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Coupling imaging mass cytometry with Alcian blue histochemical staining for a single-slide approach.Frontiers in Immunology 2024Imaging mass cytometry (IMC) is a metal mass spectrometry-based method allowing highly multiplex immunophenotyping of cells within tissue samples. However, some...
Imaging mass cytometry (IMC) is a metal mass spectrometry-based method allowing highly multiplex immunophenotyping of cells within tissue samples. However, some limitations of IMC are its 1-µm resolution and its time and costs of analysis limiting respectively the detailed histopathological analysis of IMC-produced images and its application to small selected tissue regions of interest (ROI) of one to few square millimeters. Coupling on a single-tissue section, IMC and histopathological analyses could permit a better selection of the ROI for IMC analysis as well as co-analysis of immunophenotyping and histopathological data until the single-cell level. The development of this method is the aim of the present study in which we point to the feasibility of applying the IMC process to tissue sections previously Alcian blue-stained and digitalized before IMC tissue destructive analyses. This method could help to improve the process of IMC in terms of ROI selection, time of analysis, and the confrontation between histopathological and immunophenotypic data of cells.
Topics: Staining and Labeling; Immunophenotyping; Image Cytometry; Humans; Mass Spectrometry; Animals; Single-Cell Analysis
PubMed: 38742102
DOI: 10.3389/fimmu.2024.1379154 -
Biomedicine & Pharmacotherapy =... Jun 2024Vitamin K2 (VK2) is an effective compound for anti-ferroptosis and anti-osteoporosis, and Semen sojae praeparatum (Dandouchi in Chinese) is the main source of VK2....
Vitamin K2 (VK2) is an effective compound for anti-ferroptosis and anti-osteoporosis, and Semen sojae praeparatum (Dandouchi in Chinese) is the main source of VK2. Chondrocyte ferroptosis and extracellular matrix (ECM) degradation playing a role in the pathogenesis of osteoarthritis (OA). Glutathione peroxidase 4 (GPX4) is the intersection of two mechanisms in regulating OA progression. But no studies have elucidated the therapeutic effects and mechanisms of VK2 on OA. This study utilized an in vivo rat OA model created via anterior cruciate ligament transection (ACLT) and an in vitro chondrocyte oxidative damage model induced by TBHP to investigate the protective effects and mechanisms of action of VK2 in OA. Knee joint pain in mice was evaluated using the Von Frey test. Micro-CT and Safranin O-Fast Green staining were employed to observe the extent of damage to the tibial cartilage and subchondral bone, while immunohistochemistry and PCR were used to examine GPX4 levels in joint cartilage. The effects of VK2 on rat chondrocyte viability were assessed using CCK-8 and flow cytometry assays, and chondrocyte morphology was observed with toluidine blue and alcian blue staining. The impact of VK2 on intracellular ferroptosis-related markers was observed using fluorescent staining and flow cytometry. Protein expression changes were detected by immunofluorescence and Western blot analysis. Furthermore, specific protein inhibitors were applied to confirm the dual-regulatory effects of VK2 on GPX4. VK2 can increase bone mass and cartilage thickness in the subchondral bone of the tibia, and reduce pain and the OARSI score induced by OA. Immunohistochemistry results indicate that VK2 exerts its anti-OA effects by regulating GPX4 to delay ECM degradation. VK2 can inhibit the activation of the MAPK/NFκB signaling pathway caused by reduced expression of intracellular GPX4, thereby decreasing ECM degradation. Additionally, VK2 can reverse the inhibitory effect of RSL3 on GPX4, increase intracellular GSH content and the GSH/GSSG ratio, reduce MDA content, and rescue chondrocyte ferroptosis. The protective mechanism of VK2 may involve its dual-target regulation of GPX4, reducing chondrocyte ferroptosis and inhibiting the MAPK/NFκB signaling pathway to decelerate the degradation of the chondrocyte extracellular matrix.
