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Veterinary Immunology and... May 2024Intestinal mucus barrier disruption may occur with chronic inflammatory enteropathies. The lack of studies evaluating mucus health in dogs with chronic colitis arises...
Intestinal mucus barrier disruption may occur with chronic inflammatory enteropathies. The lack of studies evaluating mucus health in dogs with chronic colitis arises from inherent challenges with assessment of the intestinal mucus layer. It is therefore unknown if reduced goblet cell (GBC) numbers and/or mucin 2 (MUC2) expression, which are responsible for mucus production and secretion, correlate with inflammation severity in dogs with granulomatous colitis (GC) or lymphocytic-plasmacytic colitis (LPC). It is undetermined if Ki-67 immunoreactivity, which has been evaluated in dogs with small intestinal inflammation, similarly correlates to histologic severity in GC and LPC. Study objectives included comparing Ki-67 immunoreactivity, GBC population and MUC2 expression in dogs with GC, LPC and non-inflamed colon; and exploring the use of ribonucleic acid (RNAscope®) in-situ hybridization (ISH) to evaluate MUC2 expression in canine colon. Formalin-fixed endoscopic colonic biopsies were obtained from 48 dogs over an eight-year period. A blinded pathologist reviewed all biopsies. Dogs were classified into the GC (n=19), LPC (n=19) or no colitis (NC) (n=10) group based on final histopathological diagnosis. Ki-67 immunohistochemistry, Alcian-Blue/PAS staining to highlight GBCs, and RNAscope® ISH using customized canine MUC2-targeted probes were performed. At least five microscopic fields per dog were selected to measure Ki-67 labelling index (KI67%), GBC staining percentage (GBC%) and MUC2 expression (MUC2%) using image analysis software. Spearman's correlation coefficients were used to determine associations between World Small Animal Veterinary Association histologic score (WHS) and measured variables. Linear regression models were used to compare relationships between WHS with KI67%, GBC%, and MUC2%; and between GBC% and MUC2%. Median WHS was highest in dogs with GC. Median KI67% normalised to WHS was highest in the NC group (6.69%; range, 1.70-23.60%). Median GBC% did not correlate with colonic inflammation overall. Median MUC2% normalised to WHS in the NC group (10.02%; range, 3.05-39.09%) was two- and three-fold higher than in the GC and LPC groups respectively. With increased colonic inflammation, despite minimal changes in GBC% overall, MUC2 expression markedly declined in the LPC group (-27.4%; 95%-CI, -49.8, 5.9%) and mildly declined in the GC and NC groups. Granulomatous colitis and LPC likely involve different pathways regulating MUC2 expression. Decreased MUC2 gene expression is observed in dogs with chronic colitis compared to dogs without colonic signs. Changes in MUC2 expression appear influenced by GBC activity rather than quantity in GC and LPC.
Topics: Animals; Dogs; Mucin-2; Goblet Cells; Ki-67 Antigen; Dog Diseases; Colitis; Female; Male; Colon; Granuloma; Immunohistochemistry
PubMed: 38537313
DOI: 10.1016/j.vetimm.2024.110740 -
Biology Feb 2024The oral cavity serves as the primary path through which substances from the outside world enter our body. Therefore, it functions as a critical component of host...
The oral cavity serves as the primary path through which substances from the outside world enter our body. Therefore, it functions as a critical component of host defense. Saliva is essential for maintaining a stable oral environment by catching harmful agents, including pathogens, allergens, and chemicals, in the air or food. CCL28, highly expressed in mucosal tissues, such as the colon and salivary glands, is a chemokine that attracts CCR10/CCR3 expressing cells. However, the role of CCL28 in salivary gland formation remains unclear. In this study, we investigated the salivary gland structure in CCL28-deficient mice. Histological analysis showed decreased staining intensity of Alcian blue, which detects acidic mucous, reduced expression of MUC2, and higher infiltration of gram-positive bacteria in the salivary glands of CCL28-deficient mice. In addition, CCL28-deficient mice contained ectopically MUC2-expressed cells in the ducts and reduced the expression of cytokeratin 18, a marker for ductal cells, within the submandibular glands, resulting in decreased duct numbers. Additionally, the submandibular glands of CCL28-deficient mice showed reduced expression of several stem cell markers. These results suggest that CCL28 regulates saliva production via proper differentiation of salivary gland stem cells and could be a valuable biomarker of salivary gland function.
