Journal of Histochemistry & Cytochemistry最新文献

Lipid signaling molecules are essential for maintaining intestinal mucosal barrier homeostasis. Over 80% of polyunsaturated fatty acids (PUFAs) are metabolized through the cytochrome P450 epoxygenase (CYP)-soluble epoxide hydrolase (sEH) axis, generating bioactive epoxy and diol fatty acids. However, the expression patterns and functional roles of these enzymes in the intestinal epithelium remain poorly defined. To address this, we used RNAscope in situ hybridization and publicly available single-cell RNA sequencing (scRNAseq) datasets to map the cell-type-specific expression of PUFA-metabolizing enzymes in the small intestine. In humans, we identified three major epithelial expression patterns: (1) stem-cell-dominant (CYP2E1), (2) enterocyte-dominant (CYP1A1), and (3) widespread expression across epithelial subsets for key CYP epoxygenases (CYP2C8, 2C9, 2C18, 2C19, 2J2, 2S1), sEH, and additional CYPs (3A4, 4A11, 4F2, 4F3, 4F8, 4F12). In mice, five distinct expression patterns were found: (1) stem cell and transit-amplifying cells (CYP2E1), (2) transit-amplifying dominant (CYP4F18), (3) transit-amplifying and enterocytes (CYP2C44), (4) enterocyte-dominant (CYP1A1), and (5) broadly expressed across all epithelial clusters (sEH, CYP2B10, 2S1, 2C55, 4F13, 4F16). Furthermore, in a radiation-induced small-bowel injury-regeneration model, we observed dynamic changes in CYP expression patterns. These findings provide the first high-resolution single-cell atlas of CYP-sEH axis enzymes in the intestinal epithelium, offering key insights into their potential roles in epithelial differentiation, injury response, and mucosal barrier integrity. This foundational work enables future studies to define the biological functions and therapeutic relevance of PUFA-derived lipid mediators in intestinal health and disease.

SummaryThis study examines the involvement of T cell autocrine hyaluronan (HA) with the immunological synapse (IS) that mediates T cell activation by antigen-presenting cells (APCs). Three-dimensional (3D) confocal images of mouse CD4⁺ T cells interacting with B cell lymphoma (A20) APCs in vitro showed HA to be primarily on the T cell side of the IS, appearing as a compact mass indenting the T cell nucleus. Similar 3D imaging of CD4⁺ T cells forming a pseudo-IS on anti-CD3 antibody-coated glass showed HA in the vicinity of the IS in dense masses or complex, arched, columnar structures. Affinity/immunofluorescence labeling studies confirmed the HA masses and columns were cytoplasmic, located beneath the cortical actin layer but outside the nucleus. In T cells forming a pseudo-IS, the HA-binding protein RHAMM was localized to cortical cytoplasm and had limited spatial overlap with cytoplasmic HA. Pre-exposure of T cells to the HA synthesis inhibitor 4-methylumbelliferone (4-MU) or to the HA-binding peptide Pep-1 inhibited IS formation with A20 APCs. Moreover, actin ring development in T cell pseudo-IS was inhibited by pre-exposure to 4-MU, but not by pre-exposure to Pep-1. Collectively, our previous and present studies suggest a complex role for cell surface and cytoplasmic T cell autocrine HA in IS formation and T cell receptor signaling.

