Mucosal mast cells of the rat intestine: a re-evaluation of fixation and staining properties, with special reference to protein blocking and solubility of the granular glycosaminoglycan

Mucosal mast cells of the gastrointestinal tract constitute a separate cell line within the mast cell system of the rat, differing in several respects from the classical connective tissue mast cells and, unlike the latter, requiring special fixation techniques for their demonstration. We have examined some histochemical properties of mucosal mast cells of the duodenum and compared them with connective tissue mast cells of the tongue or skin. The results indicate that the structural integrity of the granules of both types of mast cell is partly dependent on ionic linkages between glycosaminoglycan and protein. The so far unidentified glycosaminoglycan of mucosal mast cells appears to be more soluble than the heparin of connective tissue mast cells. The strongly fluorescent binding of Berberine to the granules of connective tissue mast cells and, depending on their content, of heparin is absent from mucosal mast cells, confirming previous findings which suggested that they contain a glycosaminoglycan with a lower degree of sulphation. Aldehyde fixation by routine procedures reversibly blocks the cationic dye binding of mucosal mast cell granules. The dye binding groups may be unmasked by trypsination or by long staining times of the order of several days. The results suggest that the blocking of staining by aldehydes is caused by a diffusion barrier of a protein nature. Mucosal and connective tissue mast cells thus differ with respect to the spatial arrangement of glycosaminoglycan and protein in their granules. As a result of the study a modified method for the demonstration of mucosal mast cells in tissue sections is described, based on normal formaldehyde fixation and staining in Toluidine Blue for a long time. It has some advantages over previous methods and preserves the structure of mucosal and connective tissue mast cells equally well.     

Biochemical reactivity of aortic connective tissue in chickens treated with cholesterol diet in combination with a nonspecific immunization or with epinephrine and thyroxine

Our previous work showed that only changes in aortic lipids and no change in connective tissue components occurred in hatched chickens, fed for four months a cholesterol atherogenic diet. The aim of this study was to test if combination of atherogenic stimuli will activate the connective tissue of aorta.Newly hatched chickens were fed a diet containing 2% cholesterol and in addition immunized with bovine serum albumen. Another group was administered epinephrine and thyroxine in excessive doses. The treatment lasted 6 to 7 weeks. In isolated aortas the amount and rate of synthesis of collagen, activity of prolyl hydroxylase and collagenase and the amount of total cholesterol were measured. With the exception of significantly increased lipid content, neither parameter reflecting the connective tissue and collagen metabolism was affected by the various atherogenic stimuli. It is concluded that in young chickens the changes in cholesterol content in aortic tissue preceded eventual activation of connective tissue components.     

Quantitative analysis of 17 amino acids in the connective tissue of patients with pelvic organ prolapse using capillary electrophoresis-tandem mass spectrometry

The simultaneous determination of 17 amino acids in connective tissue using capillary electrophoresis is described in this study. Separation was carried out on a fused silica capillary column (80 cm x 50 mm i.d.) with 1M formic acid as the running electrolyte. The detection was conducted on a mass spectrometer by selective reaction monitoring (SRM) mode via an electrospray ionization source. Tissue samples were prepared by reduction and acid hydrolysis to extract amino acids; over 84.3% recovery was seen for all compounds. The method allowed for sensitive, reproducible, and reliable quantification, and all 17 amino acids were separated using this method. Good linearity over the investigated concentration ranges was observed, with values of R higher than 0.993 for all the analytes. Precision and accuracy examined at three concentration levels ranged from 0.2% to 19.5% and 84.1% to 120.0%, respectively. Matrix effects were also tested and ranged from -9.1% to 15.4%. The validated method was applied to the quantitation of 17 amino acids in pelvic connective tissue of pelvic organ prolapsed patients. Methionine, glutamine, and histidine were significantly higher in the experimental patients compared to the controls. This suggests that changes in the amino acid concentrations within the connective tissue could be a factor in the genesis of pelvic organ prolapse. Therefore, this method is potentially applicable for amino acid analysis in tissue, providing a more complete understanding of pelvic organ prolapse.     

