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PubMed1988

Basement-membrane components associated with the extracellular matrix of the lymph node.

Lymph nodes contain an extensive array of extracellular matrix fibers frequently referred to as "reticular fibers" because of their reticular pattern and positive reaction with silver stains. These fibers are known to contain primarily type-III collagen. In the present study, frozen and plastic-embedded sections of mouse and human lymph nodes were subjected to immunostaining with a panel of monospecific antibodies directed against type-IV collagen, type-III collagen, laminin, entactin, and heparan sulfate proteoglycan. Immunofluorescent staining revealed that, in addition to being uniformly stained with antibodies to type-III collagen, these fibers also stained positively with antibodies to type-IV collagen and to other basement-membrane-specific components. Furthermore, the basement-membrane-specific antibodies stained the outer surface of individual fibers. These same type-III collagen-rich fibers were distinct from blood vascular basement membranes since they did not react with antibodies to factor VIII-related antigen, an endothelial-cell-specific marker. The role of these basement-membrane-specific components associated with the reticular fibers of lymphoid tissue is unknown. However, it is possible that the ligands promote attachment of reticular fibroblasts as well as macrophages and lymphocytes to the extracellular matrix fibers.

Animals↗
PubMed1986

Crystal-associated matrix components in rat incisor enamel. An electron-microscopic study.

Sections of glutaraldehyde-OsO4-fixed, plastic-embedded rat incisor enamel were left untreated, stained, decalcified (1% formic acid in 10% sodium citrate), or decalcified-stained. The presence of apatite crystals was monitored with electron diffraction. After brief decalcification and staining, apatite crystals and matrix components were visualized in the same field. The ghost was continuous with crystal fragments, and the coat appeared as a dense line next to crystals and ghosts. Position of ghosts and crystals at the ameloblast-enamel junction (AEJ) of the secretion zone suggested that there may be a lag of no more than 1/5 min between the elaboration of ghost and crystal. A major change in enamel morphology occurs between the AEJ and the deep enamel of the secretion zone. The ghost becomes thinner, the coat more pronounced, and the crystal enlarges. There is only little change from the deep secretion to the maturation zone enamel.

Animals↗
PubMed1987

Application of automatic image analysis for morphometric studies of peroxisomes stained cytochemically for catalase. II. Light-microscopic application.

The feasibility of the application of a television-based image analyzer, the Texture Analysis System (TAS, Leitz Wetzlar, FRG) in conjunction with a light microscope for morphometric studies of hepatic peroxisomes has been investigated. Rat liver peroxisomes were stained with the alkaline-DAB method for localization of catalase and semithin (0.25 and 1 micron) sections of plastic-embedded material were examined under an oil immersion objective. The TAS detected the peroxisomal profiles selectively and determined their morphometric parameters automatically. The same parameters were obtained also by morphometric analysis of electron micrographs from the same material. The volume density of peroxisomes determined by TAS in semithin sections of normal liver, after correction for section thickness, is quite close to the corresponding value obtained by morphometry of electron micrographs. The difference is approximately 20%. In animals treated with the hypolipidemic drug bezafibrate, which causes proliferation of peroxisomes, TAS detected readily the increase in volume density of peroxisomes in semithin sections. In comparison with electron microscopy, however, the light-microscopic approach seems to underestimate the proliferation. The lower resolution of the light microscope and overlapping of neighbouring particles in relatively thick sections used for light-microscopic analysis may account for the differences. The present study has demonstrated the usefulness of automatic image analysis in conjunction with selective cytochemical staining of peroxisomes for morphometry of this organelle in rat liver. The light-microscopic approach is not only faster but is also extremely economical by obviating the use of an electron microscope.

Animals↗
PubMed1981

Distribution of nerve growth factor in the submandibular gland of the male and female mouse. A re-examination by use of an improved immunohistochemical procedure.

