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PubMed1988

Quantitative assessment of taurine-like immunoreactivity in different cell types and processes in rat cerebellum: an electronmicroscopic study based on a postembedding immunogold labelling procedure.

Ultrathin sections of plastic-embedded rat cerebella were incubated with an antiserum against conjugated taurine and subsequently treated with a secondary antibody coupled to colloidal gold. The density of gold particles in various cellular profiles was calculated with the assistance of a computer. In the cerebellar cortex the highest density was found in the somata, dendrites, and dendritic spines of the Purkinje cells, supporting parallel light-microscopical observations in postembedding stained semithin sections from the same tissue blocks. The remaining profiles could be divided into three groups according to their immunolabelling intensity, in descending order: 1) somata and processes of granule and Golgi cells; 2) somata and processes of stellate, basket, and glial cells, and 3) mossy fiber terminals. In a representative experiment, the structures in the first and second groups showed gold particle densities in the range of 19-25%, and 4-11%, respectively, of that in the Purkinje cell somata (values corrected for background) whereas the particle density in the mossy fiber terminals was not significantly above background level. In the cerebellar nuclei, taurine-like immunoreactivity was concentrated in terminals that typically established symmetric or intermediate type contacts with weakly labelled dendrites and cell bodies. These terminals, which shared the ultrastructural features of Purkinje cell terminals, showed an average gold particle density that was about 60% higher than that of the Purkinje somata. For specificity control, ultrathin sections containing a series of different amino acid conjugates were incubated in the same drops of sera as the tissue sections. The highly selective labelling of the taurine conjugate indicated that the distribution of gold particles in the tissue was not confounded by crossreactivity with GABA, glutamate or other common amino acids but adequately reflected the distribution of fixed taurine. For additional control of specificity, the taurine antiserum was applied to the soluble fraction of a rat brain extract separated by thin layer chromatography. In this system the taurine antiserum stained a single spot that comigrated with free taurine. The present results suggest that all cell types and processes in the rat cerebellum (with the exception of the mossy fiber terminals) contain taurine. However, the concentration of taurine appears to vary considerably among the different cell types and may also differ between different parts of the same neuron.

Animals↗

PubMed1995

Temporal evolution of neuropathologic changes in an immature rat model of cerebral hypoxia: a light microscopic study.

The sequential evolution of neuropathologic changes was studied in an immature model of cerebral hypoxia-ischemia. According, 7-day postnatal rats were subjected to unilateral common carotid artery ligation combined with 2 h of hypoxia (breathing in 8% oxygen) and their brains were examined by light microscopy at recovery intervals ranging from 0 to 3 weeks. Immediately following hypoxia, a large area with a pale staining border was noted occupying most of the cerebral hemisphere ipsilateral (IL) to the occluded common carotid artery; in approximately half of the brains the dorsomedial cortex of the contralateral (CL) hemisphere was also involved. Most neurons in the pale area had nuclei containing a coarse granular condensation of chromatin. Within a few hours, the majority of neurons in the IL hemisphere had developed pyknotic nuclei and clear or eosinophilic perikarya. After 24 h these changes had evolved in the majority of brains into coagulation necrosis (infarction) in the IL hemisphere and foci of selective neuronal necrosis in the CL cortex. Within a few days infarcts became partially cavitated, and by 3 weeks a smooth-walled cystic infarct had developed. Activated microglia/macrophages and reactive astrocytes were first seen at 4 and 24 h, respectively. No parenchymal neutrophilic infiltrate was seen at any time point.

Animals↗

PubMed1981

Silver staining of the neuronal nucleus in electron microscopy: nucleolar organization in supraoptic nuclei of the rat hypothalamus.

The present paper describes a simple, efficient method for silver impregnation of supraoptic nuclei of the rat hypothalamus using a modification of the ammoniacal silver technique of Cajal (1903). This procedure, involving a silver-developer sequence in tissue blocks prior to plastic embedding, permits the simultaneous study of Ag-impregnated supraoptic neurons at both light and electron microscopic levels. Visualization of secretory magnocellular neurons impregnated by this technique using the electron microscope reveals a good preservation of nuclear structures. A selective accumulation of silver grains was observed over heterochromatin clumps and nucleoli, which allows the identification of the nucleolar fibrillar centers and also the dense fibrillar component as the main areas involved in the silver reaction. The meaning of such a silver-distribution pattern is discussed in the light of recent ultrastructural and biochemical data.