结缔组织铺片脂肪组织油红染色在组织学实验教学中的应用

目的在传统结缔组织铺片基础上开展脂肪组织油红染色方法在医学本科生组织学实验教学中的应用。方法学生先进行疏松结缔组织铺片,并施行脂肪组织油红o-甲苯胺兰-伊红三重染色,然后镜下观察。结果油红o染色把结缔组织中的脂肪细胞内脂滴保存下来并染上红色。脂肪组织中央的细胞脂滴均匀红染,充满胞浆,周边的脂肪细胞胞浆中油红染色很少,细胞呈空泡状,显示出脂肪细胞亚群存在。甲苯胺兰染色使得疏松结缔组织中肥大细胞染成紫红色,胞核染色浅,细胞数量多、成群分布。伊红可使得结缔组织内除脂肪细胞、肥大细胞意外的其他细胞的胞浆和胶原纤维染成淡红色。结论传统的组织学平铺片技术基础上引入脂肪油红o-甲苯胺兰-伊红三重染色,可增强学生动手能力,并能很好地了解输送结缔组织中细胞的不同表型和分布,丰富组织学内容,把教学、科研连接一起,达到提高实验教学质量的目的。     

Glucose gradient differences in subcutaneous tissue of healthy volunteers assessed with ultraslow microdialysis and a nanolitre glucose sensor

The abdominal subcutaneous interstitium is easily accessible for monitoring glucose for Diabetes Mellitus research and management. The available glucose sensing devices demand frequent blood sampling by finger pricking for calibration. Moreover, there is controversy about the exact relationship between the levels of glucose in the subcutis and blood. In the present study ultra-slow microdialysis was applied for subcutaneous fluid sampling, allowing continuous measurement of glucose in an equilibrated fluid using a nanolitre size sensor. The present method avoids in vivo calibration. During an oral glucose tolerance test glucose levels were measured simultaneously in blood, in adipose tissue and loose connective tissue layers of the abdominal subcutis in seven healthy subjects. Fasting glucose levels (mM) were 2.52 +/- 0.77 in adipose tissue and 4.67 +/- 0.17 in blood, this difference increasing to 6.40 +/- 1.57 and 11.59 +/- 1.52 at maximal glucose concentration. Moreover, the kinetics of glucose in blood and adipose tissue were different. In contrast, connective tissue glucose levels differed insignificantly (4.71 +/- 0.21 fasting and 11.70 +/- 1.96 at maximum) from those in blood and correlated well (r2 = 0.962). Ultra-slow microdialysis combined with a nanolitre glucose sensor could be of benefit to patients in intensive diabetes therapy. Frequent blood sampling for in vivo calibration can be avoided by monitoring glucose in the abdominal subcutaneous loose connective tissue, rather than in the adipose tissue.     

A Method for Quantifying Mechanical Properties of Tissue following Viral Infection

Viral infection and replication involves the reorganization of the actin network within the host cell. Actin plays a central role in the mechanical properties of cells. We have demonstrated a method to quantify changes in mechanical properties of fabricated model three-dimensional (3D) connective tissue following viral infection. Using this method, we have characterized the impact of infection by the human herpesvirus, cytomegalovirus (HCMV). HCMV is a member of the herpesvirus family and infects a variety of cell types including fibroblasts. In the body, fibroblasts are necessary for maintaining connective tissue and function by creating mechanical force. Using this 3D connective tissue model, we observed that infection disrupted the cell's ability to generate force and reduced the cumulative contractile force of the tissue. The addition of HCMV viral particles in the absence of both viral gene expression and DNA replication was sufficient to disrupt tissue function. We observed that alterations of the mechanical properties are, in part, due to a disruption of the underlying complex actin microfilament network established by the embedded fibroblasts. Finally, we were able to prevent HCMV-mediated disruption of tissue function by the addition of human immune globulin against HCMV. This study demonstrates a method to quantify the impact of viral infection on mechanical properties which are not evident using conventional cell culture systems.     

The vagal contribution to the rat liver innervation: a demonstration with the cobalt impregnation method

The contribution of the vagus nerves to the innervation of the liver has been studied with the cobaltous chloride impregnation method. With this method we have demonstrated that the fiber plexus in the rat hepatic parenchyma, that we had previously described and stained for acetylcholinesterase, is of a nervous nature and of vagal origin. Our results show that branches from the vagus spread abundantly with the connective tissue at the capsule. From this peripheral location, the fibres expand deeply through the parenchyma in close contact with the hepatocytes towards the central veins. Other branches run with the interlobular connective tissue, distributing to the portal veins, hepatic arteries and biliary ducts. They also have lateral branches which penetrate into the parenchyma.     