Nerve growth factor (NGF) was localized in the mouse submandibular gland by means of indirect immunofluorescence applied to 0.5 micron-thick sections of freeze-dried, plastic-embedded tissue. The antibody to NGF (IgG-fraction) was raised in rabbits immunized with pure 2.5 S NGF from submandibular glands of adult male mice. In the male gland anti-NGF bound selectively to the secretory granules was present in the cells of the granular ducts. Immunoreactive granules extended from the perinuclear region toward the apical pole. In the female gland immunoreactive cells and granules were considerably less abundant than in males. Immunofluorescence was confined to individual secretory cells located in the wall of the granular striated duct. In the present study no support was found for the hypothesis suggesting that immunoreactive NGF is formed within the secretory granules during their transport from the perinuclear region to the apical pole.

Animals↗
PubMed1977

"Tight" junctions in the sheath of normal and regenerating motor nerves of the crayfish, Orconectes virilis.

"Tight" or occluding intercellular junctions occur between adjacent glial processes in normal and regenerating crayfish motor nerve sheaths. Although infrequent, these junctions possess the ridge and groove configuration characteristics of freeze-cleaved occluding junctions. When present, nerve sheath tight junctions consist of a single, or at most a few, parallel intramembrane ridges situated on the EF membrane face of the glial plasma membrane. Consequently, such contacts are rarely recognized in thin sections of plastic-embedded nerve sheaths. Crayfish nerve sheath tight junctions are of the fascia occludens type and, therefore, do not impede solute flow across the nerve sheath. Fasciae occludentes of regenerating nerve sheaths occur in close proximity to discoid plaque-like aggregates of particles assumed to represent maculae adhaerentes. This relationship, which was not observed in normal nerve sheaths, suggests a functional association between the two types of junctions, perhaps developmental transformation of one junction type into the other. Although ridges and grooves of tight junctions occur next to cross-fractured trans-glial channels, no functional significance is proposed for this relationship. This study is the first report of tight intercellular junctions in crustacean glial nerve sheaths.

Animals↗
PubMed1977

Calcium containing particles in mitochondria of heart muscle cells as shown by cryo-ultramicrotomy and X-ray microanalysis.

Mitochondria of normal myocardial cells of the sand rat and the mouse as well as of the left ventricle of man, have been examined for their content of calcium. Ultrahistochemistry and X-ray microanalysis revealed two basically different inclusions: Osmiophilic mitochondrial granules and spherical mitochondrial particles. Osmiophilic mitochondrial granules were found in conventionally fixed and plastic embedded tissues as well as in cryosections of chemically fixed and sucrose infused tissues. Such granules lacked inert electron density and probably consisted mainly of unsaturated lipids. X-ray spectra obtained from these tissues revealed no peaks for calcium. Spherical mitochondrial particles were present in dry-cut cryo-sections of N2-frozen tissues not treated by fixatives and/or cryoprotectants. These particles were deeply electron dense in unstained, freeze-dried cryo-sections. They usually measured from 600 A-900 A in diameter in the normal myocardium of the sand rat and the mouse and from 250 A-400 A in diameter in the left ventricular myocardium of man. Significant calcium peaks could be identified in the X-ray spectra of these particles, whereas none occurred in the analyses of other tissue regions. Potassium was detected with about equal frequency in the particles and in other parts of the tissue. On the basis of the inert electron density of the particles and their absence in chemically fixed tissues as well as of the results of the X-ray analysis, it is concluded that they contain precipitates of extremely labile ions of mitochondrial calcium.

Animals↗
PubMed1990

GABA, glycine, aspartate, glutamate and taurine in the vestibular nuclei: an immunocytochemical investigation in the cat.