Topics: Animals; Ferroptosis; Extracellular Matrix; Chondrocytes; Male; Osteoarthritis; Rats, Sprague-Dawley; Rats; Phospholipid Hydroperoxide Glutathione Peroxidase; Mice; Vitamin K 2; Mice, Inbred C57BL; Cartilage, Articular; Disease Models, Animal; Signal Transduction; Cells, Cultured
PubMed: 38759289
DOI: 10.1016/j.biopha.2024.116697 -
Diabetes, Metabolic Syndrome and... 2024Obesity is a growing global problem that causes various complications such as diabetes, cognitive dysfunction, cardiovascular diseases, and hepatobiliary disease....
BACKGROUND
Obesity is a growing global problem that causes various complications such as diabetes, cognitive dysfunction, cardiovascular diseases, and hepatobiliary disease. Alpha-linolenic acid (ALA) has been reported to exhibit multiple pharmaceutical effects. This study aimed to explore the effects of ALA on obesity-induced adipose tissue accumulation, cognitive impairment, inflammation, and colonic mucosal barrier integrity.
METHODS
Mice were fed with high-fat diet (HFD) and were treated with ALA (60 or 100 mg/kg). Body weight, adipose tissue, serum glucose and lipid levels, glucose resistance, and insulin resistance were measured. Cognitive ability was analyzed using the behavior tests. PTP1B and IRS/p-AKT/p-GSK3β/p-Tau signaling were examined to evaluate inflammation and synaptogenesis. Colon mucosal barrier integrity was examined by Alcian blue staining and expression of the tight junction proteins. The production of pro-inflammatory cytokines and liver damages were evaluated. 3T3-L1 cells were used for in vitro experiments. Cell viability, migration and invasion were detected. The levels of ROS, iron, and ferrous ions were measured to assess ferroptosis. Metabolomic analysis of adipose tissues was performed.
RESULTS
ALA treatment prevented HFD-induced adipose tissue accumulation, improved glucose and lipid homeostasis and metabolism. Administration of ALA repressed the HFD-induced increase in insulin levels and insulin resistance index. Serum and colon levels of pro-inflammatory cytokines were decreased after ALA treatment. ALA elevated mitochondrial content in brown adipose tissues. ALA ameliorated obesity-induced cognitive impairment and hippocampal inflammation, enhanced colon mucosa integrity. ALA treatment ameliorated HFD-induced liver damage and lipid accumulation and inhibited differentiation of preadipocyte 3T3-L1 cells into mature adipocytes and induces ferroptosis. Metabolomic analysis suggested that ALA may target the glycerolipid metabolism pathway to ameliorate obesity. Knockdown of AGPAT2 abolished the protective effects of ALA.
CONCLUSION
ALA treatment suppressed adipose accumulation in adipocytes, improved cognitive ability and colon integrity, and alleviated liver damage by modulating the 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2).
PubMed: 38435630
DOI: 10.2147/DMSO.S434671 -
Journal of Cytology 2024In endometrial cytology, differentiating endometrial glandular stromal breakdown (EGBD) from endometrial endometrioid carcinoma (G1-EEC) is often difficult. In this...
Staining Pattern of Alcian Blue in Endometrial Cytology: Utility in Distinguishing Grade 1-Endometrial Endometrioid Carcinoma from Endometrial Glandular Stromal Breakdown.
BACKGROUND AND OBJECTIVE
In endometrial cytology, differentiating endometrial glandular stromal breakdown (EGBD) from endometrial endometrioid carcinoma (G1-EEC) is often difficult. In this study, we provided a new focus on chondroitin sulfate (CS), a major substrate component of the endometrial stroma, and assessed the diagnostic utility of Alcian Blue (AB) staining in the differential diagnosis in liquid-based cytological (LBC) samples.
MATERIALS AND METHODS
LBC specimens from 19 patients with a proliferative endometrium, 36 with EGBD, and 30 with G1-EEC who underwent endometrial cytology were stained with AB (pH 1.0), and their reactivity was observed. In addition, immunocytochemical staining of CS and CD31 was performed for five cases each to evaluate their interrelationship with blood vessels.