SummaryThis study aimed to verify the effect of peroxiredoxin 1 (PRDX1) in wound healing. PRDX1 and tumor necrosis factor receptor-associated factor 4 (TRAF4) expressions in human umbilical vein endothelial cells (HUVECs) were assessed by real-time quantitative PCR and Western blot. Cell proliferation, migration, and angiogenesis were assessed by cell counting kit-8, wound healing, and tube formation assay. The reactive oxygen species level was measured using 2'-7'-dichlorodihydrofluorescein diacetate. Senescence-associated β-galactosidase staining assay was used to detect cells senescence. The relationship among PRDX1, TRAF4, and UBE3A was determined by co-immunoprecipitation. Upregulation of PRDX1 promoted the proliferation, migration, and angiogenesis of HUVECs. Meanwhile, PRDX1 overexpression inhibited oxidative stress and senescence of HUVECs by H₂O₂-induced. Furthermore, overexpression of PRDX1 inhibited the degradation of TRAF4 to activate PI3K/AKT/VEGF axis via binding to UBE3A. The effect of PRDX1 on H₂O₂-induced oxidative stress and senescence was reversed by TRAF4 silence. The promotion of PRDX1 on proliferation, migration, and angiogenesis was canceled by knockdown of TRAF4. PRDX1 inhibited oxidative stress and senescence via restraining the degradation of TRAF4 by binding to UBE3A, eventually accelerating wound healing.

The Vibratome, or vibrating microtome allows sectioning of the mouse brain with a reliably established thickness from 50 µm, but they have a strong limitation on the size of the sample suitable for cutting. Herein is described the construction with publicly available element base (from parts for a 3D printer) a so-called 3D-vibrating microtome capable of cutting larger size brain into thin sections, and use it to investigate the limit-attainable values of the minimum slice thickness. Both of these goals have been successfully achieved. Both small mouse slices with a minimum thickness of 30 μm and large whole calf brain slices with a minimum thickness of 150 μm were obtained. Critical features of the effect of blade vibration frequency and sample feed rate on the minimum slice thickness were revealed, In the same way, a clear correlation was established between the minimum achievable thickness and its area.

SummaryOrchiectomy induces atrophy of epithelial cells in the prostate gland, stimulated by androgen deprivation. The objective of the current study was to assess the changes arising from tissue remodeling during short periods of androgen deprivation in the ventral prostate of Mongolian gerbils. Adult male gerbils were divided into groups: control and castrated, euthanized on the 3rd (Ca3d), 7th (Ca7d), and 14th (Ca14d) days post-orchiectomy (n = 7/group). The ventral lobe of the prostate was submitted for histological, stereological, morphometric, serological, and immunohistochemical analyses. Castration promoted a reduction in the weight of the ventral prostate. It altered the relative proportion of prostate tissue compartments, such as a decrease in the epithelium and non-muscular stroma and an increase in the muscular stroma. In addition, we observed that the number of Cd-68 positive cells increased in the Ca3d group, which represents a period of androgen deprivation not previously reported in the literature for Mongolian gerbils. Matrix metalloproteinase-9 quantification revealed a decrease in the Ca7d group compared to the Ca3d. In addition, the frequency of Tumor Necrosis Factor alpha increased in the Ca14d group, influenced by the duration of testosterone deficiency. The findings contribute to the understanding of prostate tissue remodeling after castration, as well as highlighting the rapid alterations in the prostate microenvironment in a short period following castration.

Glioblastoma is the most frequent and aggressive primary brain cancer in adults, and the prognosis is poor. The neurotrophic factor glial cell-derived neurotrophic factor (GDNF) and its receptors, which are involved in neuronal development, have in experimental studies been suggested to drive tumorigenic processes in glioblastoma, but the role and expression in glioblastoma in patients is under-investigated. The aim of this study was to investigate the expression of GDNF, GDNF family receptor 1-4 (GFRA1-4), and the downstream REarranged during Transfection (RET) receptor in human glioblastoma tissue by RNA in situ hybridization, immunohistochemistry, and immunofluorescence. Expression was quantified by software-based classifiers. The results showed that GDNF was expressed in approximately 10% of tumor cells. The GFRA1 receptor was widely expressed in tumor cells, often colocalizing with the astrocytic tumor cell marker glial fibrillary acidic protein (GFAP), and in a smaller fraction of tumor cells expressing the stem cell markers oligodendrocyte transcription factor 2 (OLIG2) and SRY-Box Transcription Factor 2 (SOX2). The GFRA2 receptor expression was very limited, whereas expression of GFRA3, GFRA4, and RET, respectively, was almost absent. In conclusion, GDNF and its primary receptor GFRA1 were expressed in patient glioblastoma tissue. Potential clinical value needs further investigation.

