Histochemistry and Cell Biology最新文献

Sweet taste is mediated by type II taste bud cells (TBCs), which express the heterodimeric taste receptor composed of type 1 members 2 and 3, a G protein-coupled receptor. Activating this receptor triggers phospholipase Cβ2 (PLCβ2)-dependent signaling, depolarizes cell membrane, and leads to ATP release via calcium homeostasis modulator 1 and 3 channels. However, the number of sweet-responsive cells within individual fungiform taste buds remains poorly understood. To quantify the number of sweet-responsive TBCs, we developed a novel method using biocytin uptake as an indicator of membrane depolarization. The apical side of peeled mouse lingual epithelia was stimulated with 1 M sucrose or 30 mM saccharin, while biocytin was applied to the basolateral side. Sweet stimulation significantly increased the number of biocytin-labeled cells compared to deionized-water controls. Biocytin labeling was observed primarily in PLCβ2-positive type II cells, with additional labeling in PLCβ2 and synaptosomal-associated protein 25-negative cells, suggesting the involvement of type II and, likely, type I cells. On average, 11% of type II cells per taste bud were sweet-responsive; however, this proportion varied substantially across individual taste buds. These results indicate that sweet-responsive cells form a subset of type II cells and are distributed heterogeneously among fungiform taste buds. Such heterogeneity may reflect divergent tuning properties and contribute to robust sweet taste perception. Given the short lifespan and continuous turnover of TBCs, asynchronous renewal of sweet-responsive cells across taste buds may help maintain sweet sensitivity by ensuring that some sweet-sensitive cells are consistently present.

Ovarian hyperstimulation syndrome (OHSS) is a serious complication commonly encountered in patients with infertility undergoing ovulation induction therapy. This study investigates histomorphological and biochemical effects of melatonin in an experimental OHSS model. Rats were divided into four groups: control group, controlled ovarian stimulation group (COS), ovarian hyperstimulation syndrome group (OHSS), and ovarian hyperstimulation syndrome + melatonin group (OHSS + melatonin). OHSS was induced in the OHSS and OHSS + melatonin groups by pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) injections, followed by melatonin treatment administered intraperitoneally at a dose of 50 mg/kg only in the OHSS + melatonin group. In the OHSS group, ovarian weight increased and the number of atretic follicles also rose, while melatonin treatment improved these conditions. Histological analysis showed that melatonin preserved ovarian structure and supported follicular development. Serum estradiol levels were significantly higher in the OHSS group compared with the control group, but melatonin treatment reduced these levels significantly. Peritoneal fluid levels of vascular endothelial growth factor (VEGF) and interleukin (IL)-6 were elevated in the OHSS group, but melatonin treatment decreased these levels. Additionally, melatonin reduced follicular atresia and increased the number of Graafian follicles. In conclusion, melatonin improved the biochemical and histological markers of OHSS, providing protective effects on ovarian function. These findings suggest that melatonin could be a potential therapeutic agent for the treatment of OHSS. However, further clinical and experimental studies are needed to investigate its effects.

Chronic wounds present a major clinical challenge due to impaired healing and prolonged inflammation. This present study aimed to develop and assess a platelet-rich plasma (PRP)-loaded decellularized skin-derived matrix scaffold (SDMP) for enhanced wound healing. A total of 40 male Sprague-Dawley rats were randomly planned to four groups (n = 10 per group): untreated control, PRP, decellularized skin-derived matrix (SDM), and PRP-loaded SDM (SDMP). Full-thickness excisional wounds were created on the dorsal surface of each animal and treatments were applied accordingly. Tissue sampling was performed at two time points-day 7 and day 14 post-injury-with five animals per group euthanized at each time point. Histological evaluations included assessment of newly formed epidermal length, dermal thickness, and collagen density. Biomechanical properties of the regenerated skin were analyzed on day 14 using tensile strength testing. In addition, concentrations of key regenerative (TGF-β1, VEGF) and pro-inflammatory (TNF-α, IL-1β) cytokines in wound tissues were quantified via ELISA. The SDMP-treated group showed significantly enhanced wound closure, improved re-epithelialization and dermal regeneration, greater collagen deposition, and superior tensile strength compared with other groups (p < 0.05). Moreover, cytokine analysis revealed a favorable shift in the wound microenvironment characterized by elevated growth factors and reduced inflammatory mediators (p < 0.05). In conclusion, the PRP-loaded SDMs provides a bioactive and biocompatible platform that significantly improves full-thickness skin diabetic wound healing. This approach holds promise for future translational applications in regenerative medicine.

