alcian-blue and Disease-Models--Animal

The completeness of the intervertebral disc proteome is fundamental to the integrity and functionality of the intervertebral disc.. The 20 experimental rats were placed into two groups randomly, normal group (NG) and acupuncture pathological degeneration group-2 weeks (APDG-2w). The ten 24-month-old rats were grouped into physiological degeneration group (PDG). Magnetic resonance imaging, X-ray examination, histological staining (hematoxylin & eosin, safranin-O cartilage, and alcian blue staining), and immunohistochemical examination were carried out for assessing the degree of disc degradation. Intervertebral disc was collected, and protein composition was determined by LC- MS, followed by bioinformatic analysis including significance analysis, subcellular localization prediction, protein domain prediction, GO function and KEGG pathway analysis, and protein interaction network construction. LC-PRM was done for protein quantification.. Physiological degeneration and especially needle puncture decreased T2 signal intensity and intervertebral disc height. Results from hematoxylin & eosin, safranin-O, and alcian blue staining revealed that the annulus fibrosus apparently showed the wavy and collapsed fibrocartilage lamellas in APDG-2w and PDG groups. The contents of the nucleus pulposus were decreased in physiological degeneration group and APDG-2w group compared with NG. Results from immunohistochemical analysis suggested the degeneration of intervertebral disc and inflammation in APDG-2w and PDG groups. The protein composition and expression between needle puncture rat models and the physiological degeneration group showed significant difference.. Our studies produced point-reference datasets of normal rats, physiological degeneration rats, and needle puncture rat models, which is beneficial to subsequent pathological studies. There is differential expression of protein expression in degenerative discs with aging and acupuncture, which may be used as a potential discriminating index for different intervertebral degenerations.

Topics: Alcian Blue; Animals; Disease Models, Animal; Eosine Yellowish-(YS); Hematoxylin; Intervertebral Disc; Intervertebral Disc Degeneration; Proteomics; Punctures; Rats

Ulcerative colitis (UC) is an inflammatory bowel disease with characteristic inflammation to mucosal cells in rectum and colon leading to lesions in mucosa and submucosa. Moreover, crocin is a carotenoid compound among active constituents of saffron with many pharmacological effects as antioxidant, anti-inflammatory and anticancer activities. Therefore, we aimed to investigate therapeutic effects of crocin against UC through affecting the inflammatory and apoptotic pathways. For induction of UC in rats, intracolonic 2 ml of 4% acetic acid was used. After induction of UC, part of rats was treated with 20 mg/kg crocin. cAMP was measured using ELISA. Moreover, we measured gene and protein expression of B-cell lymphoma 2 (BCL2), BCL2-associated X (BAX), caspase-3/8/9, NF-κB, tumor necrosis factor (TNF)-α and IL-1β/4/6/10. Colon sections were stained with hematoxylin-eosin and Alcian blue or immune-stained with anti-TNF-α antibodies. Microscopic images of colon sections in UC group revealed destruction of intestinal glands associated with infiltration of inflammatory cell and severe hemorrhage. While images stained with Alcian blue showed damaged and almost absent intestinal glands. Crocin treatment ameliorated morphological changes. Finally, crocin significantly reduced expression levels of BAX, caspase-3/8/9, NF-κB, TNF-α, IL-1β and IL-6, associated with increased levels of cAMP and expression of BCL2, IL-4 and IL-10. In conclusion, protective of action of crocin in UC is proved by restoration of normal weight and length of colon as well as improvement of morphological structure of colon cells. The mechanism of action of crocin in UC is indicated by activation of anti-apoptotic and anti-inflammatory effects.

Topics: Alcian Blue; Animals; Anti-Inflammatory Agents; Apoptosis; bcl-2-Associated X Protein; Carotenoids; Caspase 3; Colitis, Ulcerative; Colon; Disease Models, Animal; Inflammation; NF-kappa B; Rats; Tumor Necrosis Factor Inhibitors; Tumor Necrosis Factor-alpha

Several intestinal damage models have been developed using zebrafish, with the aim of recapitulating aspects of human inflammatory bowel disease (IBD). These experimentally induced inflammation models have utilized immersion exposure to an array of colitogenic agents (including live bacteria, bacterial products, and chemicals) to induce varying severity of inflammation. This technical report describes methods used to generate two chemically induced intestinal damage models using either dextran sodium sulfate (DSS) or trinitrobenzene sulfonic acid (TNBS). Methods to monitor intestinal damage and inflammatory processes, and chemical-genetic methods to manipulate the host response to injury are also described.

