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Whole-body imaging of mice is a key source of information for research. Organ segmentation is a prerequisite for quantitative analysis but is a tedious and error-prone task if done manually. Here, we present a deep learning solution called AIMOS that automatically segments major organs (brain, lungs, heart, liver, kidneys, spleen, bladder, stomach, intestine) and the skeleton in less than a second, orders of magnitude faster than prior algorithms. AIMOS matches or exceeds the segmentation quality of state-of-the-art approaches and of human experts. We exemplify direct applicability for biomedical research for localizing cancer metastases. Furthermore, we show that expert annotations are subject to human error and bias. As a consequence, we show that at least two independently created annotations are needed to assess model performance. Importantly, AIMOS addresses the issue of human bias by identifying the regions where humans are most likely to disagree, and thereby localizes and quantifies this uncertainty for improved downstream analysis. In summary, AIMOS is a powerful open-source tool to increase scalability, reduce bias, and foster reproducibility in many areas of biomedical research. Organ segmentation of whole-body mouse images is essential for quantitative analysis, but is tedious and error-prone. Here the authors develop a deep learning pipeline to segment major organs and the skeleton in volumetric whole-body scans in less than a second, and present probability maps and uncertainty estimates.

Whole-body imaging techniques play a vital role in exploring the interplay of physiological systems in maintaining health and driving disease. We introduce wildDISCO, a new approach for whole-body immunolabeling, optical clearing and imaging in mice, circumventing the need for transgenic reporter animals or nanobody labeling and so overcoming existing technical limitations. We identified heptakis(2,6-di- O -methyl)-β-cyclodextrin as a potent enhancer of cholesterol extraction and membrane permeabilization, enabling deep, homogeneous penetration of standard antibodies without aggregation. WildDISCO facilitates imaging of peripheral nervous systems, lymphatic vessels and immune cells in whole mice at cellular resolution by labeling diverse endogenous proteins. Additionally, we examined rare proliferating cells and the effects of biological perturbations, as demonstrated in germ-free mice. We applied wildDISCO to map tertiary lymphoid structures in the context of breast cancer, considering both primary tumor and metastases throughout the mouse body. An atlas of high-resolution images showcasing mouse nervous, lymphatic and vascular systems is accessible at http://discotechnologies.org/wildDISCO/atlas/index.php . Whole mice are immunolabeled with standard antibodies using wildDISCO.

Advances in tissue labeling and clearing methods include improvement of tissue transparency, better preservation of fluorescence signal, compatibility with immunostaining and large sample volumes. However, as existing methods share the common limitation that they can only be applied to human tissue slices, rendering intact human organs transparent remains a challenge. Here, we describe experimental details of the small-micelle-mediated human organ efficient clearing and labeling (SHANEL) pipeline, which can be applied for cellular mapping of intact human organs. We have successfully cleared multiple human organs, including kidney, pancreas, heart, lung, spleen and brain, as well as hard tissue like skull. We also describe an advanced volumetric imaging system using a commercial light-sheet fluorescence microscope that can accommodate most human organs and a pipeline for whole-organ imaging and visualization. The complete experimental process of labeling and clearing whole human organs takes months and the analysis process takes several weeks, depending on the organ types and sizes. This protocol describes labeling, clearing, imaging and cellular-level three-dimensional image reconstruction of intact human organs achieved with the help of a novel tissue clearing and labeling technology and a commercially available microscope.

In three‐dimensional (3D) space, an unbiased and systemic view of human specimens between structures and functions is required. However, conventional histological sections from specimens have made only limited progress in exploring intact information about 3D biological tissues. With significant advances in optical physics and chemical engineering, state‐of‐the‐art tissue‐clearing methods can revolutionize the intact subcellular level of human tissue histological analysis, from thick human tissues to intact human organs. The present review summarizes the principle of tissue clearing so that a trainee researcher can implement effective human tissue‐clearing protocols. Furthermore, this review highlights existing tissue‐clearing methods in specific human tissue applications, describes imaging strategies, and presents various efficient computational approaches for processing and visualizing large image data. Finally, potential future directions for developing tissue‐clearing methods for human tissues are also discussed.