The distributions of five amino acids with well-established neuroexcitatory or neuroinhibitory properties were investigated in the feline vestibular complex. Consecutive semithin sections of plastic-embedded tissue were incubated with antisera raised against protein-glutaraldehyde conjugates of GABA, glycine, aspartate, glutamate and taurine. This approach allowed us to study the relative densities of the different immunoreactivities at the level of individual cell profiles. The results indicate that in the vestibular nuclei, neuronal colocalization of two or more neuroactive amino acids is the rule rather than an exception. Colocalization was found of immunoreactivities for GABA and glycine; glycine, aspartate and glutamate; glycine and aspartate, and glutamate and aspartate. GABA immunoreactive neurons were generally small and were found scattered throughout the vestibular complex. Glycine immunoreactive neurons were similarly distributed, except in the superior nucleus where the latter type of neuron could not be detected. Neuronal profiles colocalizing immunoreactivities for GABA and glycine occurred in all nuclei, but were most numerous in the lateral nucleus. The vast majority of the neurons showed noteworthy staining for glutamate and aspartate, although the level of immunoreactivities varied (e.g., the large neurons in the lateral and descending nuclei were more intensely aspartate immunoreactive than the smaller ones). Taurine-like immunoreactivity did not occur in neuronal cell bodies but appeared in Purkinje cell axons and in glial cell profiles. The functional significance of the complex pattern of amino acid colocalization remains to be clarified. In particular it needs to be distinguished between metabolic and transmitter pools of the different amino acids. The present results call for caution when attempts are made to conclude about transmitter identity on the basis of amino acid contents alone.

Amino Acids↗
PubMed1991

Systematic presence of GABA-immunoreactivity in the tubero-infundibular and tubero-hypophyseal dopaminergic axonal systems: an ultrastructural immunogold study on several mammals.

Immunoreactivities for tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA) and, in some cases, glutamic acid decarboxylase (GAD) were detected by light and electron microscopy in axons projecting into the median eminence and pituitary gland of various mammals (rats, mice, guinea pigs, cats, rabbits and hares). Light microscope immunoperoxidase reactions were performed on adjacent semithin sections of plastic-embedded samples. In the median eminence external zone, the distributions of the TH- and GAD- or GABA-immunoreactive endings were very similar in the anterior and lateral areas, while medially the GABA-labelled endings predominated. Comparable distribution patterns were found in the various species examined. In the pituitary gland, the distributions of GABA- and TH-immunoreactivities were superimposable in the intermediate lobes of all species examined, except in the rabbit and hare in which both types of innervation were lacking. For electron microscopy, the immunogold procedure was applied to sections of lowicryl-embedded samples; simultaneous detection of GABA- and TH-immunoreactivities was enabled by recto-verso double labelling with gold particles of distinct diameters. In the median eminence, GABA-immunoreactivity occurred systematically in the TH-positive endings, while distinct GABA-positive/TH-negative axons were also detected. In the intermediate lobe, the colocalization of TH- and GABA-immunoreactivities was a constant feature of the axons innervating the melanotrophic cells in all the species examined, except in the Leporidae. The functional significance of this colocalization remains to be determined.

Animals↗
PubMed1980

Mapping stimulus-induced nervous activity in small brains by [3H]2-deoxy-D-glucose.

Nervous activity may be localized in anatomical sections of brain tissue by the autoradiographic deoxyglucose technique. The method provides sufficient structural preservation and spatial resolution for detailed functional investigation of complex but small-sized nervous systems when the original technique is modified as follows: (i) use of 3H instead of 14C as radioactive label, (ii) application of labeled deoxyglucose in concentrations close to physiological glucose levels rather than in trace amounts, (iii) stimulation for 4--9 h after deoxyglucose application instead of 20--45 min, (iv) subsequent preparation avoiding aqueous phases at all stages from fixation to autoradiography, and (v) plastic embedding of the tissue such that serial semithin sections of good structural preservation may be routinely cut. Brief aqueous fixation and dehydration at room temperature as has been described for vertebrates apparently cannot preserve stimulus-induced distribution of radioactive label in the brain of the fly Drosophila melanogaster. Aspects of the results that illustrate the potential and some limitations of the present technique are discussed.

Animals↗
PubMed1993

Immunocytochemical localization of amino acid neurotransmitter candidates in the ventral horn of the cat spinal cord: a light microscopic study.