PubMed: 38534417
DOI: 10.3390/biology13030147 -
Microscopy Research and Technique Aug 2024Aquaporins (AQPs) are important for water transport in the gastrointestinal tract. Changes in their expression and/or localization could cause in disorders and be used...
Aquaporins (AQPs) are important for water transport in the gastrointestinal tract. Changes in their expression and/or localization could cause in disorders and be used as therapeutic targets. Aquaporin-4 (AQP4) is expressed predominantly on the basolateral membrane of the parietal cells in the corpus of the murine gastric glands. Although the secretion of gastric juice is not affected in AQP4-deficient knockout, we evaluated by light microscopy whether the lack of AQP4 affects the glycopatterns of secreting gastric cells. Wild type (WT) and AQP4-deficient knockout mice (KO) were fed a standard diet ad libitum before sacrifice. Segments of stomach corpus were collected, fixed in buffered formalin, and embedded in paraffin wax. Sections, 5-μm thick, were analyzed by histochemical methods (Periodic acid-Schiff, Alcian Blue pH 2.5), and binding of lectins specific to GalNAc (SBA, DBA), Gal (PNA) GlcNAc (WGA, GSAII) mannose and/or glucose (ConA), and fucose (UEA-I, AAA, LTA). Immunohistochemical methods such as anti-Muc6 for neck cells and anti- β- H/K-ATPase for parietal cells were also performed. Compared to WT mice, in the mucous cells of KO lower amounts of glycans with galactosyl/galactosaminylated, glycosyl/glycosaminylated, and fucosylated residues were observed; lower fucosylation resulted also in the parietal cells. The observed differences of KO in respect to WT could lead to severer pathological conditions. RESEARCH HIGHLIGHTS: Glycopatterns in gastric glands were compared between wild type (WT) and AQP4-deficient knockout (KO) mice by histochemical and lectin-binding methods. In the mucous cells of KO lower amounts of glycans with galactosyl/galactosaminylated, glycosyl/glycosaminylated and fucosylated residues were observed. In the parietal cells lower fucosylation also resulted. AQP4-deficiency affects glycosylation and could result in altered functionality and pathological conditions.
Topics: Animals; Mice, Knockout; Glycosylation; Mice; Aquaporin 4; Gastric Mucosa; Parietal Cells, Gastric; Immunohistochemistry; H(+)-K(+)-Exchanging ATPase; Male; Lectins; Polysaccharides
PubMed: 38533927
DOI: 10.1002/jemt.24563 -
The American Journal of Chinese Medicine 2024Aging can cause degenerative changes in multiple tissues and organs. Gastrointestinal diseases and dysfunctions are common in the elderly population. In this study, we...
Aging can cause degenerative changes in multiple tissues and organs. Gastrointestinal diseases and dysfunctions are common in the elderly population. In this study, we investigated the effects of polysaccharide (APS) and ethanol extract (AEE) on age-related intestinal dysfunction and gut microbiota dysbiosis in naturally aging mice. The energy expenditure and physical activity of 23-month-old C57BL6/J mice were recorded using a metabolic cage system. Pathological changes in the intestine were evaluated using Alcian blue staining. The protein levels of leucine-rich repeats containing G protein-coupled receptor 5 (Lgr5) and Stat3 in the small intestine were determined using immunohistochemistry. The intestinal cell migration distance was assessed using bromodeoxyuridine (BrdU) immunofluorescence staining. The gene transcription levels of intestinal stem cell (ISC) markers and ISC-related signaling pathways were detected using quantitative real-time PCR (qRT-PCR). Microbiota analysis based on 16S rDNA was performed to evaluate the composition of the gut microbiota. APS and AEE improved a series of aging phenotypes in female but not in male aging mice. APS and AEE ameliorate intestinal dysfunction and histopathological changes in aging mice. APS had a more significant anti-aging effect than AEE, particularly on intestinal dysfunction. APS promotes ISC regeneration by activating the IL-22 signaling pathway. Cohousing (CH) experiments further confirmed that APS induced the IL-22 signaling pathway by increasing the abundance of , thereby promoting the regeneration of ISCs. Our results show that APS may serve as a promising agent for improving age-related intestinal dysfunction.
