After a crush injury in sciatic nerve fibers, dynamic changes in blood circulation and immune-cell mobilization occur during axonal regeneration. High-resolution visualization under near-physiological conditions is crucial for understanding these mechanisms. Conventional histological techniques introduce perfusion- and dehydration-induced artifacts that obscure circulation. We employed the in vivo cryotechnique (IVCT) to visualize blood flow within sciatic nerve fibers and assess temporal changes during regeneration. In uninjured mice, IVCT preserved native tissue architecture with minimal shrinkage compared to perfusion fixation, with superiority quantitatively shown by fractal analysis. In the crush model, hematoxylin-eosin, Luxol fast blue, and immunohistochemical staining of IVCT-prepared, freeze-substituted sections revealed axonal degeneration and regrowth. The close association between regenerating fibers and vascular structures, along with erythrocyte distribution, indicates a morphological link between nerves and blood vessels. Electrophysiological assessment using compound muscle action potentials and functional recovery measured by the sciatic functional index demonstrated restored nerve function at 28 days, consistent with histology. These findings suggest that IVCT is a useful method for analyzing peripheral nerve regeneration and vascular dynamics, thereby highlighting its potential as a novel approach in peripheral nerve research.

Periodontitis is a chronic inflammatory disease characterized by collagen degradation and alveolar bone loss. Although estrogen contributes to periodontal homeostasis, the role of the membrane-bound G protein-coupled estrogen receptor 30 (GPR30) in periodontitis remains unclear. This study investigated the temporal involvement of GPR30 in ligature-induced periodontitis, focusing on alveolar bone changes, collagen integrity, fibroblast activation, and epigenetic regulation. Twenty male Wistar rats were allocated to control and periodontitis groups and evaluated at days 7, 14, and 21 following placements of a stainless-steel ligature around the maxillary first molars. Alveolar bone loss was assessed radiographically, while inflammatory changes and collagen organization were examined using hematoxylin-eosin and Masson's trichrome staining. Immunohistochemistry was performed to evaluate GPR30, collagen I/III, α-smooth muscle actin (α-SMA), and histone modifications. Statistical analysis was conducted using one-way ANOVA with Tukey's post hoc test. Alveolar bone loss and collagen degradation were detectable on day 7 and peaked at day 14. GPR30 expression increased during the active inflammatory phase, accompanied by elevated α-SMA and histone H4 lysine 8 crotonylation (H4K8cr), and declined by day 21. Fibroblast transiently adopted a myofibroblast phenotype during inflammation and regressed during early tissue repair. Temporal changes in H4K8cr closely paralleled GPR30 expression. These findings indicate that GPR30 is dynamically associated with inflammatory and remodeling phases of periodontitis and may contribute to epigenetic regulation during periodontal tissue remodeling.

Cancer cells escape immune surveillance by suppressing immune responses through the binding of Programmed cell Death-Ligand 1 (PD-L1), which is abundantly expressed on the cell surface, to PD-1 on the surface of T cells. The regulation of cell surface PD-L1, one of these immune checkpoint molecules, is extremely important because it is a target for cancer immunotherapy; however, the intracellular trafficking pathway of PD-L1 has not been fully elucidated. Recently, we reported that Rab10, a small GTPase, localizes to a novel tubular endocytic pathway that evades the lysosomal degradation system. In this study, using live cells expressing GFP-PD-L1 and mScarlet-Rab10, we revealed that PD-L1 localizes in Rab10-positive endocytic tubules in some types of cancer cells. Typically, in HeLaM cells, Rab10-positive tubular structures of which membranes have PD-L1 extend from the plasma membrane toward the cell-central region. However, in Rab10-knockout HeLaM cells, no PD-L1-localized tubular structures were observed. We also found that PD-L1 dimerized by the PD-L1 inhibitor BMS-202 was removed from the cell surface and Rab10-positive tubular endosomes and transported to the lysosomal degradation system. Taken together, this study provides novel insights that the Rab10-dependent tubular endocytic pathway may play an important role in the intracellular reservoir and recycling of PD-L1 to the surface of cancer cells, possibly regulating the amount of PD-L1 on the cell surface.

