Ultrastructural examination of B-50(GAP-43) immunoreactivity in rat jejunal villi

Summary The lamina propria of rat jejunum is densely innervated with nerve fibres extending to the tips of the villi. A large number of these nerve fibres were previously shown to be B-50-immunoreactive at the light microscope level, whereas neurofilament immunoreactivity was found to be sparse in the mucosa. In this study we used immunoelectron microscopy to determine what proportion of nerve fibres in the lamina propria express B-50. Jejuna from male Lewis rats were immunolabelled for B-50 and neurofilament proteins. For electron microscopy, postembedding immunogold-silver techniques and LR White embedded tissues were used. Light microscopical immunostaining was performed by the streptavidin-biotin-peroxidase technique on deparaffinized tissue sections. We found that all ultrastructurally identifiable nerve profiles in jejunum were B-50 immunoreactive. Immunoelectron microscopy for neurofilament proteins failed to label fibres in the villi, whereas myelinated nerves in tongue sections processed in parallel (positive controls) were strongly neurofilament-protein-immunoreactive. The dominant B-50-positive and neurofilament-protein-negative phenotype supports the hypothesis of ongoing modelling or plasticity of intestinal mucosal nerves.     

Elastic fibres are an essential component of human placental stem villous stroma and an integrated part of the perivascular contractile sheath

The stroma of human placental stem villi is believed to consist only of reticular and collagen fibres. In the present study we were able to show for the first time by light (orcein staining) and electron microscopy large amounts of elastic fibres in the stem villous stroma. Electron microscopically, homogeneous elastin was found alone or in association with microfibrils. In addition, microfibrils were observed forming long bands. These three structures, generally known to form elastic connective tissue, were seen in close connection with placental extravascular smooth muscle cells, which belong to the perivascular contractile sheath (PVCS) of stem villi. Elastin was associated with these smooth muscle cells and connected to collagen fibres via microfibrils. Collagen fibres were additionally interconnected by spike-like structures. Extravascular smooth muscle cells revealed numerous adhesion plaques which occupied conspicuously long cytoplasmic faces of the plasma membrane. In cryostat sections, immunoreactivity of talin, an attachment protein of adhesion plaques linking intracellular α-actin filaments with extracellular fibronectin, was detected in extravascular and vascular (media) smooth muscle cells. The arrangement of placental extravascular smooth muscle cells, elastic and collagen fibres suggests a functional myofibroelastic unit within the PVCS, which surrounds the large foetal blood vessels possibly contributing to elasticity and supporting tensile and/or contracting forces within the stem villi. Received: 2 May 1995 / Accepted: 7 August 1995     

Pathomorphology of the Intestinal Mucosa in Diarrheic Calves

The intestinal mucosa was examined in twelve 2–5-week-old calves with a spontaneous intestinal disorder, 8 with diarrhea and 4 convalescents. The calves were fed a defined milk replacer. Light microscopy including morphometry, showed villous atrophy and crypt elongation. Villous epithelial cells had decreased height, and epithelial cells of the posterior small intestine contained an increased amount of fat droplets. Accumulation of neutrophils in crypts was frequent. Scanning electron microscopy revealed blunt villi with increased numbers of necrotic cells in the extrusion zone at the tips of the villi. The convalescents had generally milder changes, particularly in the anterior small intestine. The probable etiological factors included a rotavirus and chlamydial infection, and it is concluded that these agents together with other possible noxious influences were responsible for the increased necrobiosis of apical senescent villous epithelial cells, resulting in villous atrophy and crypt hyperplasia.     

Changes of the caecal villi during postnatal development in rats

Summary The villi of the caecal mucosa in postnatal rats were studied using both scanning electron and light microscopy.On the day of birth, numerous villi of various sizes and shapes were present on the caecal mucosa. After the 5th day, the villi decreased very rapidly in length and in number. A strong constriction was observed at the basal region of the caecal villi. During postnatal days 5 9 the villi probably separated and disappeared from the caecal mucosa. No villi were observed in rats that were over 10 days of age.     