Treatment of Raynaud's phenomenon with ketanserin in patients with connective tissue disorders.

The serotonin receptor blocker ketanserin was given orally in a double blind crossover study to 10 patients with connective tissue disorders and Raynaud''s phenomenon. Eight of the 10 patients improved clinically on ketanserin and none on placebo. Digital blood flow was assessed with laser Doppler flowmetry (LDF), photoplethysmography, and skin temperature measurements. Laser Doppler flowmetry was the most useful method, showing a significant reduction in recovery time after a standard cold provocation. Although the resting flow was not significantly improved, digital ulcers healed in four out of five patients, providing evidence of increased nutritive flow. The results of this study suggest that orally administered ketanserin may be an effective and well tolerated treatment for Raynaud''s phenomenon associated with connective tissue disorders, especially scleroderma.     

A combination Verhoeff's elastic and Masson's trichrome stain for routine histology

A method for the combined staining of elastic, muscle and connective tissue for routine use in histopathology is described. The elastica, stained black by Verhoeff's technique, is contrasted with the muscle and connective tissue stained red and green or blue respectively by a modification of Masson's trichrome. Cell nuclei stain blue-black with Weigert's iron hematoxylin. The procedure takes approximately two hours and is most suitable for the study of vascular pathology in surgical and autopsy sections.     

Association of Thy-1 cell surface differentiation antigen with certain connective tissues in vivo

Summary The cell surface differentiation antigen, Thy-1, has been demonstrated by immunofluorescence to be associated with some collagen-based connective tissue. In the rat, the basement membrane of kidney collecting tubules and of certain blood vessels, reticulin of the lamina propria, loose connective tissue in the dermis, and collagen fibres within certain nerve cell tumours, bear the antigen. Other, apparently similar, connective tissue (especially that of muscle and liver) lacked Thy-1. In the mouse, only the connective tissue of the skin was found to bear the antigen. The possible origin of this extracellular Thy-1, and its implications for the function of the molecule, are discussed.Part of this work was supported by grant no. NS10861 from the National Institute of Health and no. BNS 77-15818 from the National Science Foundation, awarded to Dr. S.E. Pfeiffer     

A Combination Verhoeffs Elastic and Masson's Trichrome Stain for Routine Histology

A method for the combined staining of elastic, muscle and connective tissue for routine use in histopathology is described. The elastica, stained black by Verhoeffs technique, is contrasted with the muscle and connective tissue stained red and green or blue respectively by a modification of Masson's trichrome. Cell nuclei stain blue-black with Weigert's iron hematoxylin. The procedure takes approximately two hours and is most suitable for the study of vascular pathology in surgical and autopsy sections.     

EDTA separation and recombination of epithelium and connective tissue of human oral mucosa. Studies of tissue transplants in nude mice

A possible epithelial-mesenchymal interaction in determining epithelial histologic features of human oral mucosa was examined. The study comprised 74 biopsies of normal buccal mucosa and 54 biopsies of normal palatal mucosa. Epithelium was separated from connective tissue by the use of 1 mM ethylenediamine tetraacetate dihydrate. Self-recombined and cross-recombined epithelial and connective tissues and connective tissue sheets alone were transplanted to subcutaneous sites of nude mice. Histologic examination of cross-recombined palatal epithelium/buccal connective tissue transplants showed a change in keratinization pattern but no major change in number of epithelial cell layers as the result of connective tissue influence. Transplanted sheets of connective tissue after growth for 14 days showed that complete separation of biopsies from buccal mucosa had been obtained. However, palatal mucosa had been incompletely separated as evidenced by re-epithelialization of most of the connective tissue transplants. The consequences of the incomplete palatal epithelium-connective tissue separation are discussed.     