Endoscopic ultrasound-guided fine-needle aspiration/biopsy (EUS-FNA/B) is critical for determining treatment strategies for patients with pancreatic cancer. However, conventional pathological examination using hematoxylin and eosin (H&E) staining is time-consuming. Microscopy with ultraviolet surface excitation (MUSE) enables rapid pathological diagnosis without requiring slide preparation. This study explores the potential of combining MUSE imaging with a cycle-consistent generative adversarial network (CycleGAN), an image generation algorithm capable of learning translations without paired images, to enhance diagnostic workflows for pancreatic EUS-FNA/B. Thirty-five pancreatic specimens were stained with Terbium/Hoechst 33342, and deep ultraviolet (DUV) fluorescence images were captured by exciting the tissue surface. These fluorescence images, along with H&E-stained formalin-fixed, paraffin-embedded (FFPE) sections from the same specimens, were divided into 256 × 256-pixel segments for CycleGAN training. The algorithm was employed to translate pseudo-H&E images from MUSE test images. The pseudo-H&E images generated by the CycleGAN showed improved inter-pathologist agreement among three pathologists compared with the original MUSE images. We established a technique to perform MUSE imaging on small pancreatic samples obtained through EUS-FNA/B and confirmed that H&E-style translation using CycleGAN simplified interpretation for pathologists. Integrating MUSE imaging with CycleGAN has the potential to offer a rapid, cost-effective, and accurate diagnostic tool.

While the rapid decrease in estrogen is well known as the main cause of postmenopausal osteoporosis in women, the precise pathogenesis of senile osteoporosis in the elderly regardless of gender is largely unknown. The age-related epigenetic regulation of receptor activator NF-κB (RANK) gene expression was investigated with the use of a high-passaged mouse osteoclast progenitor cell line, RAW264.7, as an in vitro model of aging. In the RAW264.7 cells after repeated passages, receptor RANK expression was downregulated, resulting in decreased soluble RANK ligand (sRANKL)-induced osteoclastogenesis, expression of tartrate-resistant acid phosphatase-5b (TRAcP) and cathepsin K (CTSK). Methylation-specific PCR and bisulfite mapping revealed hypermethylation of CpG-loci located in the RANK gene promoter in multiple-passaged cells. ICON probe-mediated in situ assessment of methylated-cytosine at the CpG loci revealed an increase in the percentage of methylated RAW264.7 cells in the RANK gene in a passage-dependent manner. Conversely, upon treatment with demethylating agent 5-aza-2-deoxycytidine (5-aza-dC), high-passaged RAW264.7 cells displayed restored expression of the RANK gene, osteoclastogenesis, TRAcP and CTSK. Ex vivo cultures of splenic macrophages from young (10.5 W) and aged (12 M) mice also showed that CpG methylation was predominant in the aged animals, resulting in reduced RANK expression and osteoclastogenesis. Reduced RANK expression by age-related accumulation of DNA methylation, albeit in a limited population of osteoclast precursor cells, might be, at least in part, indicative of low-turnover bone characteristic of senile osteoporosis.

Pathological hallmark of Alzheimer's disease (AD) is characterized by the accumulation and aggregation of amyloid β (Aβ) peptides into extracellular plaques of the brain. Clarification of the process of how soluble Aβ starts to assemble into amyloid fibrils is an essential step in elucidating the pathogenesis of AD. In our previous study, Aβ proteoforms including full-length Aβ40 and Aβ42/43 with N- and C-terminal truncated forms were visualized in postmortem brains from AD patients with matrix-assisted laser desorption/ionization-based mass spectrometry imaging (MALDI-MSI). In this study, Aβ proteoforms were consistently visualized by an updated protocol, and uncharacterized peptides such as Aβ1-29 and Aβ10-40 in AD brains were also visualized. To decipher neurotoxic effects of Aβ in patients' brains, here we integrate liquid chromatography tandem mass spectrometry (LC-MS/MS) based shotgun proteomics with laser microdissection (LMD) excised tissue samples as well as direct tissue imaging with MALDI-MSI. With this approach, we have highlighted dynamic alterations of microtubule associating proteins (MAPs) including MAP1A, MAP1B and MAP2 as well as AD dominant proteins including APP, UCHL1, SNCA, and APOE. Of note, as lipid dysregulation has been implicated with AD pathology, we have challenged to integrate proteomics and lipid imaging for AD and control brain tissue. Spatial multi-omics is also valid to uncover molecular pathology of white matter as well as grey matter and leptomeningeal area, for example, by visualizing heme in patients' postmortem brains.