Topics: Aged; Humans; Mice; Male; Female; Animals; Infant; Child, Preschool; Astragalus propinquus; Interleukin-22; Intestines; Signal Transduction; Intestine, Small; Stem Cells; Polysaccharides; Aging; Regeneration
PubMed: 38533568
DOI: 10.1142/S0192415X24500228 -
Scientific Reports Mar 2024Mucin protein glycosylation is important in determining biological properties of mucus gels, which form protective barriers at mucosal surfaces of the body such as the...
Mucin protein glycosylation is important in determining biological properties of mucus gels, which form protective barriers at mucosal surfaces of the body such as the intestine. Ecological factors including: age, sex, and diet can change mucus barrier properties by modulating mucin glycosylation. However, as our understanding stems from controlled laboratory studies in house mice, the combined influence of ecological factors on mucin glycosylation in real-world contexts remains limited. In this study, we used histological staining with 'Alcian Blue, Periodic Acid, Schiff's' and 'High-Iron diamine' to assess the acidic nature of mucins stored within goblet cells of the intestine, in a wild mouse population (Mus musculus). Using statistical models, we identified sex as among the most influential ecological factors determining the acidity of intestinal mucin glycans in wild mice. Our data from wild mice and experiments using laboratory mice suggest estrogen signalling associates with an increase in the relative abundance of sialylated mucins. Thus, estrogen signalling may underpin sex differences observed in the colonic mucus of wild and laboratory mice. These findings highlight the significant influence of ecological parameters on mucosal barrier sites and the complementary role of wild populations in augmenting standard laboratory studies in the advancement of mucus biology.
Topics: Mice; Female; Male; Animals; Mucins; Colon; Goblet Cells; Intestines; Estrogens; Mucin-2; Intestinal Mucosa
PubMed: 38521809
DOI: 10.1038/s41598-024-57249-x -
Journal of Orthopaedic Translation Mar 2024The musculoskeletal system contains an extensive network of lymphatic vessels. Decreased lymph flow of the draining collecting lymphatics usually occurs in clinic after...
BACKGROUND
The musculoskeletal system contains an extensive network of lymphatic vessels. Decreased lymph flow of the draining collecting lymphatics usually occurs in clinic after traumatic fractures. However, whether defects in lymphatic drainage can affect fracture healing is unclear.
METHODS
To investigate the effect of lymphatic dysfunction on fracture healing, we used a selective VEGFR3 tyrosine kinase inhibitor to treat tibial fractured mice for 5 weeks versus a vehicle-treated control. To ensure successfully establishing deceased lymphatic drainage model for fractured mice, we measured lymphatic clearance by near infrared indocyanine green lymphatic imaging (NIR-ICG) and the volume of the draining popliteal lymph nodes (PLNs) by ultrasound at the whole phases of fracture healing. In addition, hindlimb edema from day 0 to day 7 post-fracture, pain sensation by Hargreaves test at day 1 post-fracture, bone histomorphometry by micro-CT and callus composition by Alcian Blue-Hematoxylin/Orange G staining at day 14 post-fracture, and bone quality by biomechanical testing at day 35 post-fracture were applied to evaluate fracture healing. To promote fracture healing via increasing lymphatic drainage, we then treated fractured mice with anti-mouse podoplanin (PDPN) neutralizing antibody or isotype IgG antibody for 1 week to observe lymphatic drainage function and assess bone repair as methods described above.
RESULTS
Compared to vehicle-treated group, SAR-treatment group significantly decreased lymphatic clearance and the volume of draining PLNs. SAR-treatment group significantly increased soft tissue swelling, and reduced bone volume (BV)/tissue volume (TV), trabecular number (Tb.N), woven bone and biomechanical properties of fracture callus. In addition, anti-PDPN treated group significantly reduced the number of CD41 platelets in PLNs and increased the number of pulsatile lymphatic vessels, lymphatic clearance and the volume of PLNs. Moreover, anti-PDPN treated group significantly reduced hindlimb edema and pain sensation and increased BV/TV, trabecular number (Tb.Th), woven bone and biomechanical properties of fracture callus.