Scanning electron microscopy of swine lymphoid organs

The aim of this investigation was to study by scanning electron microscopy the structure of several swine lymphoid organs (lymph nodes, Peyer's patches, and tonsil). Two groups of animals were used: six-month-old pigs and six- to nine-day-old piglets. Samples were jet-washed to eliminate most free cells in order to observe the reticular framework of these organs more clearly. Peyer's patches in piglets showed two types of villi. In one of them the cellular types were absorptive cells and goblet cells. The second type of villi were shorter and wider, with M cells characterized by presenting long, thick microvilli over their surfaces. Peyer's patches of pigs did not show this second type of villi but were usually covered by absorptive villi. The soft palate tonsil was similar in both groups of animals with its surface epithelial cells full of microfolds, partially and frequently obscured by microorganisms. The appearance of the surface epithelium in the same crypt was different depending on the area. There was a large number of holes through which cells apparently passed towards the crypt lumen. The medulla in the lymph nodes was at the periphery and showed a dense reticular framework. Cortex-like lymphoid tissue was formed by lymphoid follides and diffuse lymphoid tissue with high endothelid venules and lymphatic sinuses. The serosal surface of lymphoid organs was formed either by a typical mesothelial cell layer (small intestine) or by loosely arranged connective fibers (lymph nodes).     

Staining of rabbit eosinophil and pseudoeosinophil leukocytes.

Air-dried rabbit blood was stained by HE, PAS and a modification of the Undritz II method. Eosin stained granules red in the eosinophil leukocytes. PAS was negative and the modified Undritz method failed to give consistent results. Cells with eosinophilic granules appeared in the corneal stroma 1 h after removing the corneal epithelium. They were stained red consistently by both eosin and the modified Undritz II method. Electron micrographs failed to demonstrate crystalloids in the granules. Because of the staining characteristics and the lack of crystalloids in their granules these cells were classified as pseudoeosinophil leukocytes. The electron micrographs showed some glycogen 12 h after denuding the cornea, however, glycogen was not well stained by PAS until 18 h after denuding.     

伦敦白胶和茶：一种既快又安全的用于透射电子显微镜的细菌样品制备方法

【目的】检测乌龙茶提取物是否可作为电子染色剂取代醋酸双氧铀用于细菌细胞染色,使其能在透射电子显微镜下进行观察。【方法】利用伦敦白胶对细菌样品(大肠杆菌和金黄色葡萄球菌)进行胶块的制备,再在复染铅与不复染铅这两种情况下对超薄切片样品进行3种不同染色剂的电子染色,之后在透射电子显微镜下观察比较其不同之处。这3种不同的染色剂分别是醋酸双氧铀、0.05%乌龙茶提取物以及0.1%乌龙茶提取物。首先将带有超薄切片样品的铜网悬浮于不同的待比较染液中10?15 min,若需进一步用柠檬酸铅复染,则将经3次蒸馏水冲洗过后的铜网再次悬浮于柠檬酸铅染液中8?10 min。【结果】复染铅的情况下,在透射电子显微镜下无论是大肠杆菌还是金黄色葡萄球菌,利用3种电子染色剂进行染色的结果均非常相似。【结论】实验结果表明,在观察细菌结构中,乌龙茶提取物可以替代醋酸双氧铀进行透射电子显微镜样品的电子染色。     

Isolation of villous microvessels from the human placenta

A method for isolating the microvessels of the human placental villi has been developed in order to culture perivascular cells. It consists of an initial selection of the villi by serial sieving. The villi retained by the 75 μm sieve were digested by collagenase-dispase. A Percoll gradient permitted the isolation of microvessels still surrounded by stromal fibres and cells. Another digestion by collagenase-dispase eliminated the contaminant elements and allowed, after a new Percoll gradient, microvessels with endothelium, basement membrane and a few perivascular cells to be obtained. Each step of the isolation of microvessels was monitored by light or electron microscopy. Our study confirms the isolation of microvessels embedded in their basement membrane and the preservation of endothelial and perivascular cells after digestion. This method, which has permitted the culture of placental endothelial cells and pericytes, appears of interest for studying microvascular angiogenesis and permeability.     

