[Biological structures in photoelectron emission microscope. Semithin sections of chicken liver in embedding materials Durcupan, Vestopal, Araldite, and Epon].
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Animals↗
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Oriented embedding of biological materials and accurate localization for ultrathin sectioning.
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Gelatin↗
The morphology of excitatory central synapses: from structure to function.
Synapses are the key elements for signal transduction and plasticity in the brain. For a better understanding of the functional signal cascades underlying synaptic transmission, a quantitative morphological analysis of the pre- and postsynaptic structures that represent morphological correlates for synaptic transmission is important. In particular, realistic values of the number, distribution, and geometry of synaptic contacts and the organization of the pool of synaptic vesicles provide important constraints for realistic models and numerical simulations of those parameters of synaptic transmission that, at present, are still not accessible to experiment. Although all synapses are composed of almost the same structural elements, the composition of these elements within a given synapse and the microcircuit in which they are embedded are the deciding factors determining its function. One possible way to analyze these structures is by computer-assisted three-dimensional reconstructions of synapses and their subsequent quantitative analysis based on ultrathin serial sections. The present review summarizes and discusses the morphology of five central excitatory synapses that are quantitatively well described: (1) a giant synapse, the so-called Calyx of Held, in the medial nucleus of the trapezoid body in the auditory brain stem, (2) the mossy fiber terminal establishing synapses with multiple cerebellar granule cell dendrites, (3) the mossy fiber bouton in the hippocampus predominantly terminating on proximal dendrites of CA3 pyramidal neurons, (4) the climbing fiber-Purkinje cell synapse in the cerebellum, and (5) cortical input synapses on the basal dendrites of layer 5 pyramidal cells. The detailed morphological description of these synaptic structures may help to define the morphological correlates of the functional parameters of synaptic transmission, such as the readily releasable pool of synaptic vesicles, of release, and of the variability of quantal size and might therefore explain the existing differences in the function between individual synapses embedded in different microcircuits.
Animals↗
Prevention of negative chemography in phenylenediamine stained autoradiographs of plastic semithin sections.
Negative chemography is the loss of latent image during autoradiographic exposure, due to reactive groups in the specimen. Tissue fixed with glutaraldehyde and osmium tetroxide, block stained with p-phenylenediamine and embedded in Epon for light microscope sections causes intense negative chemography when autoradiographed by dipping in Ilford K2 emulsion: this cannot be completely prevented by separating section from emulsion by means of a layer of evaporated carbon. Chemical treatment of the sections before autoradiography may reduce the chemography. Treatment with 1% hydrogen peroxide for 15 min reduced it to such an extent that subsequent coating with 5 nm carbon abolished it. Material block stained in this way gave excellent contrast, both for light and electron microscopy.
Animals↗
Embedding of bone samples in methylmethacrylate: an improved method suitable for bone histomorphometry, histochemistry, and immunohistochemistry.
Methylmethacrylate (MMA) embedding of undecalcified bone biopsies is a technique widely used for bone histomorphometry. However, conventional MMA embedding causes almost complete loss of enzyme activity and protein antigenicity in the tissues. Recently, an MMA embedding technique has been reported that preserves enzyme activity and antigenic determinants in bone tissue. We describe here a modification of this embedding method. For our modified MMA embedding process, commercially available methacrylates can be used without purification, and the histologic quality of bone sections is comparable to that of conventionally MMA-embedded bone specimens. The technique reported here can be employed for embedding of larger bone samples and is suitable for histochemical and immunohistological applications as well as for routine bone histomorphometry. By addition of methylbenzoate during infiltration and polymerization of the plastic, the antigenicity of the tissue was improved. As applications of this novel technique, demonstration of alkaline phosphatase and tartrate-resistant acid phosphatase as well as positive labeling of Kupffer cells and osteoclasts with the monoclonal antibody ED1 in sections of liver, tibiae, and vertebrae of 3-month-old rats was demonstrated. The method described here might be useful for the inclusion of histochemical and immunohistological methods into bone histomorphometry.
Animals↗
Effect of relative humidity during tabletting on matrix formation of hydrocolloids: densification behavior of cellulose ethers.