Topics: Alcian Blue; Animals; Anti-Bacterial Agents; Anti-Inflammatory Agents; Dextran Sulfate; Disease Models, Animal; Drug Discovery; Enterocolitis; Flow Cytometry; Humans; Immersion; Inflammation; Inflammatory Bowel Diseases; Interleukin-23; Intestines; Larva; Microinjections; Morpholinos; Neutral Red; Neutrophils; Nitric Oxide; Optical Imaging; Polymerase Chain Reaction; Trinitrobenzenesulfonic Acid; Zebrafish; Zebrafish Proteins

Multifunctional granular mast cells (MCs) are among targets in bronchial asthma (BA) therapy. We studied pulmonary MC population in a rat model of BA under the effect of β-adrenoreceptor antagonists and of the latter combined with the standard therapy (glucocorticoid budesonide + β2-adrenergic agonist salbutamol). MCs of different degrees of maturity were identified on paraffin section of lung stained with Alcian blue and Safranin. MC density in the lung of rats with BA increased 1.9 times. Alcian blue-positive immature cells predominated in the lungs of both intact rats and rats with BA. In response to pharmacological agents, the mean MC densities were reduced in 2-2.7 times in all the variants of experiments and were close to the norm. It allows us to suppose that MCs migration from the outside was suppressed and, in consequence of the decline of MC densities, the release of the mediators involved in the progression of BA may be diminished.

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Albuterol; Alcian Blue; Animals; Anti-Asthmatic Agents; Asthma; Bisoprolol; Budesonide; Cell Count; Disease Models, Animal; Drug Therapy, Combination; Glucocorticoids; Lung; Male; Mast Cells; Metoprolol; Phenazines; Rats; Receptors, Adrenergic, beta

Mutations in the human Shwachman-Bodian-Diamond syndrome (SBDS) gene cause defective ribosome assembly and are associated with exocrine pancreatic insufficiency, chronic neutropenia and skeletal defects. However, the mechanism underlying these phenotypes remains unclear. Here we show that knockdown of the zebrafish sbds ortholog fully recapitulates the spectrum of developmental abnormalities observed in the human syndrome, and further implicate impaired proliferation of ptf1a-expressing pancreatic progenitor cells as the basis for the observed pancreatic phenotype. It is thought that diseases of ribosome assembly share a p53-dependent mechanism. However, loss of p53 did not rescue the developmental defects associated with loss of zebrafish sbds. To clarify the molecular mechanisms underlying the observed organogenesis defects, we performed transcriptional profiling to identify candidate downstream mediators of the sbds phenotype. Among transcripts displaying differential expression, functional group analysis revealed marked enrichment of genes related to ribosome biogenesis, rRNA processing and translational initiation. Among these, ribosomal protein L3 (rpl3) and pescadillo (pes) were selected for additional analysis. Similar to knockdown of sbds, knockdown or mutation of either rpl3 or pes resulted in impaired expansion of pancreatic progenitor cells. The pancreatic phenotypes observed in rpl3- and pes-deficient embryos were also independent of p53. Together, these data suggest novel p53-independent roles for ribosomal biogenesis genes in zebrafish pancreas development.

Topics: Alcian Blue; Animals; Anthraquinones; Bone Marrow Diseases; Disease Models, Animal; Exocrine Pancreatic Insufficiency; Fluorescent Antibody Technique; Gene Expression Profiling; Gene Knockdown Techniques; In Situ Hybridization; Lipomatosis; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Pancreas; Ribosomal Protein L3; Ribosomal Proteins; Ribosomes; Shwachman-Diamond Syndrome; Tumor Suppressor Protein p53; Zebrafish; Zebrafish Proteins

Zebrafish craniofacial, skeletal, and tooth development closely resembles that of higher vertebrates. Our goal is to identify viable adult zebrafish mutants that can be used as models for human mineralized craniofacial, dental, and skeletal system disorders. We used a large-scale forward-genetic chemical N-ethyl-nitroso-urea mutagenesis screen to identify 17 early lethal homozygous recessive mutants with defects in craniofacial cartilage elements, and 7 adult homozygous recessive mutants with mineralized tissue phenotypes including craniofacial shape defects, fused sutures, dysmorphic or missing skeletal elements, scoliosis, and neural arch defects. One mutant displayed both an early lethal homozygous phenotype and an adult heterozygous phenotype. These results extend the utility of the zebrafish model beyond the embryo to study human bone and cartilage disorders.