RESULTS
Regarding the 30 G1-EEC cases, at least one of the three representative staining patterns was observed by AB staining: dot-like, microtubular, and finely branched linear patterns. Moreover, the inner portion of the tubular material observed by AB staining expressed CD31. Conversely, in the 36 EGBD cases, only five metaplastic clusters with irregular protrusions and condensed stromal clusters (CSCs) showed a dot-like positive pattern, and background CSCs did not show reactivity to AB staining in any of the cases. Furthermore, the vascular structure expressing CD31 in cell clusters was also unclear.
CONCLUSIONS
We demonstrated that AB staining shows different staining patterns in G1-EEC and EGBD, reflecting their different tissue structures. Our data provide new insights into endometrial cell diagnosis changes and demonstrate that AB staining is a potential new diagnostic aid tool for the differentiation of G1-EEC from EGBD.
PubMed: 38779603
DOI: 10.4103/joc.joc_121_23 -
Frontiers in Cell and Developmental... 2023Neural crest cells constitute a distinct set of multipotent cells that undergo migration along predefined pathways, culmination in the differentiation into a plethora...
Neural crest cells constitute a distinct set of multipotent cells that undergo migration along predefined pathways, culmination in the differentiation into a plethora of cell types, including components of the pharyngeal cartilage. The neurocranium is composite structure derived from both cranial neural crest and mesoderm cells, whereas the pharyngeal skeletal elements-including the mandibular and branchial arches-are exclusively formed by craniofacial neural crest cells. Previous studies have elucidated the critical involvement of the chemokine signaling axis Cxcl12b/Cxcr4a in craniofacial development in zebrafish (). Nonetheless, the function contribution of Cxcl12a and Cxcr4b-the homologous counterparts of Cxcl12b and Cxcr4a-remain largely unexplored. In the present study, mutant lines for and were generated employing CRISPR/Cas9 system. Temporal and spatial expression patterns of specific genes were assessed using hybridization and dual-color fluorescence hybridization techniques. High-resolution confocal microscopy was utilized for imaging to detect the pharyngeal arch or pouch patterning. Additionally, cartilage formation within the craniofacial region was analyzed via Alcian blue staining, and the proliferation and apoptosis rates of craniofacial neural crest cells were quantified through BrdU incorporation and TUNEL staining. Our data reveals that the deletion of the chemokine gene results in a marked diminution of pharyngeal cartilage elements, attributable to compromised proliferation of post-migratory craniofacial neural crest cells. Subsequent experiments confirmed that Cxcl12a and Cxcl12b exhibit a synergistic influence on pharyngeal arch and pouch formation. Collectively, the present investigation furnishes compelling empirical evidence supporting the indispensable role of Cxcl2a in craniofacial cartilage morphogenesis, albeit mutants exert a minimal impact on this biological process. We delineate that Cxcl12a is essential for chondrogenesis in zebrafish, primarily by promoting the proliferation of craniofacial neural crest cells. Furthermore, we proposed a conceptual framework wherein Cxcl12a and Cxcl12b function synergistically in orchestrating both the pharyngeal arch and pouch morphogenesis.
PubMed: 37860819
DOI: 10.3389/fcell.2023.1243265 -
International Journal of Molecular... Dec 2023Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid‑β (Aβ) in the brain. The gut/brain axis may serve a role in AD...