The distribution of immunoreactivities to six amino acids, possibly related to synaptic function, was investigated in the motor nucleus of the cat L7 spinal cord (laminae VII and IX) using a postembedding peroxidase-antiperoxidase technique. Consecutive 0.5 micron transverse sections of plastic-embedded tissue were incubated with antisera raised against protein-glutaraldehyde conjugates of gamma-aminobutyric acid (GABA), glycine, aspartate, glutamate, homocysteate, and taurine. This method allowed localization of the different immunoreactivities in individual cell profiles. The results showed that all these amino acids, except homocysteate, could be clearly detected in either neuronal or glial elements in the ventral horn. In cell bodies of neurons in lamina VII, immunoreactivity was observed for aspartate, glutamate, GABA, and glycine. Adjacent section analysis revealed that combinations of immunoreactivity for glycine/glutamate/aspartate, GABA/glycine/glutamate/aspartate and glutamate/aspartate, respectively, may occur in one and the same cell. In the motor nuclei (lamina IX), immunoreactivity to amino acids was observed in two types of neuron. Large cells, probably representing alpha-motoneurons, were harboring immunoreactivity to both glutamate and aspartate, while a few small neurons in this area displayed a colocalization of glycine, glutamate, and aspartate. Dendrites and axons in the motor nuclei contained glycine/glutamate/aspartate, GABA/glycine/glutamate/aspartate, and glutamate/aspartate immunoreactivities. In both laminae VII and IX, taurine-like immunoreactivity was absent in neuronal cell bodies, but highly concentrated in perivascular cells and small cells with a morphology resembling that of glial cells. A punctate immunolabeling, in all probability representing labeling of nerve terminals, could be demonstrated in the ventral horn for GABA, glycine, and glutamate, but not with certainty for aspartate or taurine. A quantitative estimate of the covering of cell bodies of alpha-motoneuron size by immunoreactive puncta revealed that glycine immunoreactive terminal-like structures were most abundant (covering 26-42% of the somatic membrane), while glutamate immunoreactive terminals were seen least frequently (5-9% covering). GABA-immunoreactive terminals covered from 10 to 24% of the soma surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗
PubMed1987

Catecholaminergic neurons containing GABA-like and/or glutamic acid decarboxylase-like immunoreactivities in various brain regions of the rat.

The coexistence of immunoreactivities for tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD) and/or gamma-aminobutyric acid (GABA) was revealed in various brain regions in colchicine-injected and untreated rats, using the peroxidase-antiperoxidase method. Consecutive 40 micron thick Vibratome sections were incubated in different antisera and those cells which were bisected by the plane of sectioning so as to be included at the paired surfaces of two adjacent sections were identified. The coexistence of the immunoreactivities for TH and GAD or GABA in the same cell could thus be determined by observing the immunoreactivity of the two halves of the cell incubated in two different antisera. In the olfactory bulb, retina, diencephalon, mesencephalic central grey and cerebral cortex, many TH-like immunoreactive neurons also showed GAD-like or GABA-like immunoreactivity, whereas in the substantia nigra, ventral tegmental area and locus ceruleus none of TH-like immunoreactive neurons showed either GAD-like or GABA-like immunoreactivity. In the olfactory bulb, retina and cerebral cortex, the majority of the TH-like immunoreactive neurons were also GAD-like or GABA-like immunoreactive. In the diencephalon of colchicine-injected rats, at least one-third of the TH-like immunoreactive neurons were GAD-like immunoreactive. Using serial 0.5 micron thick plastic-embedded sections, it was shown that immunoreactivities for three antigens, GAD, GABA and TH could occur in the same neurons in the olfactory bulb. These observations indicate the possible coexistence of two classical transmitters. GABA and catecholamine, in various brain regions of the rat.

Animals↗
PubMed1981

Accumulation of myocardial lipid droplets in dexamethasone-treated mice.

Conventionally fixed and plastic-embedded myocardial tissue from mice treated with 1 mg/kg body weight dexamethasone for 48 h was examined in the electron microscope. The dexamethasone-treated mice showed a marked increase in the accumulation of lipid droplets, as compared with control animals. The percent volume of lipid droplets, calculated by morphometric analysis, showed a significant increase in the dexamethasone-treated mice. No other ultrastructural difference between dexamethasone-treated mice and controls was observed.