Animals↗

PubMed1983

The "accessory body" of Cajal in the neuronal nucleus. A light and electron microscopic approach.

The present light and electron microscopic study deals with the morphology and staining properties of two intranuclear inclusions - the "accessory body" of Cajal and the "coiled body"--in the supraoptic nuclei of adult rat hypothalamus, and supports the assumption that these structures represent the same intrinsic component of the neuronal nucleus. Consequently, we propose to term it "accessory body". The structure of this body was visualized by several different staining procedures: conventional electron microscopic techniques, a silver reaction, and the regressive EDTA staining for ribonucleoproteins. The silver-impregnation method employed here, which consists of a silver development sequence on hypothalamic tissue blocks prior to plastic embedding, permitted the study of supraoptic neurons at both light and electron microscopic levels. The nature and origin of "accessory bodies" are suggested and their possible functional role is briefly discussed.

Animals↗

PubMed1986

Dendrite bundles in lamina II/III of the rabbit neocortex.

The present investigation systematically analyzes the course and arrangement of dendrites in lamina II/III of the visual and the motor cortex of the rabbit on the basis of Klüver-PAS stained 10 micron paraffin sections, 1 micron plastic-embedded semithin sections and ultrathin sections. In both areas the dendritic pattern of lamina II/III is characterized by vertical bundles reminiscent of the pattern in lamina IV/V. The bundles form in the upper half of lamina II/III. They consist mainly of apical dendrites from lamina II/III pyramidal cells and receive branches from dendrite bundles in lamina IV/V, i.e., branches from apical dendrites arising from lamina V pyramidal cells. Besides these features in common, the lamina II/III bundles in the visual cortex on the one hand and in the motor cortex on the other differ with regards to the size and shape of individual bundles as well as to the extent of connections with bundles in lamina IV/V.

Animals↗

PubMed1994

Glutamate immunoreactivity is enriched over pinealocytes of the gerbil pineal gland.

Mammalian pinealocytes have been shown to contain synaptic-like microvesicles with putative secretory functions. As a first step to elucidate the possibility that pinealocyte microvesicles store messenger molecules, such as neuroactive amino acids, we have studied the distributional pattern of glutamate immunoreactivity in the pineal gland of the Mongolian gerbil (Meriones unguiculatus) at both light- and electron-microscopic levels. In semithin sections of plastic-embedded pineals, strong glutamate immunoreactivity could be detected in pinealocytes throughout the pineal gland. The density of glutamate immunolabeling in pinealocytes varied among individual cells and was mostly paralleled by the density of immunostaining for synaptophysin, a major integral membrane protein of synaptic and synaptic-like vesicles. Postembedding immunogold staining of ultrathin pineal sections revealed that gold particles were enriched over pinealocytes. In particular, a high degree of immunoreactivity was associated with accumulations of microvesicles that filled dilated process terminals of pinealocytes. A positive correlation between the number of gold particles and the packing density of microvesicles was found in three out of four process terminals analyzed. However, the level of glutamate immunoreactivity in pinealocyte process endings was lower than in presumed glutamatergic nerve terminals of the cerebellum and posterior pituitary. The present results provide some evidence for a microvesicular compartmentation of glutamate in pinealocytes. Our findings thus lend support to the hypothesis that glutamate serves as an intrapineal signal molecule of physiological relevance to the neuroendocrine functions of the gland.

Animals↗

PubMed1981

Early morphological changes in rat cerebellum caused by a single dose of methylmercury.