Neuroinflammation is recognized as a key mechanism underlying depression, with glial cells playing a central role in regulating neuronal activity and neuroimmune interactions. However, how microglia and astrocytes in distinct brain regions respond morphologically to peripheral inflammatory stimulation and how these changes contribute to depression remain poorly understood. Here, we established a lipopolysaccharide (LPS)-induced mouse model of inflammation-related depression and observed a significant increase in c-Fos expression in emotion- and stress-related brain regions, including the bed nucleus of the stria terminalis (BST), the paraventricular nucleus of hypothalamus (PVN), the ventrolateral periaqueductal gray (vlPAG), the locus coeruleus (LC) and the solitary nucleus (Sol). Using three-dimensional (3D) reconstruction and Sholl analysis, we quantified the process complexity, spatial coverage and filamentous architecture of both microglia and astrocytes. Microglia showed hypertrophy across all examined regions. BST and PVN exhibited thicker and straighter processes, LC and vlPAG displayed decreased spatial complexity, and Sol exhibited reactive hypertrophy characterized by increased filament volume and maximal intersections. Astrocytes generally exhibited reduced filament length, process diameter, or structural simplification in the BST, PVN, LC and vlPAG, whereas Sol astrocytes displayed increased process diameter but reduced filament length, area and maximal radius. Together, these findings provide a structural basis for understanding the cellular mechanisms underlying inflammation-related depression across different brain regions and suggest potential functional roles of glial remodeling in inflammatory depression.

The primary objective of advancements in stem cell biology for reproductive medicine has been the production of artificial gametes from multipotent stem cells. We examined the efficacy of upregulating germline stem cell markers to convert mouse bone marrow mesenchymal stem cells (mBMMSCs) into late-stage germ-like cells (GLCs). The multipotent differentiation potential of mBMMSCs was investigated using oil red-O and alizarin red-S staining. The differentiation of mBMMSCs into GLCs was also investigated in relation to the effects of high concentrations of retinoic acid, ultraviolet (UV) light, and titanium nanotubes (TNTs) coated with fibrin (F). The biocompatibility and morphology of TNT, as well as the characteristics of F+TNT, were investigated through the use of MTT and scanning electron microscopy (SEM) experiments. After 14 days, the optimal TNT concentration for differentiation was 50 µg/mL. The TNT and F+TNT morphologies were verified using SEM and Raman spectra, respectively. The integrity of the cells in fibrin and the expression of the male and female germline stem cell markers Mvh/Ddx4, Dazl, and Plzf were assessed by immunofluorescence analysis, western blots, and real-time quantitative polymerase chain reaction (RT-qPCR) following multipotent mBMMSC culture in retinoic acid (RA) and F+TNT formation with RA and UV radiation, respectively. We demonstrate that a suitable two-dimensional (2D) scaffold can be obtained for germ-like cells derived from mBMMSCs through the use of F+TNT, UV radiation, and assisted reproductive technology (ART) in vitro maturation (IVM).

Diabetic wounds pose significant clinical challenges owing to delayed healing associated with chronic inflammation, impaired angiogenesis, and poor extracellular matrix (ECM) remodeling. Bioengineered scaffolds incorporating natural bioactives offer promising strategies for enhancing skin regeneration. In this study, the author developed and evaluated a collagen-hyaluronic acid (Col-HA) scaffold loaded with epigallocatechin gallate (EGCG), a polyphenolic compound known for its antioxidant, anti-inflammatory, and proangiogenic properties. EGCG was incorporated into porous Col-HA scaffolds, and their physicochemical properties, degradation rate, and drug release profile were characterized. In vitro cell viability assays were performed using mesenchymal stem cells to assess biocompatibility. A full-thickness excisional wound model was established in streptozotocin-induced diabetic rats, which were treated with control (no scaffold), Col-HA scaffold, EGCG alone, or Col-HA + EGCG scaffolds. Wound healing was evaluated on days 7 and 14 via macroscopic closure, histological stereology (epidermal/dermal volume and fibroblast and vascular density), cytokine profiling (transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α), hydroxyproline quantification, and tensile strength testing. The EGCG-loaded Col-HA scaffold exhibited a porous microstructure (~ 120 µm pore size) and a biphasic release profile, with sustained EGCG release up to 14 days. In vivo, the Col-HA + EGCG group demonstrated significantly accelerated wound closure compared with other groups (p < 0.05). Histological analysis revealed enhanced regeneration of epidermis and dermis, increased fibroblast proliferation and angiogenesis, and reduced inflammatory cell infiltration. ELISA results showed upregulated TGF-β and VEGF levels and downregulated IL-1β and TNF-α in the Col-HA + EGCG group. Moreover, collagen content and tensile strength were highest in this group, indicating superior ECM remodeling and mechanical restoration. The multifunctional Col-HA scaffold incorporated with EGCG effectively promotes diabetic wound healing by modulating inflammation, enhancing angiogenesis, and supporting tissue regeneration. This combinatorial strategy holds significant potential for advanced wound care therapies.