Study of antibodies to heterologous connective tissue, isolated from sera of patients with rheumatism]

Antibodies against connective tissue elements of various bovine organs were isolated from the sera of rheumatic fever patients with the aid of immunosorbents (bovine connective tissue extract and erythrocyte stroma). The antibody preparations obtained were not identical and contained antibodies against different antigens of bovine connective tissue. The antibody preparations failed to react with human connective tissue components.     

The influence of differing connective tissue substrates on the maintenance of adult stratified squamous epithelia

Summary The interaction between adult stratified squamous epithelium and its supporting connective tissue possibly involves both permissive and directive influences. To examine the effect of vitality and specificity of connective tissue on the maintenance of epithelial structure and histo-differentiation, specimens of skin and oral mucosa from various regions of adult mice were separated using either EDTA or trypsin. Prior to transplantation, the epithelium was recombined with either inverted homologous connective tissue or with connective tissue that had been killed either by heating or repeated freeze-thawing. Epithelial sheets were also transplanted onto the graft bed alone or in combination with striated muscle or tendon.Normal patterns of cytodifferentiation were maintained when the epithelium was recombined with inverted or frozen-thawed subepithelial connective tissue but there was a loss of spatial organization on the frozen-thawed connective tissue. In contrast, heat-killed or trypsin-treated frozen-thawed subepithelial connective tissue and non-dermal connective tissue failed to maintain a viable epithelium. These observations suggest that subepithelial connective tissues (dermis, lamina propria) but not deep connective tissues facilitate epithelial proliferation and histodifferentiation.Supported by NIH/NIDR RO1 DEO5190     

Micro-computed tomography with iodine staining resolves the arrangement of muscle fibres

We illustrate here microCT images in which contrast between muscle and connective tissue has been achieved by means of staining with iodine. Enhancement is shown to be dependent on the concentration of iodine solution (I₂KI), time in solution and specimen size. Histological examination confirms that the arrangement of individual muscle fibres can be visualised on the enhanced microCT images, and that the iodine accumulates in the muscle fibres in preference to the surrounding connective tissues. We explore the application of this technique to describe the fibrous structure of skeletal muscle, and conclude that it has the potential to become a non-destructive and cost-effective method for investigating muscle fascicle architecture, particularly in comparative morphological studies.     

Fine Structural Observations on the Turbellarians Stenostomum sp. and Microstomum lineare with Special Reference to the Extracellular Matrix and Connective Tissue Systems

Employing transmission electron microscopy, observations were made on epidermis, muscle cells and connective tissue systems, with special emphasis on extracellular matrix components (ECM), in two rather primitive turbellarians: Stenostomum sp. (Catenulida) and Microstomum lineare (Macrostomida). In Stenostomum the only ECM components found are basal laminae, predominantly situated subepidermally. In Microstomum ECM is well developed and connective tissue filaments abundant in conspicuous extracellular spaces. It is uncertain whether basal laminae exist. The finding of basal lamina structures as the only ECM component present in Stenostomum makes it now possible to establish a complete ECM and connective tissue hierarchy in turbellarians, ranging from a purely cellular type with no ECM present to systems dominated by ECM and very similar to loose connective tissue in vertebrates. Comparative aspects of ECM and connective tissue systems in turbellarians are discussed in addition to the difficulties and ambiguities regarding definition and nomenclature of basal matrices as basal laminae and subepidermal membranes.     

A histochemical comparison of methylene-blue/acid fuchsin with hematoxylin and eosin for differentiating calcification of stromal tissue

Benign and malignant connective tissue tumors consist of a fibrous component that contains varying amounts of one or more types of bone or other calcified tissue. Diagnosis of these connective tissue tumors often poses challenges for pathologists, because it is difficult to differentiate the organic matrix of osteoid from hyalinized stroma. To establish a definitive diagnosis, it sometimes is advantageous to demonstrate histologically by special staining either the type of calcification or the presence or absence of calcification. We compared the efficacy of methylene blue-acid fuchsin (MB-AF) to hematoxylin and eosin (H-E) for connective tissue tumors suspected to contain calcifications and to devise an optimal staining technique for calcification that would be specific, simple, and cost- and time-effective. We examined 50 benign and 45 malignant connective tissue tumors that were suspected to contain calcifications. Sections were stained with H-E and MB-AF and evaluated. MB-AF stained bone pink, which contrasted with blue soft tissue. After MB-AF staining, osteoid was faint pink in a blue stromal background. Osteoid was not visualized in H-E stained sections; it was stained the same shade of pink as stromal tissue. Dystrophic calcification and cementum could be identified equally well using either staining technique, but contrast was better after H-E staining. MB-AF staining of bone was comparable to H-E staining and could be used effectively to stain bone and osteoid. MB-AF is a simple, single step procedure. It also stains cementum blue with faint blue rimming and dystrophic calcification bluish-pink, but it cannot be used as a specific stain for types of calcification other than bone and osteoid.     