A single dose of 10 mg methylmercury chloride per kg body weight was given to 30 days old rats and to adult rats (180-200g)(. This resulted in brain levels of 1.4-2.2 micrograms Hg/g wet weight. In the young rats electron microscopic morphometry showed swelling of the granule cells. The extent of changes was more pronounced in the cerebellar hemispheres than in the vermis and flocculus. At 7 days after giving the methylmercury the granule cells appeared to have returned to normal. Methylmercury produced both light and electron microscopic changes in cerebellar neurons of adult (180-200 g) rats 3 days after dosing. 2.5-10% of the granule cells appeared dark and condensed in toluidine blue stained semithin sections of perfusion fixed and plastic embedded material. In control animals the comparable percentage never exceeded 1. By electron microscopic morphometry the dark cells proved to be shrunken to 70%, whereas the remaining light granule cells were swollen to 130% of the normal cell volume. The heterochromatin and mitochondrial volumes per cell remained constant in both dark and light cells from methylmercury treated animals. In the purkinje cells from both young and adult rats, geometrical changes in the cisternae of the granulated endoplasmic reticulum were evident. The swelling and shrinkage of the granule cells is supposed to be due to impaired electrolyte control and the disorganized granulated endoplasmic reticulum of the Purkinje cells may be related to the deleterious effect on protein synthesis.

Age Factors↗

PubMed1994

The nature and time course of neuronal vacuolation induced by the N-methyl-D-aspartate antagonist MK-801.

N-Methyl-D-aspartate (NMDA) antagonists cause neuronal vacuolation in the posterior cingulate and retrosplenial cortex of the rat. Because the nature of neuronal pathologic changes due to NMDA antagonists may affect the potential clinical use of this class of drugs, we undertook experiments to define the nature and time course of the vacuolation caused by high-dose (5 mg/kg) MK-801 (dizocilpine, 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine). Ultrastructural examination revealed the vacuoles to be not a form of hydropic cellular degeneration, but rather a dilatation of several intracellular compartments, chiefly endoplasmic reticulum and mitochondria. Study of the time course of the alterations revealed no light or ultrastructural features of neuronal necrosis in over 1 thousand neurons examined in layers 3 and 4 of the cingulate and retrosplenial cortex, 153 of which were vacuolated. The vacuoles resolved over time by decreasing in magnitude. Oxalate-pyroantimonate methodology revealed no redistribution of cell calcium in either vacuolated or non-vacuolated neurons. At 6 h, when vacuoles were consistently prominent in glutaraldehyde-fixed plastic-embedded tissue, a separate series of experiments was undertaken to vary methods of tissue preparation, and determine conditions under which vacuolation occurs. Frozen sections revealed no vacuoles. Subsequent paraffin embedding of the previously frozen tissue revealed no vacuoles, but vacuoles were seen in paraffin after perfusion fixation. Immersion fixation with brain refrigeration for 12 h prior to fixation revealed no vacuoles. Alcohol fixation also led to no visible vacuoles.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

The impact of renal growth, regression and regrowth in experimental diabetes mellitus on number and size of proximal and distal tubular cells in the rat kidney.

Diabetic renal growth, regression and regrowth was studied using stereological methods on perfusion-fixed rat kidneys. The study lasted 13 weeks and comprised one control group and three diabetic groups. The first diabetic group was hyperglycaemic for 13 weeks. The second group was hyperglycaemic for 10 weeks and then normoglycaemic for 3 weeks. The third group was similar to the second group except that during the last week the animals were again hyperglycaemic. Using an optical dissector on the plastic-embedded kidney slices, the number and size of proximal and distal tubular cells were estimated. The number of proximal and distal tubular cells increased by 37% and 36% during 13 weeks of experimental diabetes and the mean volume of the proximal tubular cells increased by 12% whereas the 16% increase in mean tubular cell volume was only borderline significant as compared to the control group. Normoglycaemia for 3 weeks normalized the mean volume of distal tubular cells but the proximal tubular cells tended to be 7% smaller than those in control rats. The number of proximal cells remained increased by 21% compared with the control rats and the number of distal tubular cells retained a 17% insignificant increase. After regrowth the volume of proximal tubular cells was 20% greater than in the second diabetic group and the other parameters were unchanged. In conclusion, 13 weeks of experimental diabetes induced formation of 36% more tubular cells that were enlarged only by about 14%. Normoglycaemia for 3 weeks failed to normalize the cell number. Repeated hyperglycaemia for 1 week after 2 weeks of normoglycaemia increased the size of the proximal tubular cells.