In this Tools of the Trade article, Hongcheng Mai describes the development of wildDISCO, an approach for whole-body immunolabelling, optical clearing and imaging in mice.

Blood-brain barrier (BBB) impermeability remains a major obstacle to the effective treatment of neurological disorders, particularly ischemic stroke. Here, we revealed that plant-derived extracellular vesicle-like nanoparticles (PEVs) offer a promising strategy to overcome this barrier. Using an optimized high-yield extraction protocol, we isolated PEVs from four medicinal plants: Panax ginseng , Panax notoginseng , Gastrodia elata , and Ligusticum chuanxiong. Among these, extracellular vesicles derived from Panax notoginseng (NotoEV, vesicle population) exhibited the strongest neuroprotective effects under hypoxic conditions in vitro and in vivo stroke models. Mechanistically, NotoEV delivered conserved plant microRNAs to recipient neurons, where they suppressed key stress granule nucleators GTPase-activating protein-binding protein 2 (G3bp2), Ubiquitin-associated protein 2 like (Ubap2l), and LSM14A mRNA processing body assembly factor (Lsm14a), activated mammalian target of rapamycin (mTOR) signaling, and promoted mitochondrial stabilization via the B-cell lymphoma 2 (Bcl-2)/ Translocase Of Outer Mitochondrial Membrane 20 (TOM20) axis. This cross-kingdom RNA delivery reprogrammed neuronal stress responses, reduced infarct volume, preserved neuronal morphology, and restored electrophysiological function. Collectively, our findings establish a scalable platform for plant-based nanotherapeutics and highlight the translational potential of NotoEV in treating ischemic stroke. Graphical Abstract

Many hospitals lack facilities for accurate diagnosis of acute ischemic stroke (AIS). Circular RNA (circRNA) is highly expressed in the brain and is closely associated with stroke. In this study, we examined whether the blood-borne circRNAs could be promising candidates as adjunctive diagnostic biomarkers and their pathophysiological roles after stroke. We profiled the blood circRNA expression in mice subjected to experimental focal cerebral ischemia and validated the selected circRNAs in AIS patients. We demonstrated that 128, 198, and 789 circRNAs were significantly altered at 5 min, 3 h, and 24 h after ischemic stroke, respectively. Our bioinformatics analysis revealed that the circRNA-targeted genes were associated with the Hippo signaling pathway, extracellular matrix-receptor interaction, and fatty acid metabolism at 5 min, 3 h and 24 h after ischemic stroke, respectively. We verified that many of these circRNAs existed in the mouse brain. Furthermore, we found that most of the predicted circRNA-miRNA interactions apparently exhibited functional roles in terms of regulation of their target gene expression in the brain. We also verified that many of these mouse circRNAs were conserved in human. Finally, we found that circBBS2 and circPHKA2 were differentially expressed in the blood of AIS patients. These results demonstrate that blood circRNAs may serve as potential biomarkers for AIS diagnosis and reveal the pathophysiological responses in the brain after ischemic stroke.

Hemorrhagic transformation (HT) is a serious complication that stimulates inflammation during reperfusion therapy after acute ischemic stroke. Rosuvastatin, a 3-hydroxymethyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, might improve the outcome of HT by inhibiting neuroinflammation. This study aimed to explore the protective effects of rosuvastatin against HT after recombinant tissue plasminogen activator (rt-PA) treatment in mice with experimental stroke via the attenuation of inflammation. A total of one hundred sixty-nine male BALB/c mice were used in the experiment. HT was successfully established in 70 mice that were subjected to 3 h of middle cerebral artery occlusion (MCAO) followed by a 10 mg/kg rt-PA injection over 10 min and reperfusion for 24 h. The mice were then administered rosuvastatin (1 mg/kg, 5 mg/kg) or saline (vehicle). The brain water content and neurological deficits (wire hang and adhesive removal somatosensory tests) were assessed at 24 h after rt-PA reperfusion following MCAO surgery. The morphology, blood-brain barrier (BBB) permeability and number of astrocytes and microglia were assessed by immunohistochemistry, electron microscopy and western blotting at 24 h after rt-PA reperfusion following MCAO surgery. Rosuvastatin protected against impaired neurological function and reversed the BBB leakage observed in the HT group. The increased activation of astrocytes and microglia and secretion of inflammatory factors caused by HT damage were significantly attenuated by high-dose rosuvastatin treatment vs. normal-dose rosuvastatin treatment. Related inflammatory pathways, such as the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, were downregulated in the rosuvastatin-treated groups compared with the HT group. In conclusion, our results indicate that rosuvastatin is a promising therapeutic agent for HT after rt-PA reperfusion following MCAO surgery in mice, as it attenuates neuroinflammation. Additionally, high-dose rosuvastatin treatment could have a greater anti-inflammatory effect on HT than normal-dose rosuvastatin treatment.