Mitochondrial ferritin (FtMt) is a novel ferritin that sequesters iron and plays a protective role against oxidative stress. FtMt shares a high homology with H-ferritin but is expressed only in the brain, heart, and testis. In the midbrain, FtMt expression is observed in the substantia nigra. FtMt plays a neuroprotective role in the pathology of neurodegenerative diseases such as Parkinson's disease, where excessive iron induces oxidative stress, causing cell death. Herein, we investigated FtMt immunoreactivity in the brains of patients with subarachnoid hemorrhage (SAH). Double immunofluorescence labeling of tyrosine hydroxylase (TH) and FtMt showed high colocalization in the substantia nigra pars compacta (SNc) in control and SAH cases. However, in SAH cases, FtMt immunoreactivity was observed in some TH-negative neurons. Double immunofluorescence labeling of glial cell markers and FtMt showed no apparent colocalization. The number and ratio of FtMt-positive but TH-negative neurons significantly differed between the control and SAH groups. Prussian blue staining in SAH cases showed positive iron staining over a wide surface range and the substantia nigra. Thus, FtMt may be related to iron dynamics in the substantia nigra following subarachnoid hemorrhage.

In the pathogenesis of Alzheimer's disease (AD), highly neurotoxic amyloid-β (Aβ) oligomers appear early, they are thus considered to be deeply involved in the onset of Alzheimer's disease. However, Aβ oligomer visualization is challenging in human tissues due to their multiple forms (e.g., low- and high-molecular-weight oligomers, including protofibrils) as well as their tendency to rapidly change forms and aggregate. In this review, we present two visualization approaches for Aβ oligomers in tissues: an immunohistochemical (using the monoclonal antibody TxCo1 against toxic Aβ oligomer conformers) and imaging mass spectrometry using the small chemical Shiga-Y51 that specifically binds Aβ oligomers. TxCo1 immunohistochemistry revealed Aβ oligomer distributions in postmortem human brains with AD. Using Shiga-Y51, imaging mass spectrometry revealed Aβ oligomer distributions in the brain of a transgenic mouse model for AD. These two methods would potentially contribute to elucidating the pathological mechanisms underlying AD.

In situ hybridization (ISH), which visualizes nucleic acids in tissues and cells, is a powerful tool in histology and pathology. Over 50 years since its invention, multiple attempts have been made to increase the sensitivity and simplicity of these methods. Therefore, several highly sensitive in situ hybridization methods have been developed that offer researchers a wide range of options. When selecting these in situ hybridization variants, their signal-amplification principles and characteristics must be understood. In addition, from a practical point of view, a method with good monetary and time-cost performance must be chosen. This review introduces recent high-sensitivity in situ hybridization variants and presents their principles, characteristics, and costs.

This study, it was investigated whether silymarin has a protective effect by performing histological, immunohistochemical, and biochemical evaluations on the liver damage induced by cecal ligation perforation (CLP). CLP model was established and silymarin was treated at a dose of 50 mg/kg, 100 mg/kg, and 200 mg/kg, by oral one hour before the CLP. As an effect of the histological evaluations of the liver tissues, venous congestion, inflammation, and necrosis in the hepatocytes were observed in the CLP group. A situation close to the control group was observed in the Silymarin (SM)100 and SM200 groups. As a result of the immunohistochemical evaluations, inducible nitric oxide synthase (iNOS), cytokeratine (CK)18, Tumor necrosis factor-alpha (TNF-α), and interleukine (IL)-6 immunoreactivities were intense in the CLP group. In the biochemical analysis, Alkaline Phosphatase (ALP), Aspartate Aminotransferase (AST), and Alanine Aminotransferase (ALT) levels were significantly increased in the CLP group, while a significant decrease was observed in the treatment groups. TNFα, IL-1β, and IL-6 concentrations were in parallel with histopathological evaluations. In the biochemical analysis, Malondialdehyte (MDA) level increased significantly in the CLP group, but there was a significant decrease in the SM100 and SM200 groups. Glutathione (GSH), Superoxide Dismutase (SOD), Catalase (CAT), and Glutathione Peroxidase (GSH-Px) activities were relatively low in the CLP group. According to these data, it was concluded that using silymarin reduces the existing liver damage in sepsis.