Animals↗

PubMed1977

Subchondral cancellous bone in osteoarthrosis and rheumatoid arthritis of the femoral head. A quantitative histological study of trabecular remodelling.

Thirty-three femoral heads taken from patients undergoing total hip replacement for osteoarthrosis and rheumatoid arthritis have been examined in undecalcified, plastic-embedded sections, using a quantitative histological methods. The characteristic change of trabecular remodelling in the subchonfral cancellous bone of the femoral heads was that total osteoid surface in the medial osteoarthritis (medial O.A.) and rheumatoid arthritis (R.A.) was significantly less than in the proximal osteoarthrosis (proximal O.A.) (P less than 0.01). Resorption surface was slightly greater in R.A. than in proximal O.A. (P less than 0.05). Bone area in the deep area was not different in each group. In the superficial area it was significantly less in medial O.A. than in proximal O.A. (P less than 0.05). The average width of trabeculae was slightly less in medial O.A. and R.A. It is suggested that subchondral trabecular remodelling may be a basic pathological process of general importance in the evolution of these diseases.

Adult↗

PubMed1981

Ultrastructural changes to the cochlea resulting from impulse noise.

Following impulse noise trauma to chinchillas, observation of plastic-embedded surface preparations of the organ of Corti showed no consistent relationship between cochlear hair cell loss and permanent hearing loss (Hamernik et al. 1980). In some animals there was a loss of hearing when hair cells were present. The cochleas from that experiment were examined with transmission electron microscopy to determine at the ultrastructural level if there was damage to the sensory cells that would explain the change in threshold sensitivity. Ultrastructural changes in cochlear hair cells include an increase in lysosomes, multivesicular bodies, vacuolization of subsurface cisternae, and proliferation of Hensen bodies. These changes are observed in all experimented animals. Alterations to the ultrastructure of the stereocilia vary from animal to animal and on the outer hair cells, the changes include loosening of the stereocilia membranes, loss of stiffness, fusion of the stereocilia and disintegration of the rootlets. These changes are observed only in animals that have a permanent threshold shift after noise trauma.

Animals↗

PubMed1987

Ultrastructural localization of Helix pomatia lectin-binding sites in mouse lung elastic fibers.

Helix pomatia (Snail) lectin complexed with colloidal gold (HPL-gold) recognized binding sites on elastic fibers in plastic embedded sections of lung tissue from mice of several ages. Deposition of the lectin-gold particles was examined by electron microscopy. Structures such as the elastic laminae of pulmonary vessels and elastic fibers throughout the lung was specifically and intensely decorated by the HPL-gold complex and easily visualized. The binding of the HPL-gold particles was primarily to sites on the amorphous component of elastin, to the virtual exclusion of the microfibrillar elastin elements, collagen fibers and other components of the extracellular matrix. In addition, moderate age differences in the binding of HPL-gold to elastin were apparent. These observations appear to be the first demonstration of the presence, in the amorphous component of elastin, of glycoconjugates that are specifically recognized by HPL and suggest a method by which the involvement of glycoconjugates in lung elastogenesis could be explored.

Animals↗

PubMed1989

Standardization of the Feulgen-Schiff technique. Staining characteristics of pure fuchsin dyes; a cytophotometric investigation.

Four fuchsin analogues (Pararosaniline, Rosaniline. Magenta II and New Fuchsin) usually found in Basic Fuchsin have been applied as chemically pure dyes to the Feulgen-technique. Total nuclear absorption and wavelength of the absorption maximum were measured by microspectrophotometry in Feulgen stained cytological and plastic embedded histological liver samples, and in lymphocyte nuclei in human peripheral blood smears; absorption spectra of Feulgen stained DNA-polyacrylamide films were determined by spectrophotometry. The grey value distribution of tetraploid liver cell nuclei was calculated with an image analyzer. The staining characteristics of the pure dyes were compared to commercial fuchsin samples from various suppliers. Reverse phase thin layer chromatography was used for characterization and qualitative separation of commercial batches. Pure fuchsin analogues were all equally suitable for Feulgen staining: with respect of staining intensity all pure fuchsin dyes gave nearly identical results with a bathochromic shift of the absorption maximum from Pararosaniline to New Fuchsin of about 8 microns. Differences in staining results observed among the commercial dyes were due to varying dye content, contamination with an acridine-like fluorescent compound or simply mislabelling of samples. Pure Pararosaniline is recommended for a standard Feulgen technique.

