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Intracerebral hemorrhage (ICH) is the most fatal subtype of stroke with high disability and high mortality rates, and there is no effective treatment. The predilection site of ICH is in the area of the basal ganglia and internal capsule (IC), where exist abundant white matter (WM) fiber tracts, such as the corticospinal tract (CST) in the IC. Proximal or distal white matter injury (WMI) caused by intracerebral parenchymal hemorrhage is closely associated with poor prognosis after ICH, especially motor and sensory dysfunction. The pathophysiological mechanisms involved in WMI are quite complex and still far from clear. In recent years, the neuroprotection and repairment capacity of mesenchymal stem cells (MSCs) has been widely investigated after ICH. MSCs exert many unique biological effects, including self-recovery by producing growth factors and cytokines, regenerative repair, immunomodulation, and neuroprotection against oxidative stress, providing a promising cellular therapeutic approach for the treatment of WMI. Taken together, our goal is to discuss the characteristics of WMI following ICH, including the mechanism and potential promising therapeutic targets of MSCs, aiming at providing new clues for future therapeutic strategies.

Mounting evidence suggests that distinct microbial communities reside in tumors and play important roles in tumor physiology. Recently, a previous study profiled the composition and localization of intratumoral bacteria using 16S ribosomal DNA (rDNA) sequencing and histological visualization methods across seven tumor types, including human glioblastoma. However, their results based on traditional histological examinations should be further validated considering potential sources of contamination originating from sample collection and processing.Here, we aim to propose a three-dimensional (3D) in situ intratumoral microbiota visualization and quantification protocol avoiding surface contamination and provide a comprehensive histological investigation on local bacteria within human glioma samples. We develop a 3D quantitative in situ

Due to the complexity and limited availability of human brain tissues, for decades, pathologists have sought to maximize information gained from individual samples, based on which (patho)­physiological processes could be inferred. Recently, new understandings of chemical and physical properties of biological tissues and multiple chemical profiling have given rise to the development of scalable tissue clearing methods allowing superior optical clearing of across-the-scale samples. In the past decade, tissue clearing techniques, molecular labeling methods, advanced laser scanning microscopes, and data visualization and analysis have become commonplace. Combined, they have made 3D visualization of brain tissues with unprecedented resolution and depth widely accessible. To facilitate further advancements and applications, here we provide a critical appraisal of these techniques. We propose a classification system of current tissue clearing and expansion methods that allows users to judge the applicability of individual ones to their questions, followed by a review of the current progress in molecular labeling, optical imaging, and data processing to demonstrate the whole 3D imaging pipeline based on tissue clearing and downstream techniques for visualizing the brain. We also raise the path forward of tissue-clearing-based imaging technology, that is, integrating with state-of-the-art techniques, such as multiplexing protein imaging, in situ signal amplification, RNA detection and sequencing, super-resolution imaging techniques, multiomics studies, and deep learning, for drawing the complete atlas of the human brain and building a 3D pathology platform for central nervous system disorders.

The 3D visualization based on tissue clearing technology allows us to have a deeper understanding of the 3D spatial information of deep molecules in the tissue. Tissue clearing and bacterial labeling methods have been used for in situ 3D microbiota imaging, and we have developed a pipeline for 3D visualization of in situ microbiota in human gliomas. Anti‐LPS antibodies are appropriate to label and characterize bacteria in situ within tumors. However, autofluorescence (AF) is common in biological tissues, especially in brain tissues filled with lipofuscin‐like (LF) substances. This natural fluorescent signal is usually considered to be a problem because it affects the 3D visualization of fluorescent signals in bacterial LPS staining. Here, we used Sudan Black B (SBB) to mask the AF of human glioma tissue and explored in detail the optimal quencher concentration, which allows 3D visualization of intratumoral bacteria to reduce AF and maintain the intensity of intratumoral bacteria‐specific LPS fluorescent signals.

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Mounting evidence suggests that distinct microbial communities reside in tumors and play important roles in tumor physiology. Recently, Nejman et al. profiled the composition and localization of intratumoral bacteria using 16S DNA sequencing and histological visualization methods across seven tumor types, including human glioblastoma. However, considering potential contamination in their sample origins and processing, the results based on traditional histological methods need to be validated. Here, we propose a three-dimensional (3D) intratumoral microbiota visualization and quantification protocol to observe microbiota in intact tumor tissues on the premise of avoiding possible contamination in the surface of tissues, based on tissue clearing, immunofluorescent labeling, microscopy imaging, and image processing. For the first time, we have achieved 3D quantitative imaging of bacterial LPS fluorescent signals deep in gliomas in a contamination-free manner, which was founded mostly localized near nuclear membranes or in the intercellular space. Through an automated statistical algorithm, reliable signals can be distinguished for further analysis of their sizes, distribution, and fluorescence intensities. Combining two-dimensional images from multiple thin-section histological methods, including immunochemistry and fluorescence in situ hybridization, we provide a comprehensive histological investigation of the morphology and distribution of these signals on human glioma samples. We expect that this multi-evidence chain will provide supporting proof for the presence of intratumoral bacteria in human glioma and that the integrated pipeline can be applied to investigate the native bacteria within diverse tumors and contribute to the interpretation of their direct roles in the tumor microenvironment.