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid‑β (Aβ) in the brain. The gut/brain axis may serve a role in AD pathogenesis. The present study investigated deposition of Aβ in the intestinal epithelium and its potential effects on intestinal barrier function in a transgenic mouse model of AD. To investigate alterations in the structure and functionality of the intestinal mucosal barrier in AD model mice, hematoxylin and eosin staining for Paneth cell count, Alcian blue‑periodic acid Schiff staining for goblet cells, immunohistochemistry and immunofluorescence for mucin (MUC)2 and wheat germ agglutin expression, transmission electron microscopy for mucosal ultrastructure, FITC‑labeled dextran assay for intestinal permeability, quantitative PCR for goblet cell precursor expression and western blot analysis for tight junction proteins, MUC2 and inflammatory cytokine detection were performed. The results showed that AD model mice exhibited excessive Aβ deposition in the intestinal epithelium, which was accompanied by increased intestinal permeability, inflammatory changes and decreased expression of tight junction proteins. These alterations in the intestinal barrier led to an increased proliferation of goblet and Paneth cells and increased mucus synthesis. Dysfunction of gut barrier occurs in AD and may contribute to its etiology. Future therapeutic strategies to reverse AD pathology may involve early manipulation of gut physiology and its microbiota.
Topics: Mice; Animals; Mucins; Alzheimer Disease; Tight Junctions; Intestinal Mucosa; Mice, Transgenic; Permeability; Tight Junction Proteins
PubMed: 37830152
DOI: 10.3892/ijmm.2023.5316 -
Clinical Oral Investigations Dec 2023Salivary proteins, acidic glycoproteins, and free calcium might take part in oral mucosal defence against inflammation in oral lichen planus (OLP). The study aimed to...
OBJECTIVES
Salivary proteins, acidic glycoproteins, and free calcium might take part in oral mucosal defence against inflammation in oral lichen planus (OLP). The study aimed to investigate whether the levels of sulfated and sialylated glycoproteins, total protein, and free calcium in saliva from patients with OLP differ from those of individuals without oral mucosal diseases.
MATERIAL AND METHODS
Patients diagnosed with OLP (n = 25) and two control groups without any oral mucosal disease; age- and gender-matched controls (n = 25, 65.6 ± 2.9 years), and younger controls (n = 25, 41.8 ± 2.5 years) were included. Subjective dry mouth (xerostomia) was assessed by asking a single-item question. Chew-stimulated whole saliva was collected to measure sulfated and sialylated glycoproteins by the Alcian Blue method. The total protein was determined spectrophotometrically, and the free calcium measured using an electrode.
RESULTS
The output of salivary sulfated and sialylated glycoproteins in the OLP group (21.8 ± 2.4 µg/min) was lower than in the age- and gender-matched controls (43.0 ± 2.9 µg/min, p = 0.0002), whereas the total protein and calcium output did not differ between the three groups (p > 0.05). The prevalence of xerostomia was significantly higher in the OLP group compared to both control groups (p = 0.038).
CONCLUSIONS
Patients with OLP showed a high prevalence of xerostomia and lower levels of salivary acidic type glycoproteins compared to the individuals without oral mucosa disease.
CLINICAL RELEVANCE
It is relevant to investigate the role of acidic glycoproteins in the pathogenesis of OLP.
Topics: Humans; Lichen Planus, Oral; Calcium; Saliva; Xerostomia; Glycoproteins; Case-Control Studies
PubMed: 38114810
DOI: 10.1007/s00784-023-05411-6 -
JOR Spine Mar 2024Intradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile...
BACKGROUND
Intradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ-MSC)-derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate-bead culture model.
METHODS
After EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 μg/mL WJ-MSC-EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9-dimethylmethylene blue assays. hNPCs in alginate beads were paraffin-embedded and stained for histological analysis (hematoxylin-eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (), anabolic (), and hNPC marker () genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis.
RESULTS
Treatment with WJ-MSC-EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment.
CONCLUSIONS
WJ-MSC-derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.
PubMed: 38222813
DOI: 10.1002/jsp2.1274 -
Journal of Orthopaedic Translation Sep 2023Dedifferentiated fat cells (DFATs) are highly homogeneous and multipotent compared with adipose-derived stromal cells (SCs). Infrapatellar fat pad (IFP)-SCs have...