The aim of this research was to investigate the elastic-plastic deformation behavior of the cellulose ethers hydroxypropylmethylcellulose (HPMC), hydroxyethylmethylcellulose (HEMC), and sodium carboxymethylcellulose (NaCMC) at relative humidities (RH) of 38, 57, and 75% and assess how the release of drugs embedded in such matrices is affected by the inner structure of the tablets formed during tabletting. Sorption and desorption isotherms and glass transition temperature were determined between 32 and 75% RH. The materials were equilibrated at 38, 57, and 75% RH and tabletted to a range of graded maximum relative densities. Pressure-time and displacement-time curves were analyzed by use of the Heckel function and a modified Weibull function (pressure-time only). After equilibration at the different RHs, all materials were in the glassy state. The respective degrees of polymerization had negligible effect on the absolute content of water, the sorption isotherms, and finally the densification behavior. At 38% RH, NaCMC contains the same amount of water as HPMC and HEMC, but deforms less plastically than the latter. This is attributed to tight binding of the water of hydration in the former. With increasing RH, NaCMC becomes only a little more plastic than both HPMC and HEMC, although it contains more than twice the amount of water. The binding strength of water and its molecular mobility, not the amount, seems to determine the readiness for volume reduction under load.
Carboxymethylcellulose Sodium↗
Co-culture with human fibroblasts increases the radiosensitivity of MCF-7 mammary carcinoma cells in collagen gels.
The growth and differentiation of normal and neoplastic epithelial cells may be regulated by the presence of adjacent normal tissues and cells, particularly stromal fibroblasts. However, the influence of normal fibroblast-tumor cell interactions on the response of malignant epithelial cells to radiation has not been adequately investigated nor has the possible role played by a 3-D environment in such modulation. We addressed this question by embedding MCF-7 mammary carcinoma cells into a collagen lattice, alone or mixed with HSF human dermal fibroblasts, and kept the gels anchored to the plastic surface or suspended in the culture medium. Some gels served as controls and others were irradiated with 6 MV photons fractionated into 3 daily doses totaling 5 or 10 Gy. After 2 or 7 days from the last treatment (7 or 12 days in culture, respectively), gels were processed in 1 of 2 ways: overall cell survival was determined by the MTT assay, while the survival of MCF-7 cells was selectively detected by a clonogenicity assay. Under these experimental conditions, we found that, in the presence of HSF fibroblasts, the growth of MCF-7 cells was restrained and radiosensitivity increased compared with MCF-7 cells cultured alone. For example, while the average number of MCF-7 foci/gel recovered from control gels with MCF-7 cells alone was 2,460 on day 7 and 3, 290 on day 12 of culture, it was 4 to 5 times lower (p < 0.001) in control gels with mixed MCF-7 and HSF cells. Radiation affected severely the survival of MCF-7 cells in all experimental groups but not sufficiently to mask the differences. For example, following exposure to the low dose of 5 Gy, the average number of MCF-7 foci/gel recovered from MCF-7-containing gels was 590 on day 7 and 329 on day 12 of culture, whereas numbers from the gels containing mixed MCF-7 and HSF cells were only 218 and 73, respectively (p < 0. 003 in both cases). HSF fibroblasts did not grow in our system, but they contracted strongly anchored and floating gels.
Breast Neoplasms↗
A simple technique to facilitate the ultrastructural analysis of cells in soft agar culture systems: demonstration of the development in vitro of morphologically mature mast cells and phagocytic macrophages from the bone marrow cells of genetically mast cell-deficient W/Wv or congenic normal mice.
We developed a simple technique that greatly facilitates the ultrastructural examination of cells growing in small, widely dispersed colonies in agar medium, and used the method to examine the development of morphologically mature mast cells and actively phagocytic macrophages in agar cultures of mouse bone marrow cells. The bone marrow cells of genetically mast cell-deficient WBB6F1-W/Wv or congenic normal (+/+) mice were cultured in semisolid agar medium supplemented with supernatants of concanavalin A-stimulated splenocytes. To prepare the colonies of hematopoietic cells for transmission electron microscopy, all the colonies within the agar-containing medium in a 96-well culture plate were removed with a Pasteur pipette and placed in a dilute, mixed aldehyde fixative. After fixation, the agar still enmeshing and separating individual colonies of cells was melted at 94 degrees C, rapidly mixed with molten 2% agar in a microfuge tube, centrifuged for 1 minute, and then the plastic tube was cooled in ice for 30 minutes. The plastic was removed with a razor blade, the agar block was hemisected from top to bottom, and then the blocks were processed for electron microscopy, embedded flat, and sectioned for light and electron microscopy. The culture conditions tested resulted in the development of morphologically mature mast cells and actively phagocytic macrophages, whether cultures were initiated with bone marrow cells from WBB6F1-W/Wv or congenic +/+ mice.
