechanisms of brain function. However, no efficient, systematic approach is available for describing the molecular phenotypes of specific neural circuits at the whole brain scale. In this study, we developed a rapid whole-brain optical tomography method and devised an efficient approach to map brain-wide structural and molecular information in the same brain: rapidly imaging and sectioning the whole brain as well as automatically collecting all slices; conveniently selecting slices of interest through quick data browsing and then performing post hoc immunostaining of selected slices. Using this platform, we mapped the brain-wide distribution of inputs to motor, sensory and visual cortices and determined their molecular phenotypes in several subcortical regions. Our platform significantly enhances the efficiency of molecular phenotyping of neural circuits and provides access to automation and industrialization of cell type analyses for specific circuits.

A Quantitative Analysis of the Distribution of CRH Neurons in Whole Mouse Brain.

Corticotropin-releasing hormone (CRH), with widespread expression in the brain, plays a key role in modulating a series of behaviors, including anxiety, arousal, motor function, learning and memory. Previous studies have focused on some brain regions with densely distributed CRH neurons such as paraventricular hypothalamic nucleus (PVH) and bed nuclei of the stria terminalis (BST) and revealed some basic structural and functional knowledge of CRH neurons. However, there is no systematic analysis of brain-wide distribution of CRH neurons. Here, we performed a comprehensive study of CRH neurons in CRH-IRES-Cre;Ai3 mice via automatic imaging and stereoscopic cell counting in a whole mouse brain. We acquired four datasets of the CRH distributions with co-localized cytoarchitecture at a voxel resolution of 0.32 µm × 0.32 µm × 2 µm using brain-wide positioning system (BPS). Next, we precisely located and counted the EYFP-labeled neurons in different regions according to propidium iodide counterstained anatomical reference using Neuronal Global Position System. In particular, dense EYFP expression was found in piriform area, BST, central amygdalar nucleus, PVH, Barrington's nucleus, and inferior olivary complex. Considerable CRH neurons were also found in main olfactory bulb, medial preoptic nucleus, pontine gray, tegmental reticular nucleus, external cuneate nucleus, and midline thalamus. We reconstructed and compared the soma morphology of CRH neurons in 11 brain regions. The results demonstrated that CRH neurons had regional diversities of both cell distribution and soma morphology. This anatomical knowledge enhances the current understanding of the functions of CRH neurons. These results also demonstrated the ability of our platform to accurately orient, reconstruct and count neuronal somas in three-dimension for type-specific neurons in the whole brain, making it feasible to answer the fundamental neuroscience question of exact numbers of various neurons in the whole brain.

Association between persistent tachycardia and tachypnea and in-hospital mortality among non-hypotensive emergency department patients admitted to the hospital.

OBJECTIVE: Vital sign trends are used in clinical practice to assess treatment response and aid in disposition, yet quantitative data to support this practice are lacking. This study aimed to determine the prognostic value of vital sign normalization. METHODS: Secondary analysis of a prospective cohort of adult emergency department (ED) patients admitted a single urban tertiary care hospital. A random sample of 182 days was chosen, and a manual review of all admissions was undertaken. Persistent tachycardia or tachypnea was defined as failure to decrease to a normal value in the ED. Elevated upon admission was defined as an abnormal value at the last set of vital signs documented. The primary outcome was in-hospital mortality. RESULTS: 4,878 patients were enrolled and 4.5 (±3.8) sets of vital signs were checked per patient. 1,770 patients were tachycardic and 1,499 were tachypneic. Among tachycardic patients, 941 (53%) were persistently tachycardic and 1,074 (61%) were tachycardic upon admission. Among tachypneic patients 639 (42%) were persistently tachypneic and 768 (51%) were tachypneic upon admission. Mortality was higher in patients persistently tachycardic (5.7% vs. 3.1%, P=0.008) or tachycardic upon admission (5.5% vs. 3.0%, P=0.014). Similar results were found in tachypneic patients (8.3% vs. 4.5%, P=0.003; 7.8% vs. 4.4%, P=0.006). CONCLUSION: Persistent tachycardia and tachypnea are associated with an increased risk of mortality in ED patients admitted to the hospital. Further study is necessary to determine if improved recognition or earlier interventions can affect outcomes.

Early-stage reduction of the dendritic complexity in basolateral amygdala of a transgenic mouse model of Alzheimer's disease.

Alzheimer's disease is a representative age-related neurodegenerative disease that could result in loss of memory and cognitive deficiency. However, the precise onset time of Alzheimer's disease affecting neuronal circuits and the mechanisms underlying the changes are not clearly known. To address the neuroanatomical changes during the early pathologic developing process, we acquired the neuronal morphological characterization of AD in APP/PS1 double-transgenic mice using the Micro-Optical Sectioning Tomography system. We reconstructed the neurons in 3D datasets with a resolution of 0.32 × 0.32 × 1 µm and used the Sholl method to analyze the anatomical characterization of the dendritic branches. The results showed that, similar to the progressive change in amyloid plaques, the number of dendritic branches were significantly decreased in 9-month-old mice. In addition, a distinct reduction of dendritic complexity occurred in third and fourth-order dendritic branches of 9-month-old mice, while no significant changes were identified in these parameters in 6-month-old mice. At the branch-level, the density distribution of dendritic arbors in the radial direction decreased in the range of 40-90 µm from the neuron soma in 6-month-old mice. These changes in the dendritic complexity suggest that these reductions contribute to the progressive cognitive impairment seen in APP/PS1 mice. This work may yield insights into the early changes in dendritic abnormality and its relevance to dysfunctional mechanisms of learning, memory and emotion in Alzheimer's disease.