Animals↗
PubMed1990

An immunohistochemical study of localization of type I and type II collagens in mandibular condylar cartilage compared with tibial growth plate.

Immunohistochemical localization of type I and type II collagens was examined in the rat mandibular condylar cartilage (as the secondary cartilage) and compared with that in the tibial growth plate (as the primary cartilage) using plastic embedded tissues. In the condylar cartilage, type I collagen was present not only in the extracellular matrix (ECM) of the fibrous, proliferative, and transitional cell layers, but also in the ECM of the maturative and hypertrophic cell layers. Type II collagen was present in the ECM of the maturative and hypertrophic cell layers. In the growth plate, type II collagen was present in the ECM of whole cartilaginous layers; type I collagen was not present in the cartilage but in the perichondrium and the bone matrices. These results indicate that differences exist in the components of the ECM between the primary and secondary cartilages. It is suggested that these two tissues differ in the developmental processes and/or in the reactions to their own local functional needs.

Animals↗
PubMed1983

Renal enlargement: comparative autoradiographic studies of 3H-thymidine uptake in diabetic and uninephrectomized rats.

Incorporation of 3H-thymidine into renal cortical tissue has been studied by light microscopic autoradiography in streptozotocin-diabetic rats, uninephrectomized rats, uninephrectomized diabetic rats, insulin-treated diabetic rats and control rats. The percentage of labelled cortical nuclei (the labelling index) was determined separately in glomeruli, proximal tubules and distal tubules after 2, 4 and 6 days on autoradiographs from 1 micron thick plastic embedded sections. The incorporation of thymidine in glomerular nuclei was consistently low (less than 1%) and no differences were found between the control and experimental groups. In both proximal and distal tubules an increase in thymidine incorporation was seen on day 2 followed by a decline on days 4 and 6. The maximal labelling on day 2 in proximal tubules was 9.1% in the uninephrectomized diabetic group, 3.7% in the diabetic group and 1.4% in the uninephrectomized group. In distal tubules the corresponding values were 5.2, 3.5 and 1.1%. The increase in kidney weight after 6 days was 83, 62 and 37%, respectively. Estimates of the net increase in the number of cortical tubular cells in the different experimental groups showed that the kidney enlargement followed different patterns with respect to the extent of cellular hyperplasia and hypertrophy. The kidney growth in uninephrectomized diabetic rats was dominated by tubular cellular hyperplasia, in the diabetic group hyperplasia and hypertrophy participated to approximately the same extent, whereas cellular hypertrophy was most pronounced in the uninephrectomized animals. Nuclear labelling in the insulin-treated diabetic rats was not different from that of control rats and consequently a hyperplastic effect of streptozotocin can be ruled out.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Morphology and transcriptional activity of mouse oocyte chromosomes.

Lampbrush chromosomes of growing amphibian oocytes carry thousands of lateral loops each of which consists of a chromatin fiber heavily encrusted with nascent ribonucleoprotein fibrils. These are believed to be responsible for the accumulation and maintenance of RNA transcripts found stored in the egg. In the case of mammalian oocytes, lampbrush chromosomes are most likely to occur during the major growth phase and also possibly during pachytene-early diplotene stages of meiosis. We have examined pachytene and early diplotene mouse oocyte chromosomes through the light microscope using sections of plastic-embedded material and air dried spreads stained with either silver nitrate or methyl green pyronin. Our results indicates that the projections radiating from the chromosomal axis are bundles of chromatin fibers rather than single fibers covered with an ribonucleoprotein matrix. These bundles may represent partially unfolded chromomeres. The axis itself could be partially dispersed revealing threads surrounding a fine linear element. -- Little is known about chromosome structure in growing mammalian oocytes, the stage when transcriptional activity is likely to be most rapid. In our preparations chromosomes at this stage appear as partially condensed fuzzy threads of relatively uniform width. In some cases, the fuzzy thread is seen to contain a dense linear core in the center. Thus, during the growth phase, the chromosomes retain a relatively condensed axis, a characteristic of meiotic chromosomes in general. RNA-containing material is found diffusely spread within the nucleus but not specifically associated with the chromosomes. Electron microscopic analysis of spread chromatin from growing oocytes demonstrates that most transcription units possess only one or two nascent ribonucleoprotein fibrils while a few have more. These and other published data indicate that mouse oocytes do not have true lampbrush chromosomes at any stage of their development.