Chemical Phenomena↗

PubMed1985

Subcellular localization of serotonin immunoreactivity in rat enterochromaffin cells.

Serotonin immunoreactive material was localized to rat enterochromaffin cells (EC cells) at the subcellular level using antibodies to serotonin (5-HT) raised in rabbits. Ultrathin sections from paraformaldehyde fixed plastic embedded tissues were directly labelled with the 5-HT antiserum, using the protein A-gold technique to visualize the immunoreaction. The 5-HT immunoreactivity (5-HT-IR) in the rat gastrointestinal mucosa was exclusively localized to epithelial EC cells with a low background over other epithelial non-enterochromaffin cells. Quantitative evaluation of the immunoreaction revealed that most of the 5-HT-IR in the cytoplasm of EC cells (60%) was located over the dense cores of the secretory granules. However, a significant part of the cytoplasmic 5-HT-IR (40%) was located outside the dense cores of the secretory granules which suggests that different forms of 5-HT storage may exist.

Animals↗

PubMed1989

Glutamate-like immunoreactivity revealed in rat olfactory bulb, hippocampus and cerebellum by monoclonal antibody and sensitive staining method.

Although there is good evidence favoring L-glutamate as a major excitatory amino acid transmitter, relatively little is known about the distribution of nerve terminals using this substance. A method visualizing glutamate-like immunoreactivity at the light microscopic level by means of a monoclonal antibody, mAb 2D7, is described. --The antigen used for immunization was a glutaraldehyde-linked glutamate-BSA conjugate, and hybridomas were differentially screened by ELISA for production of antibodies recognizing glutamate- but not aspartate-BSA. The crossreactivity of 'anti-glutamate' mAb 2D7 as estimated in absorption tests was low even with conjugates closely related to glutamate-BSA.--Semithin sections from rapidly perfusion-fixed, plastic-embedded rat brain tissues were etched and stained by a combination of the peroxidase-antiperoxidase method and silver enhancement of the diaminobenzidine reaction product. Only this amongst several other immunohistochemical methods tried produced labeling patterns which showed terminal-like elements in brain regions such as olfactory bulb, hippocampus and cerebellum, and which were mostly consistent with already available information on systems using glutamate as neurotransmitter. Particularly striking was the staining of elements reminiscent of mossy fiber terminals in hippocampus and cerebellum as well as of cerebellar parallel fiber terminals.

Animals↗

PubMed1979

Correlation of individual skeletal muscle fibres from "semithin" sections stained with p-phenylene-diamine and histochemical sections incubated for myofibrillar ATP-ase.

Muscle fibres in transverse "semithin" sections of osmium-fixed and plastic embedded muscle tissue stained with p-phenylene-diamine has been classified into three types (M, INT and H) and correlated to the same fibres in neighboring sequential preincubated myofibrillar ATP-ase sections. A close correlation, but no equality was found between the fibre types classified according to the ATP-ase and the p-phenylene-diamine methods. On the average, the content of mitochondria and fat droplets was highest in the ATP-ase classified type I fibres (dominated by M fibres), and higher in the endurance trained than in the untrained subjects.

Adenosine Triphosphatases↗

PubMed1983

Tetracycline cathodoluminescence in bone, dentine and enamel.

The emission of light under electron bombardment (cathodoluminescence-CL) in an SEM has been demonstrated from tetracycline and tetracycline labelled areas of bone, dentine and secretory and maturation zone enamel. CL images could be compared with backscattered (BSE) and secondary (SE) electron images in (a) fresh, air-dried or freeze-dried samples taken without fixation at a short term after labelling: (b) polished block faces of fixed, plastic embedded tissue and (c) cut section surfaces cut through fixed and unfixed tissue. These results indicate a considerable future potential for the tetracycline-CL-SEM mode in experimental studies on teeth and histopathological and growth studies of bone.

Aging↗

PubMed1978

An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues.

Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described. To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixature is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.

Animals↗