Macrophages have multiple roles in the heart including immune surveillance and extracellular matrix remodeling. Aging increases both collagen deposition and macrophage number in the heart; however, rodent models used to study cardiac macrophages have age-related comorbidities such as atherosclerosis and hypertension. The Fischer 344 rat does not develop these conditions with aging; therefore, the purpose of this study was to evaluate macrophage number and polarization in the hearts of aged (24-month) and young (6-month) Fischer 344 rats. Paraffin-embedded hearts were assessed for collagen deposition and immunolabeled for CD68, CD163, CD206, and galectin-3. Compared with young rats, significantly greater collagen deposition was observed in the old rats. There were no significant differences in CD68⁺ or CD163⁺ cells between age groups, but both CD206⁺ and galectin-3⁺ cells were more numerous in the aged animals. Double-immunofluorescence studies demonstrated that galectin-3 colocalized with both CD68 and CD163, suggesting that galectin-3 is found in cardiac macrophages. Further colocalization studies demonstrated similar proportions of CD68⁺/CD163^-, CD68⁺/CD163⁺, and CD68^-/CD163⁺ cells between age groups, suggesting that aging does not affect macrophage polarization. As CD206⁺ and galectin-3⁺ cells promote fibrosis, these results warrant future studies that delineate the specific roles of these cells in the aged heart.

Probably the most sensitive and frequently performed stain for calcium deposits in mammalian tissues is the von Kossa, a stain used for over a century. When originally incorporated into daily use in the histopathology laboratory on paraffin tissue sections, the procedure called for immersion of tissue sections in an aqueous 5% silver nitrate solution and placed in direct sunlight for 20-30 min to make calcium deposits appear black. Although this effective method continues to be used in select locations, the modern laboratory often does not have access to bright sunlight. This study revisited the traditional von Kossa staining method with alternatives in light sources, silver nitrate concentrations, and timing of silver solution exposure and determined that a 1% silver nitrate solution is equally effective as a 5% solution in bright sunlight for 30-60 min. In addition, a 1% silver nitrate solution can be successfully used with exposure to a 3.5W light-emitting diode (LED) daylight lamp for 1 hr, sunlight through a tinted glass window for 1 hr, or overhead room lighting for 2 hr, and the staining reaction is unaffected by temperatures ranging from 1C to 40C.

Alendronate (ALN), a nitrogen-containing bisphosphonate, is widely used to treat bone disorders. While its inhibitory effect on osteoclast activity is well-established, its impact on the release of extracellular vesicles (EVs) is less understood. This study investigated the effect of ALN on the quantity and size distribution of EVs released by osteoclasts cultured on bovine bone slices pretreated with 10-µM ALN, 100-µM ALN, or vehicle. Raw 264.7 cells were differentiated into osteoclasts using RANK-ligand, and EVs were isolated from conditioned media. Tartrate-resistant acid phosphatase (TRAP) staining, phalloidin staining for actin rings, and nanoparticle tracking analysis (NTA) were performed. TRAP staining showed a significant reduction in the number of TRAP-positive multinucleated cells in the 100-µM ALN group, confirming that high-concentration ALN also impairs osteoclast formation. Phalloidin staining showed a significant decrease in actin ring formation in the 100-µM ALN group, confirming ALN's inhibitory effect on osteoclast activity. NTA revealed a lower total EV concentration in the 100-µM ALN group, with a distinct peak of smaller EVs (<100 nm), suggestive of exosomes. These findings indicate that ALN, especially at higher concentrations, alters the release profile of osteoclast-derived EVs, potentially affecting intercellular communication and bone remodeling beyond its direct inhibition of resorption.