Agar↗
Araldite F as injection material for quantitative morphology of cerebral vascularization.
This study deals with the applicability of Araldite F as injection medium for quantitative morphology of the cerebral vascularization. The physical properties of the plastic that are essential for obtaining optimal results have been determined and tested by injection into the cerebral vessels of ten dog brains and five human brains. The dynamic viscosity (at 20 degrees C) of the plastic is 1.5 times that of blood. The polymerization time (+/- 45 min) is long enough to fill the whole vascular tree in a large organ such as the human brain. The plastic shows minimal volume shrinkage (3.90% +/- 0.05%), and the final cast is suitable for scanning electron microscopy (SEM) investigation after corrosion and histological morphometrical examination after embedding in 2-hydroxyethyl-methacrylate (GMA). Special attention has been paid to the conditions under which injection of Araldite F into the cerebral arteries must take place to obtain the most accurate quantitative data on cerebral vascularization and vascular area distribution. Simultaneous injection of the major cerebral arteries of fresh autopsy material, after having removed blood clots by perfusing Kabikinase, is recommended.
Animals↗
The in vitro effect of pulpal pressure and luting agent on tensile bond strength of complete cast crowns.
STATEMENT OF PROBLEM: The degree to which pulpal pressure may affect bond strength of complete cast crowns is unknown. PURPOSE: The aim of this in vitro study was to evaluate the effect of simulated pulpal pressure on the tensile bond strength of complete cast crowns luted with 2 different cements. MATERIAL AND METHODS: Forty-eight human mandibular canine teeth were cleaned and stored in saline solution. The crowns were prepared by 1 investigator, and standardization of the preparation was accomplished by fixing a dental handpiece in a parallelometer. Uniform grooves, 0.5 mm in depth, were prepared with burs with depth guides. The complete crowns were prepared with a 0.5-mm shoulder margin. Teeth were randomly divided into 2 groups of 24 teeth each (Group I and Group II). In Group I, bonding and tensile test procedures of the teeth were carried out under simulated pulpal pressure (15 cm of saline solution). In Group II, simulated pulpal pressure was not used. The roots were removed 1 mm below the cementoenamel junction to create direct communication with the pulp chamber. The remaining pulpal tissues were carefully removed, and crowns were embedded in acrylic resin. The acrylic resin was then penetrated by a stainless steel tube that connected the pulp chamber and the barrel of a disposable plastic 5-ml syringe. The pulp chambers were filled with physiological saline solution under elevated pressure to locate the area of greatest permeability on the dentinal surface. Crowns were cast (Co-Cr alloy) with a 20-mm bar to allow testing of the bond strength. Each of the 2 groups were further divided into 2 luting cement groups of 12 each (Group I, A and B, and Group II, A and B). Group IA/IIA and Group IB/IIB specimens were luted with a polycarboxylate luting cement (Poly-F Plus) and an adhesive luting cement (Superbond C&B), respectively. After storage in distilled water for 24 hours, all specimens were subjected to a tensile bond test in a universal testing machine at a crosshead speed of 0.5 mm/min until failure. The maximum load at fracture (Newton) was recorded. The results were then evaluated with 2-way analysis of variance and Tukey's honestly significant difference tests (alpha=.05). RESULTS: Simulated pulpal pressure increased the bond strength of cast complete crowns cemented with an adhesive luting agent (P=.01). No significant difference was found in the bond strength of complete cast crowns cemented with polycarboxylate cement with or without pulpal pressure. Superbond C&B adhesive luting agent showed significantly higher bond strength values for Group I (388.9+/-32.7) and Group II (300.9+/-66.8), when compared with polycarboxylate cement for Group I (221.3+/-17.3) and Group II (186.8+/-38.5) (P=.001). CONCLUSION: Simulated pulpal pressure had a positive effect on the retention of complete cast crowns when cemented with Superbond C&B adhesive luting agent. Superbond C&B significantly increased the retention of crowns in either the presence or absence of pulpal pressure.