The Gastrointestinal Mucosa in Young Milk-Fed Calves

Gastrointestinal segments from 4 healthy, 17-, 21-, 22- and 23-day-old calves fed on whole cow’s milk were examined. Scanning electron microscopy showed that the anterior duodenum had short villi varying in shape from leaf-shaped to nodular; the middle duodenum had broad, tongue-shaped villi and the anterior, middle, and parts of the posterior jejunum had slender, finger-shaped or leaf-shaped villi. The villi of the mucosa covering Peyer’s patches in the posterior jejunum were short and either conical or tongue-shaped; there were also small “pseudovilli” caused by bulges in the lymphoid tissue. Morphometry showed that the villi were longer in the anterior jejunum than in the duodenum and the posterior parts of the jejunum (P < 0.005). Morphologically fat absorption was most heavy in the anterior third of the small intestine. Moderate amounts of fat were also found in the epithelium of the posterior jejunum and of the abomasum. Large fat droplets were seen in apical duodenal enterocytes, in contrast to the small epithelial droplets in other areas with fat absorption. Nile blue staining indicated that the fat in the large droplets was esterified.     

Fenestrations of the basal lamina of intestinal villi of the rat

Fenestrations of the basal lamina of rat intestinal villi were revealed by scanning electron microscopy after removal of the overlying epithelial cells by osmic acid maceration. These fenestrations are circular to oval in shape and are 0.5 micron to 5 microns in diameter. They are richly distributed at a density of 1-2 X 10(4)/mm2 in the upper two thirds of the villi, except at the very tips. Roughly 500 fenestrations are found on each side of an average sized tongue-shaped villus. Transmission electron-microscopic observations showed that these fenestrations were passages for migrating cells of the immune system such as lymphocytes, eosinophils and macrophages. Protrusions from the basal parts of epithelial cells were also observed passing through these fenestrations. These findings are discussed with respect to their immunological implications and to the passage of nutrients.     

Static magnetic fields affect cell size, shape, orientation, and membrane surface of human glioblastoma cells, as demonstrated by electron, optic, and atomic force microscopy.

BACKGROUND: It is common knowledge that static magnetic fields (SMF) do not interact with living cells; thus, fewer studies of SMF compared with variable magnetic fields are carried out. However, evidence demonstrated that SMF affect cellular structures. To investigate the effect of exposure to increasing doses of SMF on cell morphology, human glioblastoma cells were exposed to SMF ranging between 80 and 3,000 G (8 and 300 mT). METHODS: Cell morphology of human glioblastoma cells, derived from a primary culture, was studied by electron and optic microscopy. FITC-phalloidin staining of actin filaments was also investigated. Finally, cell surface structure changes were detected by atomic force microscopy. RESULTS: Scanning electron microscopy demonstrated a dose-dependent cell shape modification, progressive cell detachment, loss of the long villi, and appearance of membrane roughness and blebs. FITC-phalloidin staining confirmed the villi retention and cell dimension decrease. At 3,000 G, the appearance of apoptotic morphology was also observed by transmission electron microscopy. Cell exposed to SMF showed different orientation and alignment when compared with nonexposed cells. The atomic force microscopy of the exposed cells' membrane surfaces demonstrated the disappearance of the ordered surface ripples and furrows typical of the unexposed cells, and the occurrence of surface membrane corrugation at increasing dose exposure CONCLUSIONS: Our experimental procedures demonstrated that exposure to SMF affects not only cell size, shape, and orientation but also human glioblastoma cells' membrane surfaces.     