Animals↗
PubMed1991

Gold-labelled dystrophin molecule in muscle plasmalemma of mdx control mice as seen by electron microscopy of deep etching replica.

The Duchenne muscular dystrophy product 'dystrophin' has been shown to be located at the inner surface of normal muscle plasma membrane. This study was undertaken to visualize the shape of dystrophin molecules and their topographical distribution at the inner surface of murine skeletal muscle plasma membrane. The immunogold electron microscopy of plastic-embedded quadriceps femoris muscles of six mdx mice and six control mice showed the presence of gold particles along the muscle plasma membrane undercoat of all muscle samples from the control mice without any antibody reaction in the mdx mice muscles. The gold-labelled muscles of six mdx and six control mice were quickly frozen by liquid helium in a rapid-freeze apparatus. High magnification electron microscopy of the quick-freeze, deep-etch, rotary-shadow replicas of the gold-labelled muscles demonstrated the presence of dystrophin molecules associated with gold particles at the cytoplasmic surface of mdx control mice. The dystrophin molecules displayed a variety of shapes, such as rods with a reduction in diameter from one end to the other end and/or with the enlargement of their end(s). These dystrophin molecules were incorporated in the meshwork of muscle plasma membrane-associated cytoskeletons.

Animals↗
PubMed1981

Presence of a smooth muscle system in aortic valve leaflets.

The location and the spatial arrangement of smooth muscle cells in aortic valves have been assessed by a systematic analysis of serial semithin sections of plastic embedded porcine and human aortic leaflets, combined with an electron microscope study. The investigation showed that smooth muscle cells, either single and arranged in thin bundles, and other cell types such as myofibroblasts are constantly present in the aortic valve leaflets. In addition, it was possible to devise a model of the three dimensional, specific organization of the smooth muscle bundles which can be interpreted as an intrinsic muscle system of the leaflets. As the muscular elements might play an active role in the normal functioning of the valve, their presence should be taken into account in designing (bio)prosthetic leaflets and in the evaluation of valve pathology.

Animals↗
PubMed1982

Development of the early human ovary and role of the mesonephros in the differentiation of the cortex.

The morphogenesis of the primary gonadal differentiation, of the sexual differentiation and the growth of the ovarian cortex during its early period have been studied on 10 human embryos between 12 and 95 mm CR-length. Semithin sections of glutaraldehyde-OsO4-fixed and plastic-embedded material were used to demonstrate the structural events on a cellular level. The primary gonadal blastema within the genital ridge is formed by two types of somatic cells; cells segregated from the mesonephros and cells of the proliferating coelomic epithelium. The two types of cells show a tendency to intermingle and they enclose the immigrating primordial germ cells. In the female gonad the indifferent period terminates between day 40 and 42 of ovulation age (20 to 23 mm CR-length). Between day 40 and 50 the blastemal content of the indifferent gonad is remodelled and an ovarian cortex differentiates. Cellular strands extending from the primary blastema and strands from the superficial blastemal layer contribute to the formation of the cortex. Within the newly formed medulla, remnants of the disintegrating primary blastema differentiate into medullary cords. Cells of mesonephric origin which invade the growing cortex via the rete blastema interact with cells deriving from the superficial epithelium, and both exert their opposite influence on the germ cells. While female sexual differentiation is characterized by failure of the dark mesonephric cells to completely penetrate the gonadal blastema, the morphogenetic process resulting in the formation of the ovarian cortex shows a strong invasion of the cortex by the dark mesonephric cells. Dark cells advance at the most superficial layer of the cortex and increase in number at the deeper level of the cortex. Onset of oogonial proliferation and meiotic prophase seems to depend on the numerical proportion between the activating dark and the inhibiting light supporting cells.

Cell Differentiation↗