Actin is a pivotal cytoskeletal protein that also regulates chromatin remodeling, transcription, and RNA processing within the nucleus. These nuclear functions are regulated by post-translational modifications (PTMs), but the roles of specific PTMs of nuclear actin remain poorly understood. Of these, the O-GlcNAcylation of Ser199 (gS199) is of particular interest, because this residue can also be phosphorylated (pS199) and is adjacent to the Thr201-203 cluster, a known promoter of filament elongation. In this study, we aimed to elucidate the role of Ser199 O-GlcNAcylation in nuclear actin organization and function. We demonstrate that O-GlcNAcylation at Ser199 actin is associated with actin localization to nuclear speckles and suppresses filament formation. In vivo and in vitro assays revealed that gS199- and pS199-actin have a punctate distribution within the nucleus and colocalize with the speckle marker SRSF2 (SC35). Immunoelectron microscopy showed that this localization was markedly enhanced under diabetic conditions. Furthermore, the introduction of an anti-gS199-actin antibody induced nuclear filament formation, directly linking Ser199 O-GlcNAcylation to the inhibition of actin polymerization. Immunoprecipitation and mass spectrometry identified glyceraldehyde 3-phosphate dehydrogenase and histone H1.4 as nuclear binding partners of modified Ser199-actin. These findings suggest a mechanism by which Ser199 O-GlcNAcylation restricts actin polymerization, anchors actin to nuclear speckles, and thereby influences RNA processing. Dysregulation of this pathway in diabetes may destabilize nuclear speckle organization and contribute to the transcriptional defects that underlie diabetic complications.

Histopathological diagnosis relies on careful and expert assessment of tissue as guided by multiple criteria relevant to specific immunohistochemical (IHC) markers. Computer-aided detection or diagnosis systems have recently been deployed to detect abnormalities in histological samples, transforming many areas of research and medicine such as pathology. These software packages can provide a helpful decision support tool for accelerating analysis, but they would need to capture information from the sample in a manner that facilitates the multicriteria assessment/interpretation demanded by the IHC markers and other histochemical stains. As a result of this potential, and the limited assessment of the performance of software utilized for automated analysis of histological samples, we conducted this study. We aimed to provide a technical assessment of two analysis approaches that are utilized in two commercially available image analysis software platforms, namely positive pixel count analysis approach and cell-by-cell analysis approach. These two approaches are used in many digital histopathological slide analysis software packages including ImageScope (Leica Biosystems) and HALO (Indica Labs), which respectively deploy the aforementioned algorithms and thus were used as proxies for the comparison in this study. Thirty-seven whole slide images of immunohistochemically stained tumor samples from breast, colon, and endometrium were analyzed using three different sampling methods recording percentage of antibody marker positivity. The pixel-based software was better able to identify color intensity, offering the option for grading the IHC marker. However, the object-based software outperformed pixel-based software, having more consistent positivity estimates across the three sampling methods. These results are limited by the small number of clinical samples, IHC marker heterogeneity, and the lack of ground-truth data. Nonetheless, neither of the software packages' metrics performed in a manner required for comprehensive assessment of the IHC markers in this study, yet they can be used to address specific questions related to quantitative expression of tumor diagnostic markers.

Fetal sex influences gene expression in the healthy feto-placental endothelium, potentially contributing to sex-dependent developmental programming and disease risk. Gestational diabetes mellitus (GDM) alters maternal-fetal homeostasis and placental vascular function. Building on previous findings of sex-biased gene expression in healthy feto-placental endothelial cells (fpEC), we investigated whether these biases persist or change following GDM exposure. We first identified sex-biased gene expression in fpEC from GDM pregnancies, then analyzed GDM-induced changes separately in male and female fpEC. Gene ontology enrichment was performed using the PANTHER database. Proliferation and network formation were assessed by BrdU incorporation assay and Matrigel assay, respectively. Female fpEC exhibited a greater transcriptional response to GDM, with more differentially expressed genes than male cells. Functionally, GDM reduced proliferation and increased network formation in female fpEC, while male cells were comparatively unaltered. In healthy conditions, male and female fpEC showed clear transcriptomic and functional dimorphism, which was abolished by GDM. Interestingly, GDM amplified sex-biased gene expression despite convergence in cellular behavior. These findings highlight fetal sex as a key modifier of the placental endothelial response to GDM and support its relevance in sex-specific pregnancy outcomes.