Stress and matrix‐responsive cytoskeletal remodeling in fibroblasts

In areolar “loose” connective tissue, fibroblasts remodel their cytoskeleton within minutes in response to static stretch resulting in increased cell body cross‐sectional area that relaxes the tissue to a lower state of resting tension. It remains unknown whether the loosely arranged collagen matrix, characteristic of areolar connective tissue, is required for this cytoskeletal response to occur. The purpose of this study was to evaluate cytoskeletal remodeling of fibroblasts in, and dissociated from, areolar and dense connective tissue in response to 2 h of static stretch in both native tissue and collagen gels of varying crosslinking. Rheometric testing indicated that the areolar connective tissue had a lower dynamic modulus and was more viscous than the dense connective tissue. In response to stretch, cells within the more compliant areolar connective tissue adopted a large “sheet‐like” morphology that was in contrast to the smaller dendritic morphology in the dense connective tissue. By adjusting the in vitro collagen crosslinking, and the resulting dynamic modulus, it was demonstrated that cells dissociated from dense connective tissue are capable of responding when seeded into a compliant matrix, while cells dissociated from areolar connective tissue can lose their ability to respond when their matrix becomes stiffer. This set of experiments indicated stretch‐induced fibroblast expansion was dependent on the distinct matrix material properties of areolar connective tissues as opposed to the cells' tissue of origin. These results also suggest that disease and pathological processes with increased crosslinks, such as diabetes and fibrosis, could impair fibroblast responsiveness in connective tissues. J. Cell. Physiol. 228: 50–57, 2013. © 2012 Wiley Periodicals, Inc.     

Connective tissue mast cells in contact with fibroblasts express IL-3 mRNA. Analysis of single cells by polymerase chain reaction.

Mast cell-fibroblast interactions have been extensively investigated in the last few years. Fibroblasts support the in vitro survival but not proliferation of mouse connective-tissue type mast cells. However, the factor(s) that allow their survival on fibroblast monolayers has not been identified. We have investigated the presence of mRNA for IL-3 and granulocyte-macrophage-CSF in single mouse mast cells, before and after co-culture with 3T3 fibroblasts, using the polymerase chain reaction technique. The system was calibrated first by using in vitro generated population of mouse bone-marrow derived mast cells (BMMC). Significant differences in the amplification of IL-3 cDNA were observed in each of the BMMC cells examined, whereas the amplification of cDNA for the alpha-subunit of the Fc epsilon RI were similar. Inasmuch as murine cultured IL-3-dependent mast cells differentiate into connective tissue-like mast cells when co-cultured with 3T3 fibroblasts without any exogenous supply of growth factors, it was of interest to determine whether these connective tissue-like mast cells produce IL-3 message. Separation of the differentiated BMMC from the fibroblast monolayer, by either trypsinization or by single cell manipulation revealed the synthesis of a detectable amount of IL-3 mRNA in these mast cells. Whether this IL-3 mRNA was induced by fibroblasts was further investigated using connective tissue mast cells freshly purified from the mouse peritoneal cavity. Only about 20% of these connective tissue mast cells produced detectable amount of granulocyte-macrophage-CSF mRNA whereas in less than 10% of the cells IL-3 mRNA was detected. However, when these connective tissue mast cells were co-cultured with 3T3 fibroblasts for 18 hours and then separated, IL-3 mRNA were detected in most of the cells whereas no such mRNA was detected in tissue mast cells incubated for 18 h with medium derived from 3T3 fibroblasts. Therefore we conclude that fibroblasts induce the accumulation of IL-3 mRNA in connective tissue mast cells. The production of IL-3 may play a role in the survival of this type of mast cells on the fibroblast monolayer.