CONCLUSIONS
Inhibition of proper lymphatic drainage function delayed fracture healing. Use of a anti-PDPN neutralizing antibody reduced lymphatic platelet thrombosis (LPT), increased lymphatic drainage and improved fracture healing.
THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE
(1) We demonstrated lymphatic drainage function is crucial for fracture healing. (2) To unblock the lymphatic drainage and prevent the risk of bleeding and mortality by blood thinner, we demonstrated PDPN neutralizing antibody is a novel and safe way forward in the treatment of bone fracture healing by eliminating LPT and increasing lymphatic drainage.
PubMed: 38511124
DOI: 10.1016/j.jot.2024.02.001 -
Non-coding RNA Research Jun 2024Spinal facet joint osteoarthritis (FJOA) is an OA disease with pathogenesis and progression uncovered. Our present study was performed to elucidate the role of DNM3OS on...
Spinal facet joint osteoarthritis (FJOA) is an OA disease with pathogenesis and progression uncovered. Our present study was performed to elucidate the role of DNM3OS on spinal FJOA. In this study, spine facet joint tissue of patients were collected. and models were constructed with SW1353 cells and rats. Hematoxylin and eosin (HE) staining, Safranin O-fast Green, Alcian blue staining, and Tolueine blue O (TBO) staining were employed for histology analyses. Quantitative PCR, western blotting, and Immunofluorescence were performed to evaluate the expression of genes. The levels of inflammatory cytokines were measured by enzyme-linked immunosorbent assay analysis. Cell Counting Kit-8 and flow cytometry were used for cell activity and apoptosis evaluation. The targeting sites between microRNA (miR)-127-5p and cadherin 11 (CDH11) were predicted TargetScan and miRbase database and confirmed by Dual-luciferase reporter assays. CHIP and EMS assay were employed to confirm the binding of LEF1and DNM3OS promoter. Our results showed that DNM3OS was found to upregulated, while miR-127-5p was downregulated in severe FJOA patients and inflammation-induced chondrosarcoma SW1353 cells. DNM3OS reduced cell activity, induced cell apoptosis and extracellular matrix (ECM) degradation by sponging miR-127-5p . miR-127-5p targeted CDH11 and inhibited wnt3a/β-catenin pathway to regulate OA . LEF1 promoted DNM3OS transcription to form a positively feedback in activated wnt3a/β-catenin pathway. rat model also confirmed that DNM3OS aggravated FJOA. In summary, DNM3OS/miR-127-5p/CDH11 enhanced Wnt3a/β-Catenin/LEF-1 pathway to form a positive feedback and aggravate spinal FJOA.
PubMed: 38505310
DOI: 10.1016/j.ncrna.2024.01.008 -
In Vitro Cellular & Developmental... Apr 2024MicroRNAs (miRNAs) play an important role in articular cartilage damage in osteoarthritis (OA). However, the biological role of miRNAs in the chondrogenic...
MicroRNAs (miRNAs) play an important role in articular cartilage damage in osteoarthritis (OA). However, the biological role of miRNAs in the chondrogenic differentiation of bone marrow mesenchymal stem cell (BMSC) remains largely unclear. Rabbit bone marrow mesenchymal stem cells (rBMSCs) were isolated, cultured, and identified. Afterwards, rBMSCs were induced to chondrogenic differentiation, examined by Alcian Blue staining. Differentially expressed miRNAs were identified in rBMSCs between induced and non-induced groups by miRNA sequencing analysis, part of which was validated via PCR assay. Cell viability and apoptosis were assessed by CCK-8 assay and Hoechst staining. Saffron O staining was utilized to assess chondrocyte hyperplasia. The expression of specific chondrogenic markers, including COL2A1, SOX9, Runx2, MMP-13, Aggrecan, and BMP-2, were measured at mRNA and protein levels. The association between beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC) and miR-10a-5p in the miRNA family from rabbit (ocu-miR-10a-5p) was determined by luciferase reporter assay. A total of 76 differentially expressed miRNAs, including 52 downregulated and 24 upregulated miRNAs, were identified in rBMSCs from the induced group. Inhibition of ocu-miR-10a-5p suppressed rBMSC viability and chondrogenic differentiation, as well as downregulated the expression of β-catenin, SOX9, COL2A1, MMP-13, and Runx2. BTRC was predicted and confirmed as a target of ocu-miR-10a-5p. Overexpression of BTRC rescued the promoting impacts of overexpressed ocu-miR-10a-5p on chondrogenic differentiation of rBMSCs and β-catenin expression. Taken together, our data suggested that ocu-miR-10a-5p facilitated rabbit BMSC survival and chondrogenic differentiation by activating Wnt/β-catenin signaling through BTRC.
