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Methods in Molecular Biology (Clifton,... 2014The first step in almost every investigation of skeletal phenotypes is analysis of whole-mount skeletal preparations. Whole-mount skeletal staining permits evaluation of...
The first step in almost every investigation of skeletal phenotypes is analysis of whole-mount skeletal preparations. Whole-mount skeletal staining permits evaluation of the shapes and sizes of skeletal elements in their appropriate locations. The technique is thus the major method for detecting changes in skeletal patterning. Because cartilage and bone can be distinguished by differential staining, this technique is also a powerful means to assess the pace of skeletal maturation. This protocol covers staining of the pre- and postnatal mouse skeleton using Alcian blue and Alizarin red to identify cartilage and bone, respectively.
Topics: Animals; Bone and Bones; Female; Fetus; Histocytological Preparation Techniques; Mice; Microscopy; Pregnancy; Staining and Labeling
PubMed: 24482169
DOI: 10.1007/978-1-62703-989-5_9 -
Molecular Genetics and Metabolism 2017Glycosaminoglycans (GAGs) are long blocks of negatively charged polysaccharides. They are one of the major components of the extracellular matrix and play multiple roles... (Review)
Review
Glycosaminoglycans (GAGs) are long blocks of negatively charged polysaccharides. They are one of the major components of the extracellular matrix and play multiple roles in different tissues and organs. The accumulation of undegraded GAGs causes mucopolysaccharidoses (MPS). GAGs are associated with other pathological conditions such as osteoarthritis, inflammation, diabetes mellitus, spinal cord injury, and cancer. The need for further understanding of GAG functions and mechanisms of action boosted the development of qualitative and quantitative (alcian blue, toluidine blue, paper and thin layer chromatography, gas chromatography, high pressure liquid chromatography, capillary electrophoresis, 1,9-dimethylmethylene blue, enzyme linked-immunosorbent assay, mass spectrometry) techniques. The availability of quantitative techniques has facilitated translational research on GAGs into the medical field for: 1) diagnosis, monitoring, and screening for MPS; 2) analysis of GAG synthetic and degradation pathways; and 3) determination of physiological and pathological roles of GAGs. This review provides a history of development of GAG assays and insights about the use of tandem mass spectrometry and its applications for GAG analysis.
Topics: Chromatography, Thin Layer; Early Diagnosis; Glycosaminoglycans; Humans; Mucopolysaccharidoses; Tandem Mass Spectrometry; Translational Research, Biomedical
PubMed: 27746032
DOI: 10.1016/j.ymgme.2016.09.005 -
ELife May 2022Proliferating cells undergo metabolic changes in synchrony with cell cycle progression and cell division. Mitochondria provide fuel, metabolites, and ATP during...
Proliferating cells undergo metabolic changes in synchrony with cell cycle progression and cell division. Mitochondria provide fuel, metabolites, and ATP during different phases of the cell cycle, however it is not completely understood how mitochondrial function and the cell cycle are coordinated. CLUH (clustered mitochondria homolog) is a post-transcriptional regulator of mRNAs encoding mitochondrial proteins involved in oxidative phosphorylation and several metabolic pathways. Here, we show a role of CLUH in regulating the expression of astrin, which is involved in metaphase to anaphase progression, centrosome integrity, and mTORC1 inhibition. We find that CLUH binds both the mRNA and its product astrin, and controls the synthesis and the stability of the full-length astrin-1 isoform. We show that CLUH interacts with astrin-1 specifically during interphase. Astrin-depleted cells show mTORC1 hyperactivation and enhanced anabolism. On the other hand, cells lacking CLUH show decreased astrin levels and increased mTORC1 signaling, but cannot sustain anaplerotic and anabolic pathways. In absence of CLUH, cells fail to grow during G1, and progress faster through the cell cycle, indicating dysregulated matching of growth, metabolism, and cell cycling. Our data reveal a role of CLUH in coupling growth signaling pathways and mitochondrial metabolism with cell cycle progression.
Topics: Alcian Blue; Cell Cycle; Mechanistic Target of Rapamycin Complex 1; Metaphase; Mitochondria; Mitochondrial Proteins; Phenazines; Phenothiazines; RNA, Messenger; Resorcinols
PubMed: 35559794
DOI: 10.7554/eLife.74552 -
Veterinary Journal (London, England :... Jul 2022The endothelial glycocalyx (eGlx) lines the luminal surface of endothelial cells. It is critical in maintaining vascular health and when damaged contributes to many...
The endothelial glycocalyx (eGlx) lines the luminal surface of endothelial cells. It is critical in maintaining vascular health and when damaged contributes to many diseases. Its fragility makes studying the eGlx technically challenging. The current reference standard for eGlx visualisation, by electron microscopy using glutaraldehyde/Alcian blue perfusion fixation, has not been previously reported in dogs. Established techniques were applied to achieve visualisation of the eGlx in the microvasculature of reproductive tissue in five healthy dogs undergoing elective neutering. Uterine and testicular artery samples underwent perfusion fixation, in the presence of Alcian blue, prior to transmission electron microscopy imaging. Image processing software was used to determine eGlx depth. EGlx was visualised in the arteries of two dogs, one testicular and one uterine, with median (range) eGlx depths of 68.2 nm (32.1-122.9 nm) and 47.6 nm (26.1-129.4 nm) respectively. Study of the eGlx is technically challenging, particularly its direct visualisation in clinical samples. Further research is needed to develop more clinically applicable techniques to measure eGlx health.