家鸽小肠绒毛微血管铸型的扫描电镜观察

用扫描电镜观察了ＡＢＳ丁酮溶液灌注的家鸽小肠绒告发同血管构筑情况。家鸽小肠绒毛血管丛由输入沁动脉、毛细血管网和输出小静脉组成,小肠绒毛血管丰富,并相到吻合成单层密集网;办入小动脉既可从肠腺周围血管丛发出,也可直接由粘膜下去一发出,绒毛下部血管表现为微直血管形态,可能部分具有门静脉性质。     

DIFFERENTIAL LOCALIZATION OF CELL SURFACE AND SECRETORY COMPONENTS IN RAT INTESTINAL EPITHELIUM BY USE OF LECTINS

Sections through various levels of small intestine from adult male rats were examined by fluorescence microscopy after treatment with fluorescein isothiocyanate-labeled lectins from Dolichos biflorus, Lotus tetragonolobus, Ricinus communis, and Triticum vulgare (wheat germ). The latter three lectins reacted with the microvillar portion of the epithelial cells lining the crypts and villi in sections of intestine adjacent to the pylorus. This pattern of reactivity was sharply altered along the first 15 cm of intestine so that in sections distal to this point the luminal surfaces of only those epithelial cells in the crypts and at the base of the villi reacted with the L. tetragonolobus and R. communis lectins, whereas the wheat germ lectin reacted with the surfaces of the cells lining the villi. In sections from the distal end of the small intestine, all three lectins reacted with the surfaces of cells only at the base of the villi and in the crypts. These results show a difference in surface components in cells at various portions on the villi and the dependence of these differences on the region of intestine. The D. biflorus lectin reacted with approximately 25% of the goblet cells at each level of intestine studied whereas the reactivities of the goblet cells with the other three lectins were dependent upon the region of intestine.     

Morphological effects of a large single dose of cycloheximide on the intestinal epithelium of the rat.

Adult male rats received 15 mg/kg cycloheximide and the subsequent morphological effects at three and six hours after injection were evaluated using histometry, light and electron microscopy, histological demonstration of terminal web and acid phosphatase, and radioautography with tritiated thymidine. Rapid atrophy of the villi took place, progressing from the villus tip by premature exfoliation of epithelial cells. The crypts also diminished by random exfoliation of many crypt cells and by partial or complete disintegration. Mitosis and epithelial cell migration were absent. By six hours, the area occupied by the villi and the crypts per unit length of histological section was decreased by about 70-90% in most of the small intestine but only by about 40-60% in the duodenum and the terminal ileum. In the upper half of the villi, the epithelium was strongly positive for acid phosphatase and contained large numbers of round bodies resembling primary lysosomes. In the lower half, the microvillous border and terminal web were found to be disrupted. Animals receiving only 5 mg/kg cycloheximide also showed the atrophy of villi and crypts, and the round bodies resembling lysosomes. Evidence from several sources has indicated that protein synthesis in normal villus epithelial cells subsides toward the villus tip and becomes minimal at exfoliation. At exfoliation, proteins responsible for epithelial cohesion probably fail because they are no longer replenished. Cycloheximide appears to accelerate this process.     

The mechanism of epithelial shedding after ischemic damage to the small intestinal mucosa

The intestinal mucosa of the rat was examined by light and electron microscopy 15, 30, 60 and 120 min after complete ligation of the vessel arcades of the proximal jejunum. The characteristic sign of ischemic damage to the small intestinal mucosa and the reason for epithelial shedding is the appearance of membrane enclosed cytoplasmic blebs which arise at the cell base of the enterocytes and detach the epithelium from the basement membrane. This process begins at the tip of the villi before the enterocytes display signs of irreversible damage and progress to the base of the villi with continuation of the ischemia.     

Back-Scattered Electron Imaging of Sections Through the Cochlea: A New Technique for Studying Cochlear Morphology

A new technique for studying the morphology of the cochlea is described. The development of back-scattered electron (BSE) detectors has allowed the examination of heavy-metal stained tissues by scanning electron microscopy. Comparison with light microscopy on adjacent resin sections through whole decalcified cochleae demonstrated that the back-scattered electron technique provides equal or superior clarity and resolution throughout the light microscope range of magnification, allows identification of lysosomes, mitochondria and endoplasmic reticulum, and extends useful magnification into the range previously associated only with transmission electron microscopy. Back-scattered electron imaging enables the study of sections of the undissected cochlea at high magnifications and resolution.     

