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.

Stress urinary incontinence presents a condition not only found in female elderlies, but also in young athletes participating in high-impact sports such as volleyball or trampolining. Repeated jumps appear to be a predisposing factor. Yet the pathophysiology remains incompletely elucidated to date; especially with regard to the influence of the surrounding buttock tissues including gluteus maximus. The present study assessed the morpho-mechanical link between gluteus maximus and the pelvic floor female bodies. 25 pelves obtained from Thiel embalmed females were studied in a supine position. Strands of tissues connecting gluteus maximus with the pelvic floor obtained from 20 sides were assessed mechanically. Plastinates were evaluated to verify the dissection findings. In total, 49 hemipelves were included for data acquisition. The fascia of gluteus maximus yielded connections to the subcutaneous tissues, the fascia of the external anal sphincter and that of obturator internus and to the fascia of the urogenital diaphragm. The connection between gluteus maximus and the urogenital diaphragm withstood an average force of 23.6 ± 17.3 N. Cramér φ analyses demonstrated that the connections of the fasciae connecting gluteus maximus with its surroundings were consistent in the horizontal and sagittal planes, respectively. In conclusion, gluteus maximus is morphologically densely linked to the pelvic floor via strands of connective tissues investing the adjacent muscles. Though gluteus maximus has also been reported to facilitate urinary continence, the here presented morpho-mechanical link suggests that it may also have the potential to contribute to urinary stress incontinence. Future research combining clinical imaging with in-situ testing may help substantiate the potential influence from a clinical perspective.

Analysis of entire transparent rodent bodies after clearing could provide holistic biological information in health and disease, but reliable imaging and quantification of fluorescent protein signals deep inside the tissues has remained a challenge. Here, we developed vDISCO, a pressure-driven, nanobody-based whole-body immunolabeling technology to enhance the signal of fluorescent proteins by up to two orders of magnitude. This allowed us to image and quantify subcellular details through bones, skin and highly autofluorescent tissues of intact transparent mice. For the first time, we visualized whole-body neuronal projections in adult mice. We assessed CNS trauma effects in the whole body and found degeneration of peripheral nerve terminals in the torso. Furthermore, vDISCO revealed short vascular connections between skull marrow and brain meninges, which were filled with immune cells upon stroke. Thus, our new approach enables unbiased comprehensive studies of the interactions between the nervous system and the rest of the body.

Triggering Receptor Expressed on Myeloid cells 2 (TREM2), which is expressed on myeloid cells including microglia in the CNS, has recently been identified as a risk factor for Alzheimer's disease (AD). TREM2 transmits intracellular signals through its transmembrane binding partner DNAX-activating protein 12 (DAP12). Homozygous mutations inactivating TREM2 or DAP12 lead to Nasu-Hakola disease; however, how AD risk-conferring variants increase AD risk is not clear. To elucidate the signaling pathways underlying reduced TREM2 expression or loss of function in microglia, we respectively knocked down and knocked out the expression of TREM2 in in vitro and in vivo models. We found that TREM2 deficiency reduced the viability and proliferation of primary microglia, reduced microgliosis in Trem2-/- mouse brains, induced cell cycle arrest at the G1/S checkpoint, and decreased the stability of β-catenin, a key component of the canonical Wnt signaling pathway responsible for maintaining many biological processes, including cell survival. TREM2 stabilized β-catenin by inhibiting its degradation via the Akt/GSK3β signaling pathway. More importantly, treatment with Wnt3a, LiCl, or TDZD-8, which activates the β-catenin-mediated Wnt signaling pathway, rescued microglia survival and microgliosis in Trem2-/- microglia and/or in Trem2-/- mouse brain. Together, our studies demonstrate a critical role of TREM2-mediated Wnt/β-catenin pathway in microglial viability and suggest that modulating this pathway therapeutically may help to combat the impaired microglial survival and microgliosis associated with AD.SIGNIFICANCE STATEMENT Mutations in the TREM2 (Triggering Receptor Expressed on Myeloid cells 2) gene are associated with increased risk for Alzheimer's disease (AD) with effective sizes comparable to that of the apolipoprotein E (APOE) ε4 allele, making it imperative to understand the molecular pathway(s) underlying TREM2 function in microglia. Our findings shed new light on the relationship between TREM2/DNAX-activating protein 12 (DAP12) signaling and Wnt/β-catenin signaling and provide clues as to how reduced TREM2 function might impair microglial survival in AD pathogenesis. We demonstrate that TREM2 promotes microglial survival by activating the Wnt/β-catenin signaling pathway and that it is possible to restore Wnt/β-catenin signaling when TREM2 activity is disrupted or reduced. Therefore, we demonstrate the potential for manipulating the TREM2/β-catenin signaling pathway for the treatment of AD.