Topics: Alcian Blue; Animals; Dogs; Endothelial Cells; Glycocalyx; Perfusion
PubMed: 35640795
DOI: 10.1016/j.tvjl.2022.105844 -
Ocular Oncology and Pathology Sep 2021The goal of this study was to histopathologically evaluate the appearance of degrading MIRAgel scleral buckles so that they can be reliably distinguished by ophthalmic...
INTRODUCTION
The goal of this study was to histopathologically evaluate the appearance of degrading MIRAgel scleral buckles so that they can be reliably distinguished by ophthalmic pathologists from other foreign materials.
METHODS
Retrospective chart review and histopathologic study with special stains, including Alcian blue, periodic acid-Schiff, Masson's trichrome, and Perls' Prussian blue for iron, of 4 cases between 2017 and 2019.
RESULTS
Hydrolyzed MIRAgel scleral buckles from 4 patients had a consistent histopathologic appearance. They had a honeycomb structure with the walls of the lattice ranging from distinct to poorly defined. The walls of the lattice were positive for Alcian blue, while the contents of each cell contained periodic acid-Schiff-positive material. Other special stains were not as valuable in highlighting the material.
DISCUSSION
Although the capsules of MIRAgel scleral buckles have been well studied, the material itself has not been. While clinical history and radiographic appearance are often diagnostic of a hydrolyzed MIRAgel scleral buckle, there are instances of preoperative ambiguity where histopathologic confirmation can be useful.
CONCLUSIONS
MIRAgel scleral buckles have a distinct histopathologic appearance that can be readily distinguished from that of other foreign materials.
PubMed: 34604200
DOI: 10.1159/000514299 -
Frontiers in Bioscience (Landmark... Feb 2022Although autogenous bone implantation is considered to be the gold standard for the reconstruction of bone defects, this approach remains challenging when treating...
BACKGROUND
Although autogenous bone implantation is considered to be the gold standard for the reconstruction of bone defects, this approach remains challenging when treating extensive bone defects (EBDs). Therefore, artificial materials (AMs) such as artificial bone and scaffolds are often used for treating EBDs. Nevertheless, complications such as material failure, foreign body reaction, and infection are common. To overcome these issues, we aimed to develop a new treatment for an EBD using scaffold-free adipose-derived stromal cells (ADSCs) to fabricate chondrogenic/osteogenic-induced constructs without AMs.
METHODS
ADSCs were obtained from the subcutaneous adipose tissue of 8-week-old female Wistar rats (n = 3) and assessed to determine their potential for multilineage differentiation into adipocytes (Oil Red O staining), chondrocytes (hematoxylin and eosin, Alcian blue, and Safranin O staining), and osteoblasts (Alizarin red and von Kossa staining). Spheroids (n = 320), each containing 3.0 × 104 ADSCs, were then used to fabricate scaffold-free cell constructs using a bio-3D printer with a needle array. The spheroids and constructs were stimulated with induction medium to induce chondrogenic and osteogenic differentiation. The induced cartilage- and bone-like constructs were finally evaluated using micro-computed tomography (μCT) and histological analysis.
RESULTS
The collected ADSCs were capable of trilineage differentiation, and were successfully used to produce scaffold-free constructs. The fabricated constructs (n = 3) exhibited equivalent strength (load, 195.3 ± 6.1 mN; strength, 39.1 ± 1.2 kPa; and stiffness, 0.09 ± 0.01 N/mm) to that of soft tissues such as the muscles in the uninduced condition. In chondrogenic induction experiments, Alcian blue and Safranin O staining confirmed the differentiation of the constructs into cartilage, and cartilage tissue-like structures were produced. In the osteogenic induction experiment, Alizarin Red and von Kossa staining showed calcium salt deposition, and μCT images confirmed the same calcification level as that of the cortical bone.
CONCLUSIONS
Scaffold-free constructs consisting of ADSCs without an AM were fabricated, and cartilage- and bone-like tissues were successfully generated, demonstrating their potential for bone reconstruction.