Analysis of Variance↗
GABAergic innervation of corticotropin-releasing hormone (CRH)-secreting parvocellular neurons and its plasticity as demonstrated by quantitative immunoelectron microscopy.
GABA has been identified as an important neurotransmitter in stress-related circuitry mediating inhibitory effects on neurosecretory neurons that comprise the central limb of the hypothalamo-pituitary-adrenocortical axis. Using combinations of pre-embedding immunostaining and postembedding immunogold methods at the ultrastructural level, direct synaptic contacts were revealed between GABA-containing terminals and neurosecretory cells that were immunoreactive for corticotropin-releasing hormone (CRH) in the hypothalamic paraventricular nucleus (PVN). The vast majority of axo-dendritic GABA synapses was symmetric (inhibitory) type, and 46% of all synaptic boutons in the medial parvocellular subdivision of the PVN were immunoreactive to GABA. Using the disector method, an unbiased stereological method on serial ultrathin sections, the total calculated number of synaptic contacts within the medial parvocellular subdivision of the PVN was 55.4 x 10(6)/mm(3). On CRH-positive profiles 20.1 x 10(6) GABAergic synaptic boutons were detected per mm(3) in control, colchicine-treated rats. In the medial parvocellular subdivision, 79% of GABAergic boutons terminated on CRH neurons. Following adrenalectomy, which increases the synthetic and secretory activities of CRH neurons, the number of GABAergic synapses that terminate on CRH-positive profiles was increased by 55%. GABA-containing boutons appeared to be swollen, while the contact surfaces of cellular membranes between GABAergic boutons and CRH-positive profiles were shorter in adrenalectomized animals than in controls. Our data provide ultrastructural evidence for direct inhibitory GABAergic control of stress-related CRH neurons and suggest a pivotal role of GABA-containing inputs in the functional plasticity of parvocellular neurosecretory neurons seen in response to adrenalectomy.
Adrenalectomy↗
The demonstration of acetylcholinesterase in plastic sections. Its application as a marker of early neuronal development.
In this paper a histotechnical procedure with respect to the demonstration of acetylcholinesterase in Technovit 7100 (a recently developed GMA) sections is described. From fixation up to embedding several procedural steps were varied and tested, resulting in a suitable procedure. The embedded tissue or sections can be stored for months at 7 degrees C with only a slight reduction of the enzymic activity. The enzyme also appears relatively temperature resistant and independent of section thickness (2-10 microns). The enzymic activity remains constant during incubation. The technique has been applied to our subject of study in order to qualify and quantify several aspects of the development of neuroblasts.
Acetylcholinesterase↗
Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow.
In the presence of beta-glycerophosphate and vitamin C, cultures of normal mouse bone marrow cells form three-dimensional structures that stain positive with the Von Kossa technique and express alkaline phosphatase (ALP), collagen type I, and osteocalcin. Little is known about the characteristics and frequency of the cells that contribute to this phenomenon. Most likely, mature osteoblastic cells do not contribute to the nodule formation because no osteocalcin expressing cells are detected in the flushed marrow by in situ hybridization. Limiting dilution analysis shows that, in normal bone marrow, 1 of 2.2 x 10(5) cells has the potency to form a bone nodule and to express ALP, collagen, and osteocalcin in a temporal fashion. Upon in vivo treatment with 5-fluorouracil (5-FU), this frequency increases 12-fold, eg, 1 in 1.75 x 10(4) cells shows osteogenic activity. In comparison, fibroblast colony forming cells occur at a frequency of 1 of 2.5 x 10(4) or 1 of 5 x 10(3) plated cells in normal or 5-FU-treated marrow, respectively. Using density centrifugation, the majority of the osteoprogenitor cells in 5-FU marrow are found in the low-density (1.066 to 1.067 g/mL) fractions. In addition, these cells bind to nylon wool but not to plastic and aggregate in the presence of wheat germ agglutinin and soybean agglutinin. Scanning and transmission electron microscopy shows that the bone nodules in 5-FU marrow cultures are composed of fibroblastoid cells embedded in a mineralized collagen matrix. In conclusion, our results show that a quiescent cell population in the murine bone marrow with fibroblastoid characteristics contributes to the formation of bone-like nodules in vitro.