The femoral nerve stretch test is an essential part of clinical neurological examinations. This test is performed alongside Magnetic Resonance Imaging (MRI) to determine if there is any evidence of nerve root irritation, usually as a consequence of disc prolapse. The test occasionally gives false positive results. Why such false positives can occur, is subject to continued research, however, no obvious reason has yet emerged. We hypothesize that connectives of the femoral nerve may explain such a phenomenon. To see these connectives, we approached the femoral nerve from dorsal in 12 cases. With the use of ink injection into the subparaneural compartment of the femoral nerve and dissections, a thin transparent structure can clearly be seen that is separate from the epineurium, perineurium, and a paraneural sheath. A continuation of the paraneural sheath produces a fascia plate approximately 1.5 cm in width and with a thickness of around 3 mm, which not only circumnavigates the nerve but projects into the surrounding tissues. Our qualitative observations show that not only does this femoral nerve fascia plate exists, but it also contains nerves and vessels. Furthermore, we show that the femoral nerve is connected to the myofascial complex of the iliopsoas, and in a separate fascia plate from the iliopsoas fascia. This plate is a hitherto neglected connective which extends as far as the spinal dura mater. Evidence from our plastinates and histological sections suggests that when tension is applied to the femoral nerve during the femoral nerve stretch test, tension is also applied to the femoral nerve fascia plate. The femoral nerve fascia plate could be a specific factor that contributes to pain resulting in a false positive femoral nerve stretch test.

Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the pre-clinical stage. VIDEO ABSTRACT.

Triggering receptor expressed on myeloid cells-2 (TREM2) and colony-stimulating factor 1 receptor (CSF1R) are crucial molecules for microgliopathy, which is characterized by microglia dysfunction and has recently been proposed as the neuropathological hallmark of neurological disorders. TREM2 and CSF1R are receptors expressed primarily in microglia in the brain and modulate microglial activation and survival. They are thought to be in close physical proximity. However, whether there is a direct interaction between these receptors remains elusive. Moreover, the physiological role and mechanism of the interaction of TREM2 and CSF1R remain to be determined. Here, we found that TREM2 interacted with CSF1R based on a co-immunoprecipitation assay. Additionally, we found that CSF1R knockdown significantly reduced the survival of primary microglia and increased the Trem2 mRNA level. In contrast, CSF1R expression was increased in Trem2-deficient microglia. Interestingly, administration of CSF1, the ligand of CSF1R, partially restored the survival of Trem2-deficient microglia in vitro and in vivo. Furthermore, CSF1 ameliorated Aβ plaques deposition in Trem2-/-; 5XFAD mouse brain. These findings provide solid evidence that TREM2 and CSF1R have intrinsic abilities to form complexes and mutually modulate their expression. These findings also indicate the potential role of CSF1 in therapeutic intervention in TREM2 variant-bearing patients with a high risk of Alzheimer's disease (AD).

Pain over the superior angle of the scapula is a common musculoskeletal symptom. It is often accompanied by radiating pain to the neck, head, and shoulder. The aetiologies can be varied but may also be idiopathic in nature. To explore the fascial connections of this region, we studied 26 unembalmed, -two Thiel and one alcohol body-donors of science, by dissection, histological probes, and plastinations. When removing the descending and transverse fibres of the trapezius, a large prominent triangular area of white connectives is revealed, varying in mass. A subdivision of these connectives can be further dissected to prove that the rhomboid minor and levator scapulae muscles are interconnected and enclosed by connectives. Between these two muscles a bridge of connective tissue, containing fat, is observed. These connectives end cranially at the surface of the splenius capitis, and at the midline, containing vessels and nerves, as supported by histology and plastinations. This unification is separate from the rhomboid major muscle but overlaps with the latter dorsally. It connects to the superior angle of scapula and its upper medial borders, respectively, and cranially to the root of the spine of the scapula. Beneath the united levator scapulae and rhomboid minor, described here, the serratus posterior superior and possibly serratus anterior form a hypomochlion or fulcrum at the superior angle of the scapula. Any tension on this unified entity can unbalance this fulcrum. Investigating the connections between these two unified muscles may help explain the often idiopathic nature of superior scapular pain, and the success or failure of surgery, and other treatments.

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.

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.

Epilepsy is a common neurological disease with recurrent seizures and neurobehavioral comorbidities, including cognitive impairment and psychiatric disorders. Recent studies suggest that L-3-n-butylphthalide (NBP), an extract from the seeds of Apium graveolens Linn. (Chinese celery), ameliorates cognitive dysfunction in ischemia and/or Alzheimer's disease animal models. However, little is known about the role of NBP in epilepsy and the associated comorbidities. Here, using a pilocarpine-induced chronic epileptic mouse model, we found that NBP supplement not only alleviated seizure severity and abnormal electroencephalogram, but also rescued cognitive and emotional impairments in these epileptic mice. The possible underlying mechanisms may be associated with the protective role of NBP in reducing neuronal loss and in restoring the expression of neural synaptic proteins such as postsynaptic density protein 95 (PSD95) and glutamic acid decarboxylase 65/67 (GAD65/67). In addition, NBP treatment increased the transcription of neuroprotective factors, brain-derived neurotrophic factor and Klotho. These findings suggest that NBP treatment may be a potential strategy for ameliorating epileptogenesis and the comorbidities of cognitive and psychological impairments.