Topics: Adipose Tissue; Animals; Cell Differentiation; Cells, Cultured; Female; Osteogenesis; Printing, Three-Dimensional; Rats; Rats, Wistar; Stromal Cells; Tissue Engineering; Tissue Scaffolds; X-Ray Microtomography
PubMed: 35226995
DOI: 10.31083/j.fbl2702052 -
Autopsy & Case Reports 2017Gelatinous transformation of the bone marrow (GTBM) is a rare hematologic entity, which was first described by Paul Michael in 1930. GTBM is mostly associated with... (Review)
Review
Gelatinous transformation of the bone marrow (GTBM) is a rare hematologic entity, which was first described by Paul Michael in 1930. GTBM is mostly associated with caloric intake/anorexia nervosa, although it also has been described accompanying other pathologic conditions, such as malignancy, systemic lupus erythematosus, HIV infections. Even though the diagnostic features of the hematopoietic tissue, such as hypoplasia, adipose cell atrophy, and deposition of a gelatinous substance in the bone marrow (which stains with Alcian blue at pH 2.5) are quite specific, the underlying pathogenic mechanisms remain poorly understood. Considering the evidence of reversibility-notably in cases of malnutrition and anorexia-this entity should be kept high on cards as a possible differential diagnosis of patients presenting with cytopenias and associated weight loss or starvation, especially in developing countries with nutritionally deprived populations. On an extensive review of the literature aimed at comprehensively addressing the evolution of the GTBM from the past century until now, we conclude that the lack of clinical suspicion and awareness regarding this pathologic entity has led to misdiagnosis and delayed diagnosis.
PubMed: 29259927
DOI: 10.4322/acr.2017.039 -
Journal of Oral Biology and... 2021Hyalinization is a process of conversion of stromal connective tissue into a homogeneous, acellular translucent material. Nevertheless, hyalinization could provide... (Review)
Review
BACKGROUND
Hyalinization is a process of conversion of stromal connective tissue into a homogeneous, acellular translucent material. Nevertheless, hyalinization could provide insights into the biologic behaviour and prognosis of pathological lesions. Few studies with limited sample size have intended to assess the correlation of hyalinization and biologic behaviour in oral lesions.
AIM
The current review aims to comprehensively appraise the mechanism of hyalinization in pathological oral hyalinizing lesions (OHL) and its clinical implications with emphasis on differential stains employed.
METHODS
An electronic search was performed in the PubMed database (from year 2000-2020) using the keywords "special stains in oral hyalinizing lesions", "significance of hyalinization in oral lesions" and "hyalinization and biologic behaviour". Original research articles analyzing the effect of hyalinization on biologic characteristics of the lesion were evaluated in this review. Narrative review articles that provided insights into the mechanism of hyalinization and maturity of collagen fibers were also considered for analysis.
CONCLUSION
The presence of hyalinization does seem to have a significant effect on the biologic behaviour of pathological lesions. There is substantial scope to further investigate the process of hyalinization on larger samples and its correlation with the aggressive behaviour of OHLs. Special stains and advanced investigations such as immunohistochemistry for stromal markers would define the nature of hyalinized material and validate the correlation.
CLINICAL SIGNIFICANCE
The prediction of the biologic behaviour of a lesion established through assessment of hyalinization would prevent unwanted over or under treatment leading to a better prognosis.
PubMed: 34094841
DOI: 10.1016/j.jobcr.2021.05.002 -
Sovremennye Tekhnologii V Meditsine 2021Mast cells play an important role in the body defense against allergens, pathogens, and parasites by participating in inflammation development. However, there is... (Review)
Review
Mast cells play an important role in the body defense against allergens, pathogens, and parasites by participating in inflammation development. However, there is evidence for their contributing to the pathogenesis of a number of atopic, autoimmune, as well as cardiovascular, oncologic, neurologic, and other diseases (allergy, asthma, eczema, rhinitis, anaphylaxis, mastocytosis, multiple sclerosis, rheumatoid arthritis, inflammatory gastrointestinal and pulmonary diseases, migraine, etc.). The diagnosis of many diseases and the study of mast cell functions in health and disease require their identification; so, the knowledge on adequate imaging techniques for mast cells in humans and different species of animals is of particular importance. The present review summarizes the data on major methods of mast cell imaging: enzyme histochemistry, immunohistochemistry, as well as histochemistry using histological stains. The main histological stains bind to heparin and other acidic mucopolysaccharides contained in mast cells and stain them metachromatically. Among these are toluidine blue, methylene blue (including that contained in May-Grünwald-Giemsa stain), thionin, pinacyanol, and others. Safranin and fluorescent dyes: berberine and avidin - also bind to heparin. Longer staining with histological dyes or alcian blue staining is needed to label mucosal and immature mast cells. Advanced techniques - enzyme histochemistry and especially immunohistochemistry - enable to detect mast cells high-selectively using a reaction to tryptases and chymases (specific proteases of these cells). In the immunohistochemical study of tryptases and chymases, species-specific differences in the distribution of the proteases in mast cells of humans and animals should be taken into account for their adequate detection. The immunohistochemical reaction to immunoglobulin E receptor (FcεRI) and c-kit receptor is not specific to mast cells, although the latter is important to demonstrate their proliferation in normal and malignant growth. Correct fixation of biological material is also discussed in the review as it is of great significance for histochemical and immunohistochemical mast cell detection. Fluorescent methods of immunohistochemistry and a multimarker analysis in combination with confocal microscopy are reported to be new technological approaches currently used to study various mast cell populations.
Topics: Animals; Chymases; Humans; Immunohistochemistry; Mast Cells; Tryptases
PubMed: 34603768
DOI: 10.17691/stm2021.13.4.10