Topics: Alcian Blue; Animals; Anthraquinones; Cartilage; Craniofacial Abnormalities; Disease Models, Animal; Ethylnitrosourea; Genes, Recessive; Mutagenesis; Phenotype; Zebrafish

The COL2A1 gene encodes the α1(II) chain of the homotrimeric type II collagen, the most abundant protein in cartilage. In humans, COL2A1 mutations create many clinical phenotypes collectively termed type II collagenopathies; however, the genetic basis of the phenotypic diversity is not well elucidated. Therefore, animal models corresponding to multiple type II collagenopathies are required. In this study we identified a novel Col2a1 missense mutation--c.44406A>C (p.D1469A)--produced by large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis in a mouse line. This mutation was located in the C-propeptide coding region of Col2a1 and in the positions corresponding to a human COL2A1 mutation responsible for platyspondylic lethal skeletal dysplasia, Torrance type (PLSD-T). The phenotype was inherited as a semidominant trait. The heterozygotes were mildly but significantly smaller than wild-type mice. The homozygotes exhibited lethal skeletal dysplasias, including extremely short limbs, severe spondylar dysplasia, severe pelvic hypoplasia, and brachydactyly. As expected, these skeletal defects in the homozygotes were similar to those in PLSD-T patients. The secretion of the mutant proteins into the extracellular space was disrupted, accompanied by abnormally expanded rough endoplasmic reticulum (ER) and upregulation of ER stress-related genes, such as Grp94 and Chop, in chondrocytes. These findings suggested that the accumulation of mutant type II collagen in the ER and subsequent induction of ER stress are involved, at least in part in the PLSD-T-like phenotypes of the mutants. This mutant should serve as a good model for studying PLSD-T pathogenesis and the mechanisms that create the great diversity of type II collagenopathies.

Topics: Alcian Blue; Animals; Anthraquinones; Bone and Bones; Chromosome Mapping; Collagen Type II; Disease Models, Animal; DNA Primers; Ethylnitrosourea; Genotype; Immunohistochemistry; Mice; Mice, Mutant Strains; Microscopy, Electron, Transmission; Mutagenesis; Mutation, Missense; Osteochondrodysplasias; Reverse Transcriptase Polymerase Chain Reaction

Utilization of a combined Alcian Blue and Pyronine Y histochemical method for the assessment of multiple parameters in the respiratory tract of various species is described. Acidic mucins were deep blue (sialylated mucins), red (sulfated mucins), or variably purple (mixture of sialylated/sulfated mucins), and differential mucus production was readily detected in a murine respiratory syncytial virus vaccine model of pulmonary inflammation. Elastic fibers stained red in the walls of pulmonary arteries, connecting airways, alveolar septa, and subpleural interstitium. Mast cells had red to red-purple granular cytoplasmic staining. Nuclei were ubiquitously counterstained pale blue. Representative staining was detected in tissues from multiple species, including inbred mice, rats, ferrets, cats, dogs, sheep, and pigs. The fluorescent property of the stained tissues offers additional modalities with which to analyze tissue sections. This histochemical technique detects multiple critical parameters in routine paraffin sections of lung tissue, reduces the need for repeated serial sectioning and staining, and is cost-effective and simple to perform.

Topics: Alcian Blue; Animals; Carnivora; Disease Models, Animal; Lung Diseases; Mice; Pyronine; Rabbits; Rats; Respiratory System; Sheep; Staining and Labeling; Swine

Whereas the alterations of diverse tissues in cellular and molecular levels have been investigated during leg lengthening via microscopy and biochemical studies, little is known about the response of deep fascia. This study aims to investigate the changes of the extracellular matrix in deep fascia in response to leg lengthening.. Animal model of leg lengthening was established in New Zealand white rabbits. Distraction was initiated at a rate of 1 mm/day and 2 mm/day in two steps, and preceded until increases of 10% and 20% in the initial length of tibia had been achieved. Alcian blue stain and picrosirius-polarization method were used for the study of the extracellular matrix of deep fascia samples. Leica DM LA image analysis system was used to investigate the quantitative changes of collagen type I and III.. Alcian blue stain showed that glycosaminoglycans of fascia of each group were composed of chondroitin sulphate and heparin sulphate, but not of keratan sulphate. Under the polarization microscopy, the fascia consisted mainly of collagen type I. After leg lengthening, the percentage of collagen type III increased. The most similar collagen composition of the fascia to that of the normal fascia was detected at a 20% increase in tibia length achieved via a distraction rate of 1 mm/d.. The changes in collagen distribution and composition occur in deep fascia during leg lengthening. Although different lengthening schemes resulted in varied matrix changes, the most comparable collagen composition to be demonstrated under the scheme of a distraction rate of 1 mm/day and 20% increase in tibia length. Efficient fascia regeneration is initiated only in certain combinations of the leg load parameters including appropriate intensity and duration time, e.g., either low density distraction that persist a relatively short time or high distraction rates.