Alkaline Phosphatase↗
A new method for isolation of polyethylene wear debris from tissue and synovial fluid.
Sub-micron-sized ultrahigh molecular-weight polyethylene (PE) debris is generated in the joint space as a result of articulation and cyclic loading of an orthopaedic implant. Its characterization requires isolation and subsequent analysis by ultra-structural methods. An innovative method based on the digestion of paraffin-embedded tissue samples was proposed. Tissue slices were digested with sodium hypochlorite directly on polycarbonate filter. The same procedure could be applied also to fresh synovial fluid. Plastic particles were not lost or damaged during treatment. Chemical identification of particles was done by micro-Raman spectroscopy that confirmed purity of retrieved PE particles. Size and shape of PE particles were characterised using scanning electron microscopy and were comparable in number and morphology to the retrieval by other authors. Equivalent diameter ranged from 0.48 to 0.95microm and particle number ranged from 9 to 23x10(9)/cm(3).
Aged↗
Early pain in preterm infants. A model of long-term effects.
There are multiple lines of evidence suggesting that in vulnerable prematurely born infants, repeated and prolonged pain exposure may affect the subsequent development of pain systems, as well as potentially contribute to alterations in long-term development and behavior. Multiple factors cumulatively contribute to altered developmental trajectories in such infants. These include characteristics of the developing organism (low tactile threshold, sensitization, rapid brain development), characteristics intrinsic to the infant (gestation, illness severity), characteristics of the experience in the neonatal intensive care unit (pain exposure and cumulative stress), and characteristics of the caregivers within their family and social context. This article provides a model for examining long-term effects of pain in the newborn period embedded in a developmental context framework.
Animals↗
Optical analysis of surface changes on early retrievals of highly cross-linked and conventional polyethylene tibial inserts.
Retrieved tibial liners of highly cross-linked and conventional polyethylene were examined for articular and backside surface damage. Surfaces were graded for pitting, machine-mark loss, scratching, abrasion, delamination, and embedded debris. Whereas no difference existed in the damage score for the 2 groups, the highly crosslinked group showed significantly less elimination of machine marks. Wear, surface plastic deformation, or a combination, could account for the damage on these components. Only 1 of the highly crosslinked polyethylene inserts was available for destructive testing. That insert was melted to activate the shape memory, and thus differentiate, between wear versus plastic deformation. Nearly all changes on the articular and backside surfaces disappeared upon melting, and original machining marks reappeared, suggesting that the surface changes for that component were primarily the result of plastic deformation and not material removal.
Arthroplasty, Replacement, Knee↗
Technology-based vs. traditional instruction. A comparison of two methods for teaching the skill of performing a 12-lead ECG.
The purpose of this study was to compare the effectiveness of an interactive, multimedia CD-ROM with traditional methods of teaching the skill of performing a 12-lead ECG. A randomized pre/posttest experimental design was used. Seventy-seven baccalaureate nursing students in a required, senior-level critical-care course at a large midwestern university were recruited for the study. Two teaching methods were compared. The traditional method included a self-study module, a brief lecture and demonstration by an instructor, and hands-on experience using a plastic manikin and a real 12-lead ECG machine in the learning laboratory. The second method covered the same content using an interactive, multimedia CD-ROM embedded with virtual reality and supplemented with a self-study module. There were no significant (p < .05) baseline differences in pretest scores between the two groups and no significant differences by group in cognitive gains, student satisfaction with their learning method, or perception of self-efficacy in performing the skill. Overall results indicated that both groups were satisfied with their instructional method and were similar in their ability to demonstrate the skill correctly on a live, simulated patient. This evaluation study is a beginning step to assess new and potentially more cost-effective teaching methods and their effects on student learning outcomes and behaviors, including the transfer of skill acquisition via a computer simulation to a real patient.
Adult↗
A device for serially mounting 0.5-2.0 micrometer sections of 1-2 millimeter epon embedded tissue blocks for light microscopy.
The construction and operation of a simple device for serially mounting small Epon sections for light microscopy are described. This device allows the middle portion of the slide to serve both as a shallow reservoir behind the knife edge for flotation of serially oriented sections, and as a miniature hot plate for the subsequent flattening and adhesion of the sections. The advantages of this device, which gave consistently good results with Epon embedded Argas (Persicargas) arboreus tick tissues, are discussed.
Animals↗