Frontotemporal dementia includes a large spectrum of neurodegenerative disorders. SQSTM1, coding for p62 protein, plays a vital role in the pathogenesis of FTD. Here, we report a case of a female patient with SQSTM1 mutation S224X, who was 59 years old when she initially exhibited memory decline, mild personality changes, and subtle atrophy of frontal/temporal lobes in magnetic resonance imaging (MRI). Genetic testing revealed a nonsense mutation of the SQSTM1 gene (S224X), resulting in premature termination of protein synthesis and a predicted truncated protein 217 amino acids shorter than the normal protein. Moreover, neither intact nor truncated SQSTM1 proteins was detectable in SQSTM1 S224X mutant overexpressing HEK-293T cells. We assayed for SQSTM1 cDNA in samples from the patient's peripheral leucocytes, and did not detect its mutation. The test of quantitative PCR showed significant decreased level of SQSTM1 mRNA from peripheral leucocytes of the patient compared to five dementia controls. Our results identify a novel pathogenic SQSTM1 S224X mutation in an atypical FTD patient accompanied with loss of SQSTM1/p62 protein expression probably due to SQSTM1 gene haploinsufficiency.

Optical tissue transparency permits scalable cellular and molecular investigation of complex tissues in 3D. Adult human organs are particularly challenging to render transparent because of the accumulation of dense and sturdy molecules in decades-aged tissues. To overcome these challenges, we developed SHANEL, a method based on a new tissue permeabilization approach to clear and label stiff human organs. We used SHANEL to render the intact adult human brain and kidney transparent and perform 3D histology with antibodies and dyes in centimeters-depth. Thereby, we revealed structural details of the intact human eye, human thyroid, human kidney, and transgenic pig pancreas at the cellular resolution. Furthermore, we developed a deep learning pipeline to analyze millions of cells in cleared human brain tissues within hours with standard lab computers. Overall, SHANEL is a robust and unbiased technology to chart the cellular and molecular architecture of large intact mammalian organs.

The ability of muscle to generate force depends on its architecture and health condition. MR-based diffusion tensor imaging of muscle (mDTI) is an innovative approach for showing the fiber arrangement for the whole muscle volume. For accurate calculations of fiber metrics, muscle segmentation prior to tractography is regarded as necessary. Since segmentation is known to be operator dependent, it is important to understand how segmentation affects tractography. The aim of this study was to compare the results of deterministic fiber tracking based on muscle models generated by two independent operators. In addition, this study compares the results with a segmentation-free approach. Fifteen subjects underwent mDTI of the right shoulder. The results showed that mDTI can be successfully applied to complex joints such as the human shoulder. Furthermore, operator segmentation did not influence the results of fiber tracking and fascicle length (FL), fiber volume (FV), fractional anisotropy (FA), axial diffusivity (AD), radial diffusivity (RD), and mean diffusivity (MD) showed excellent intraclass correlation estimates (≥ 0.975). As an exploratory approach, the segmentation-free fiber tracking showed significant differences in terms of mean fascicle length. Based on these findings, we conclude that tractography is not sensitive to small deviations in muscle segmentation. Furthermore, it implies that mDTI and automatic segmentation approaches or even a segmentation-free analysis can be considered for evaluation of muscle architecture.

Mutation of Triggering receptor expressed on myeloid cells 2 (TREM2) impairs the response of microglia to amyloid-β (Aβ) pathology in Alzheimer's disease (AD), although the mechanism governing TREM2-regulated microglia recruitment to Aβ plaques remains unresolved. Here, we confirm that TREM2 mutation attenuates microglial migration. Then, using Trem2-/- mice and an R47H variant mouse model for AD generated for this study, we show that TREM2 deficiency or the AD-associated R47H mutation results in inhibition of FAK and Rac1/Cdc42-GTPase signaling critical for cell migration. Intriguingly, treatment with CN04, a Rac1/Cdc42-GTPase activator, partially enhances microglial migration in response to oligomeric Aβ42 in Trem2-/- or R47H microglia both in vitro and in vivo. Our study shows that the dysfunction of microglial migration in the AD-associated TREM2 R47H variant is caused by FAK/Rac1/Cdc42 signaling disruption, and that activation of this signaling ameliorates impaired microglial migration response to Aβ42 , suggesting a therapeutic target for R47H-bearing patients with high risk of AD.

The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.

The aim of the study was to assess the relationship between the expression of elastin, collagen type I, II,III and the degenera- tion of the facet joint capsule and the ligamentum flavum.