Topics: Alcian Blue; Animals; Biomarkers; Bone Lengthening; Chondroitin Sulfates; Collagen Type I; Collagen Type III; Coloring Agents; Disease Models, Animal; Extracellular Matrix; Fascia; Fasciotomy; Heparin; Hindlimb; Image Processing, Computer-Assisted; Microscopy, Polarization; Proteoglycans; Rabbits; Stress, Mechanical

This report describes an inhibitory effect of Gahmi-Shini-San (GSS) on mast cell-mediated immediate-type allergic reactions. GSS is an Oriental herbal medication, which has been successfully used in Korea for the treatment of allergic disorders, mainly skin anaphylactic diseases. GSS inhibited the ear swelling response induced by intradermal injection of compound 48/80 in a mouse model on a concentration-dependent basis. The mast cells in mouse ear tissue were stained by alcian blue/nuclear fast red. GSS significantly inhibited the compound 48/80-induced degranulation from mast cells in ear tissue. GSS dose-dependently inhibited the histamine release from the rat peritoneal mast cells by compound 48/80. We also studied the effect of GSS on mast cell-dependent passive cutaneous anaphylaxis activated by dinitrophenyl IgE antibody. GSS showed inhibition of passive cutaneous anaphylaxis following oral administration. These results indicated that GSS has inhibitory effect on mast cell-dependent immediate type cutaneous reactions.

Topics: Alcian Blue; Animals; Cell Degranulation; Disease Models, Animal; Dose-Response Relationship, Drug; Ear; Histamine; Hypersensitivity; Mast Cells; Mice; Neutral Red; p-Methoxy-N-methylphenethylamine; Passive Cutaneous Anaphylaxis; Peritoneal Cavity; Plant Extracts; Rats

We have developed a new method that is applicable for a macroscopic and objective evaluation of erosion in the large intestine in the experimental ulcerative colitis model induced by dextran sulfate sodium (DSS) in rats. The large intestine (without cecum) fixed in 10% neutral buffered formalin solution for more than one week was opened and stained with 1% alcian blue solution. The mucosal surface was stained in light and shade-blue. Histopathological examination revealed that the dark blue area on the mucosal surface had no epithelia and that the connective tissues in the lamina propria were stained with alcian blue. Salazosulfapyridine at 15 and 50 mg/kg twice a day inhibited the erosion area (dark blue area) by 29.6% and 50.2%, respectively. Also, prednisolone at 0.5 mg/kg, twice a day inhibited the erosion by 53.3%. Thus, by measuring the dark blue area stained with 1% alcian blue solution, we could estimate macroscopically and objectively the area of erosions. This method seems to be a useful index for assessing the damages produced in the experimental ulcerative colitis model in rats.

Topics: Alcian Blue; Animals; Colitis, Ulcerative; Dextran Sulfate; Disease Models, Animal; Intestinal Mucosa; Prednisolone; Rats; Rats, Sprague-Dawley; Staining and Labeling; Sulfasalazine

The etiology of the eye defects in myelencephalic blebs (my) mutant mice has been poorly understood for almost seventy years. Embryos from 9 to 14 1/2 days of gestation were subjected to Alcian blue 8GX staining for acidic glycosaminoglycan deposition in basement membrane structures of the developing eye in my stock and control specimens. In addition 12 day embryos were subjected to avidinbiotin-peroxidase labelling for laminin. At 9-9 1/2 days of gestation more Alcian blue positive extracellular matrix was found in the region between the optic vesicle and the overlying putative lens ectoderm in the my stock embryos. By 12 days, there was an irregular and lesser amount of deposition of glycosaminoglycans in the len's capsule and in the "inner limiting membrane" of the presumptive neural retina; however, the deposition of laminin appeared to be greater in the inner limiting membrane of the my eye. By 14 days, the damage to the eye in the my embryos can be quite extensive, and the deposition of glycosaminoglycans was very meager in this situation. It appears that irregular deposition of glycosaminoglycans in the extracellular matrix and possible increase in the amount of laminin in basement structures in my embryos indicate disruption of the normal histochemistry involved in the development of the eye. Altered histochemistry may in turn indicate changes in permeability between cells of the developing tissues which result in the blebbing.

Topics: Alcian Blue; Animals; Basement Membrane; Disease Models, Animal; Eye; Gestational Age; Glycosaminoglycans; Laminin; Mice; Mice, Mutant Strains