Topics: Animals; Rabbits; MicroRNAs; Mesenchymal Stem Cells; Cell Differentiation; Chondrogenesis; Wnt Signaling Pathway; Chondrocytes; Ubiquitin-Protein Ligases; Apoptosis; Cell Survival; beta Catenin; Base Sequence; Gene Expression Regulation
PubMed: 38504085
DOI: 10.1007/s11626-024-00888-1 -
International Journal of Molecular... Feb 2024Intracranial aneurysms are common, but only a minority rupture and cause subarachnoid haemorrhage, presenting a dilemma regarding which to treat. Vessel wall imaging... (Meta-Analysis)
Meta-Analysis Review
Intracranial aneurysms are common, but only a minority rupture and cause subarachnoid haemorrhage, presenting a dilemma regarding which to treat. Vessel wall imaging (VWI) is a contrast-enhanced magnetic resonance imaging (MRI) technique used to identify unstable aneurysms. The pathological basis of MR enhancement of aneurysms is the subject of debate. This review synthesises the literature to determine the pathological basis of VWI enhancement. PubMed and Embase searches were performed for studies reporting VWI of intracranial aneurysms and their correlated histological analysis. The risk of bias was assessed. Calculations of interdependence, univariate and multivariate analysis were performed. Of 228 publications identified, 7 met the eligibility criteria. Individual aneurysm data were extracted for 72 out of a total of 81 aneurysms. Univariate analysis showed macrophage markers (CD68 and MPO, = 0.001 and = 0.002), endothelial cell markers (CD34 and CD31, = 0.007 and = 0.003), glycans (Alcian blue, = 0.003) and wall thickness ( = 0.030) were positively associated with enhancement. Aneurysm enhancement therefore appears to be associated with inflammatory infiltrate and neovascularisation. However, all these markers are correlated with each other, and the literature is limited in terms of the numbers of aneurysms analysed and the parameters considered. The data are therefore insufficient to determine if these associations are independent of each other or of aneurysm size, wall thickness and rupture status. Thus, the cause of aneurysm-wall enhancement currently remains unknown.
Topics: Humans; Intracranial Aneurysm; Aneurysm, Ruptured; Magnetic Resonance Imaging; Subarachnoid Hemorrhage; Image Enhancement
PubMed: 38473947
DOI: 10.3390/ijms25052700 -
Current Protocols Mar 2024Embryonic limb bud-derived micromass cultures are valuable tools for investigating cartilage development, tissue engineering, and therapeutic strategies for...
Embryonic limb bud-derived micromass cultures are valuable tools for investigating cartilage development, tissue engineering, and therapeutic strategies for cartilage-related disorders. This collection of fine-tuned protocols used in our laboratories outlines step-by-step procedures for the isolation, expansion, and differentiation of primary mouse limb bud cells into chondrogenic micromass cultures. Key aspects covered in these protocols include synchronized fertilization of mice (Basic Protocol 1), tissue dissection, cell isolation, micromass formation, and culture optimization parameters, such as cell density and medium composition (Basic Protocol 2). We describe techniques for characterizing the chondrogenic differentiation process by histological analysis (Basic Protocol 3). The protocols also address common challenges encountered during the process and provide troubleshooting strategies. This fine-tuned comprehensive protocol serves as a valuable resource for scientists working in the fields of developmental biology, cartilage tissue engineering, and regenerative medicine, offering an updated methodology for the study of efficient chondrogenic differentiation and cartilage tissue regeneration. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synchronized fertilization of mice Basic Protocol 2: Micromass culture of murine embryonic limb bud-derived cells Basic Protocol 3: Qualitative assessment of cartilage matrix production using Alcian blue staining.
Topics: Animals; Mice; Cells, Cultured; Chondrogenesis; Cell Differentiation; Cartilage; Mammals
PubMed: 38465381
DOI: 10.1002/cpz1.1005