Precise Cerebral Vascular Atlas in Stereotaxic Coordinates of Whole Mouse Brain.

Understanding amazingly complex brain functions and pathologies requires a complete cerebral vascular atlas in stereotaxic coordinates. Making a precise atlas for cerebral arteries and veins has been a century-old objective in neuroscience and neuropathology. Using micro-optical sectioning tomography (MOST) with a modified Nissl staining method, we acquired five mouse brain data sets containing arteries, veins, and microvessels. Based on the brain-wide vascular spatial structures and brain regions indicated by cytoarchitecture in one and the same mouse brain, we reconstructed and annotated the vascular system atlas of both arteries and veins of the whole mouse brain for the first time. The distributing patterns of the vascular system within the brain regions were acquired and our results show that the patterns of individual vessels are different from each other. Reconstruction and statistical analysis of the microvascular network, including derivation of quantitative vascular densities, indicate significant differences mainly in vessels with diameters less than 8 µm and large than 20 µm across different brain regions. Our precise cerebral vascular atlas provides an important resource and approach for quantitative studies of brain functions and diseases.

Rapid decomposition and visualisation of protein-ligand binding free energies by residue and by water.

Recent advances in computational hardware, software and algorithms enable simulations of protein-ligand complexes to achieve timescales during which complete ligand binding and unbinding pathways can be observed. While observation of such events can promote understanding of binding and unbinding pathways, it does not alone provide information about the molecular drivers for protein-ligand association, nor guidance on how a ligand could be optimised to better bind to the protein. We have developed the waterswap (C. J. Woods et al., J. Chem. Phys., 2011, 134, 054114) absolute binding free energy method that calculates binding affinities by exchanging the ligand with an equivalent volume of water. A significant advantage of this method is that the binding free energy is calculated using a single reaction coordinate from a single simulation. This has enabled the development of new visualisations of binding affinities based on free energy decompositions to per-residue and per-water molecule components. These provide a clear picture of which protein-ligand interactions are strong, and which active site water molecules are stabilised or destabilised upon binding. Optimisation of the algorithms underlying the decomposition enables near-real-time visualisation, allowing these calculations to be used either to provide interactive feedback to a ligand designer, or to provide run-time analysis of protein-ligand molecular dynamics simulations.

Computational assay of H7N9 influenza neuraminidase reveals R292K mutation reduces drug binding affinity.

The emergence of a novel H7N9 avian influenza that infects humans is a serious cause for concern. Of the genome sequences of H7N9 neuraminidase available, one contains a substitution of arginine to lysine at position 292, suggesting a potential for reduced drug binding efficacy. We have performed molecular dynamics simulations of oseltamivir, zanamivir and peramivir bound to H7N9, H7N9-R292K, and a structurally related H11N9 neuraminidase. They show that H7N9 neuraminidase is structurally homologous to H11N9, binding the drugs in identical modes. The simulations reveal that the R292K mutation disrupts drug binding in H7N9 in a comparable manner to that observed experimentally for H11N9-R292K. Absolute binding free energy calculations with the WaterSwap method confirm a reduction in binding affinity. This indicates that the efficacy of antiviral drugs against H7N9-R292K will be reduced. Simulations can assist in predicting disruption of binding caused by mutations in neuraminidase, thereby providing a computational 'assay.'

Updated treatments for neonatal phimosis and redundant prepuce / 中华男科学杂志

Phimosis and redundant prepuce, as common problems in andrology, are found in the majority of male neonates. Early treatment of neonatal phimosis and redundant prepuce has the advantages of better cost--effectiveness and higher safety, and contributes much to the prevention of childhood urinary tract infection and adult sexually transmitted diseases, as well as to the improvement of genital hygiene. Neonatal circumcision is commonly performed in some countries, but remains at a low rate in China. This article updates surgical and non-surgical treatments of neonatal phimosis and redundant prepuce, introduces the benefits of neonatal circumcision, and appeals for more attention to the management of neonatal phimosis and redundant prepuce.

The need for high-quality training and surgical standards for adult male circumcision in China / 中华男科学杂志

Although HIV is a significant problem in Africa, HIV infection rates are rising rapidly in other regions such as Asia and South America. International health organizations have recognized the need to develop effective strategies to check the worldwide transmission of HIV. Studies have demonstrated the significant reduction of HIV, HPV, HSV-2 and other STD infection rates with male circumcision (MC). Although numerous MC techniques are available, there are no standardized protocols and surgical training programs. Studies have shown that the standardization of MC techniques coupled with training programs can significantly reduce complication rates. High complication rates have been a primary obstacle to the implementation of MC services. We recommend the establishment of surgical standards and training protocols prior to the promotion of MC services in China.

Male circumcision is an effective "surgical vaccine" for HIV prevention and reproductive health / 中华男科学杂志

Recent randomized controlled clinical trials in Africa have demonstrated that adult male circumcision (MC) efficiently decreases the rate of HIV, HPV and HSV-2 infections. Many studies have clearly shown that MC is a simple, safe, and cost-effective method for the prevention of sexually transmitted diseases and urinary tract infection, and for improving genital hygiene. While a 30% MC prevalence exists worldwide, only 5% or less of the Chinese males have undergone circumcision. In this review, we report recent trends in international MC and HIV prevention efforts, as well as the potential benefits and importance of promoting MC in China. We appeal to medical and public health authorities to pay close attention to the international experience in MC and HIV prevention.