Small extracellular vesicles (sEVs) contain abundant circular RNAs (circRNAs) and are involved in cellular processes, particularly hypoxia. However, the process that packaging of circRNAs into neuronal sEVs under hypoxia is unclear. This study revealed the spatial mechanism of the Fused in Sarcoma protein (FUS) that facilitates the loading of functional circRNAs into sEVs in hypoxia neurons. It is found that FUS translocated from the nucleus to the cytoplasm and is more enriched in hypoxic neuronal sEVs than in normal sEVs. Cytoplasmic FUS formed aggregates with the sEVs marker protein CD63 in cytoplasmic stress granules (SGs) under hypoxic stress. Meanwhile, cytoplasmic FUS recruited of functional cytoplasmic circRNAs to SGs. Upon relief of hypoxic stress and degradation of SGs, cytoplasmic FUS is transported with those circRNAs from SGs to sEVs. Validation of FUS knockout dramatically reduced the recruitment of circRNAs from SGs and led to low circRNA loading in sEVs, which is also confirmed by the accumulation of circRNAs in the cytoplasm. Furthermore, it is showed that the FUS Zf_RanBP domain regulates the transport of circRNAs to sEVs by interacting with hypoxic circRNAs in SGs. Overall, these findings have revealed a FUS‐mediated transport mechanism of hypoxia‐related cytoplasmic circRNAs loaded into sEVs under hypoxic conditions. This study aimed to unravel the role of FUS in the loading of functional circRNAs into neuronal sEVs under hypoxic conditions, which involves their aggregations in SGs and specific interaction between the FUS Zf_RanBP domain to regulate circRNA sorting in sEVs.

Ischemic stroke is usually followed by inflammatory responses mediated by microglia. However, the effect of statins on directly preventing posthypoxia microglia inflammatory factors to prevent injury to surrounding healthy neurons is unclear. Atorvastatin and rosuvastatin, which have different physical properties regarding their lipid and water solubility, are the most common HMG-CoA reductase inhibitors (statins) and might directly block posthypoxia microglia inflammatory factors to prevent injury to surrounding neurons. Neuronal damage and microglial activation of the peri-infarct areas were investigated by Western blotting and immunofluorescence after 24 hours in a middle cerebral artery occlusion (MCAO) rat model. The decrease in neurons was in accordance with the increase in microglia, which could be reversed by both atorvastatin and rosuvastatin. The effects of statins on blocking secretions from posthypoxia microglia and reducing the secondary damage to surrounding normal neurons were studied in a coculture system in vitro. BV2 microglia were cultured under oxygen glucose deprivation (OGD) for 3 hours and then cocultured following reperfusion for 24 hours in the upper wells of transwell plates with primary neurons being cultured in the bottom wells. Inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and cyclooxygenase-2 (COX2), which are activated by the nuclear factor-kappa B (NF-κB) signaling pathway in OGD-induced BV2 microglia, promoted decreased release of the anti-inflammatory cytokine IL-10 and apoptosis of neurons in the coculture systems according to ELISA and Western blotting. However, pretreatment with atorvastatin or rosuvastatin significantly reduced neuronal death, synaptic injury, and amyloid-beta (Aβ) accumulation, which might lead to increased low-density lipoprotein receptors (LDLRs) in BV2 microglia. We concluded that the proinflammatory mediators released from postischemia damage could cause damage to surrounding normal neurons, while HMG-CoA reductase inhibitors prevented neuronal apoptosis and synaptic injury by inactivating microglia through blocking the NF-κB signaling pathway.