Back-scattered electron imaging of sections through the cochlea: a new technique for studying cochlear morphology

A new technique for studying the morphology of the cochlea is described. The development of back-scattered electron (BSE) detectors has allowed the examination of heavy-metal stained tissues by scanning electron microscopy. Comparison with light microscopy on adjacent resin sections through whole decalcified cochleae demonstrated that the back-scattered electron technique provides equal or superior clarity and resolution throughout the light microscope range of magnification, allows identification of lysosomes, mitochondria and endoplasmic reticulum, and extends useful magnification into the range previously associated only with transmission electron microscopy. Back-scattered electron imaging enables the study of sections of the undissected cochlea at high magnifications and resolution.     

The effect of hydroxyurea on ciliogenesis in ciliary epithelium of the mollusk Lymnaea stagnalis

Electron-microscopy study of the ciliary epithelium structure of the mollusk Lymnaea stagnalis was carried out under the action of hydroxyurea. By the method of radioautography, a high proliferative activity of the ciliary epithelium was established as the norm; a cluster distribution of cells, including the label, was noted. The presence of hydroxyurea in the mollusk organism was shown to inhibit proliferation. Scanning electron microscopy of the molluskan foot surface revealed clusters of nonciliated cells and of cells with short villi in control epithelial folds. Under hydroxyurea treatment for 24 h, such sites disappeared completely and ciliary epithelium looked uniform and was composed of cells with long cilia. By transmission electron microscopy, it was established that hydroxyurea did not affect the formation of the basal body and course of ciliogenesis. It has been suggested that hydroxyurea not only inhibits proliferative activity of epithelial cells, but also induces differentiation of unciliated into the ciliated cells.     

Immunohistochemical investigation on the presence of chondroitin sulfate in calcification nodules of epiphyseal cartilage.

Chondroitin sulfate localization in mouse epiphyseal cartilage was studied using CS-56 monoclonal antibody immunospecific for the glycosaminoglycan portion of the molecule. For light and fluorescence microscopy, decalcified specimens were embedded in paraffin, Lowicryl, or were frozen and cryostat-sectioned, and the antigen-antibody reaction was demonstrated by treating sections with IgM-peroxidase, IgM-alkaline phosphatase, or IgM-fluorescein conjugates. For electron microscopy, decalcified and undecalcified specimens were embedded in Lowicryl; ultrathin sections from undecalcified specimens were decalcified by flotation on EDTA; sections from both types of specimens were treated with IgM-immunogold conjugate for demonstration of CS-56 reaction. Before immunoreaction, part of all decalcified sections were digested with Streptomyces or testicular hyaluronidase. Control sections were treated with either mouse and goat non-immune serum, or mouse monoclonal antiserum to human dendritic reticulum cells. Both light and electron microscopy show CS-56 reaction with cytoplasmic components of maturing and hypertrophic chondrocytes. Under the light microscope, immunoreaction was not visible in calcified matrix, and was visible in uncalcified matrix only after hyaluronidase digestion. Under the electron microscope, it was evident both in uncalcified and calcified matrix, although the latter showed few immunogold particles, usually placed on areas which appeared incompletely calcified. Gold particles were chiefly distributed at the periphery of calcification nodules and fully calcified matrix. These results show that CS-56, besides reacting with cytoplasm of maturing and hypertrophic chondrocytes, binds to crystal ghosts and other components of cartilage matrix, immunoreactivity decreasing as calcification increases. This suggests that chondroitin sulfate molecules are either degraded during calcification, or segregated into macromolecular complexes, or both degraded and segregated. The second possibility is supported by the increase of immunosensitivity induced by hyaluronidase digestion.