BACKGROUND
Dedifferentiated fat cells (DFATs) are highly homogeneous and multipotent compared with adipose-derived stromal cells (SCs). Infrapatellar fat pad (IFP)-SCs have advanced chondrogenic potency; however, whether IFP-DFATs could serve as better cell material remains unclear. Here, we aimed to examine the influence of age and body mass index (BMI) on the features of IFPs and IFP-derived cells (IFP-SCs and IFP-DFATs) with exploration of the clinical utilization of IFP-DFATs.
METHODS
We collected IFPs with isolation of paired IFP-SCs and IFP-DFATs from individuals aged 65 years and older with distinct body weights who underwent total knee replacement for osteoarthritis (OA). Flow cytometry was used to characterize the cellular immunophenotypes. Adipogenesis and chondrogenesis were performed . Real-time qPCR, western blotting, and Oil Red O or Alcian blue staining were performed to evaluate inflammation, adipogenesis, and chondrogenesis. RNA sequencing and Seahorse analyses were conducted to explore the underlying mechanisms.
RESULTS
We found that IFPs from old or normal-weight individuals with knee OA were pro-inflammatory, and that interleukin-6 (IL-6) signaling was associated with multiple immune-related molecules, whereas IFP-derived cells could escape the inflammatory properties. Aging plays an important role in diminishing the chondrogenic and adipogenic abilities of IFP-SCs; however, this effect was avoided in IFP-DFATs. Generally, IFP-DFATs presented a steady state of chondrogenesis (less influenced by age) and consistently enhanced adipogenesis compared to paired IFP-SCs in different age or BMI groups. RNA sequencing and Seahorse analysis suggested that the downregulation of eukaryotic initiation factor 2 (EIF2) signaling and enhanced mitochondrial function may contribute to the improved cellular biology of IFP-DFATs.
CONCLUSIONS
Our data indicate that IFP-DFATs are superior cell material compared to IFP-SCs for cartilage differentiation and adipogenesis, particularly in advanced aging patients with knee OA.
THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE
These results provide a novel concept and supportive evidence for the use of IFP-DFATs for cell therapy or tissue engineering in patients with knee OA. Using Ingenuity Pathway Analysis (IPA) of RNA-seq data and Seahorse analysis of mitochondrial metabolic parameters, we highlighted that some molecules, signaling pathways, and mitochondrial functions are likely to be jointly coordinated to determine the enhanced biological function in IFP-DFATs.
PubMed: 37680904
DOI: 10.1016/j.jot.2023.08.001 -
International Journal of Fertility &... Nov 2023Men's infertility and lack of production of healthy and active sperm are concerns of recent years in most countries. Studies on the preparation of extracellular matrix...
BACKGROUND
Men's infertility and lack of production of healthy and active sperm are concerns of recent years in most countries. Studies on the preparation of extracellular matrix (ECM) from decellularization of testis tissue and spermatogenesis could provide proper results to solve some of the men's infertility problems. This study aims to decellularize calf testis by different methods to reach a suitable scaffold and introduce it in spermatogenesis studies.
MATERIALS AND METHODS
In this experimental study, calf testis were decellularized by a freeze-de freeze, 1% sodium deoxycholate (SD), 0.1% sodium dodecyl sulfate (SDS), 0.1% SDS-vacuum, 1% SDS, 1% SDS-vacuum, and Triton- X100 methods. The content of DNA, collagen, and glycosaminoglycan (GAG) was analyzed using the kit and staining with Hematoxylin-Eosin, Masson's trichrome, Alcian blue, and Orcein methods. The morphology of the scaffolds was analyzed with a scanning electron microscope (SEM).
RESULTS
Methods of 1% SDS, 1% SDS-vacuum, and 1% SD completely removed the cells. The preservation of collagen and GAG was confirmed using the staining kit and methods. The use of a vacuum showed greater porosity in the SEM images. Toxicity and hemolysis were not observed in the scaffolds.
CONCLUSION
Testis decellularization with 1% SDS and 1% SD, in addition to cell removal, could maintain the ECM structure to a large extent without having cytotoxic and hemolysis effects.
PubMed: 38041457
DOI: 10.22074/ijfs.2023.1989173.1433