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PubMed1998

The axonal transmission of herpes simplex virus to epidermal cells: a novel use of the freeze substitution technique applied to explant cultures retained on cover slips.

Retaining the ultrastructural arrangement of a mixed-cell culture on a solid support while processing for immunocytochemical study is a technical challenge. We developed a technique to study the axonal transport of the Herpes simplex virus from dorsal root ganglia sensory neurones to epidermal cells. Autologous explants of human foetal dorsal root ganglia and skin were cultured on plastic cover slips. Axon fascicles grew from the ganglia to the epidermal cells and the ganglia were inoculated selectively with virus. The whole preparation, retained on the cover slip, was fixed with formaldehyde 4% (freshly prepared from paraformaldehyde)/glutaraldehyde 0.1%, processed by freeze substitution, and embedded in Lowicryl HM20 resin. The edges of the cover slip in the block were trimmed, allowing clean and complete separation from the resin block, which retained the tissue. The resin block was placed in fresh HM20 and repolymerized. The polymerizing resin bonded strongly to the existing block. After trimming, serial sections were easily obtained and successfully immunolabelled for viral proteins. This is a convenient technique for immunolabelling tissue grown on cover slips in which the preservation of the ultrastructural interactions between different cells is important. It should be adaptable to a number of cell-culture applications and has a number of advantages over other techniques.

Axons↗
PubMed2006

Nuclear pore complex structure and plasticity revealed by electron and atomic force microscopy.

To study the ultrastructure of nuclear pore complexes (NPCs), a wide spectrum of different electron microscopy (EM) or atomic force microscopy (AFM) techniques can be employed. The combination of these methods can reveal new insights into the structural and functional organization of this important supramolecular machine through which nucleocytoplasmic transport occurs. Negative staining, quick freezing/freeze-drying/rotary metal shadowing, embedding and thin sectioning, cryoelectron microscopy and tomography, scanning electron microscopy, or combination with immunolabeling techniques are tools for collecting data and information about the three-dimensional structure and architecture of the NPCs. AFM enables investigation of the functional dynamics of native NPCs under physiological conditions.

Animals↗
PubMed2005

Glial remodeling and neural plasticity in human retinal detachment with proliferative vitreoretinopathy.

PURPOSE: To investigate glial remodeling and neuronal plasticity in adult human retinal detachment complicated by proliferative vitreoretinopathy (PVR) and to grade pathologic changes with a severity scoring system. METHODS: Sixteen full-thickness retinectomy specimens obtained at retinal relaxing surgery for PVR were fixed in 4% paraformaldehyde immediately after excision and compared to similarly processed normal donor retinas. Agarose-embedded sections (100-microm-thick) were double labeled for immunohistochemistry by confocal microscopy, with antibodies against rod opsin and GFAP; vimentin and M/L-cone opsin; calbindin D and S-cone opsin; and cytochrome oxidase and synaptophysin. These staining patterns formed the basis of a retinal pathology scoring system, and immunohistochemistry was also used to detect CD68, neurofilaments, protein kinase C, growth-associated protein-43, and a pan-cone-specific enzymatic marker. Morphology was also assessed by light microscopy of resin-embedded semithin sections. RESULTS: Prolonged detachment was characterized by photoreceptor degeneration and intracellular redistribution of opsin proteins to the plasma membrane in the outer nuclear layer (ONL). Remodeling of rod synaptic terminals was characterized by terminal retraction and also by axon extension to the inner retina in most specimens. Rod bipolar cell dendrites extended into the ONL, as did fine, horizontal cell processes. Large ganglion cells showed upregulated neurofilament and GAP-43 expression, with neurites sprouting from somata and axon collaterals. Anti-cytochrome oxidase labeling of surviving inner segments was reduced but detectable in all specimens, as was anti-calbindin D labeling of horizontal and amacrine cells. All specimens demonstrated a marked upregulation of Muller cell and astrocyte expression of GFAP and vimentin. More severe degenerative changes correlated with trauma and prolonged detachment duration when scored according to this system. CONCLUSIONS: The neural and glial components of detached neurosensory retina complicated by PVR exhibit pathology that changes characteristically with increasing detachment severity. Even in advanced degeneration, most of the structural motifs necessary for functional recovery are retained. Evidence of remodeling in the first-, second-, and third-order neurons of detached adult human retina may represent an attempt to re-establish synaptic connectivity.

Adolescent↗
PubMed1987

A new technique for reconstruction of the auricle in acquired deformities.

A technique for reconstruction of the ear in patients with acquired deformities is described. The auricular framework is excavated using rib cartilage and embedded through a subcutaneous tunnel undermined on the mastoid region. Carefully applied dressings are necessary for a successful result.

Bandages↗
PubMed1988

Localization of alpha-casein gene transcription in sections of epoxy resin-embedded mouse mammary tissues by in situ hybridization.

The objective of our study was to evaluate the suitability of aldehyde-fixed, epoxy resin-embedded tissue for efficient and reproducible detection of casein mRNA in mouse mammary tissue by in situ hybridization. We used mouse alpha-casein-specific, 35S-labeled riboprobes generated from a Gemini-3 vector. Both complementary (anti-sense) and homologous (sense) RNA probes were utilized in our study (specific activity ranged from 5-7 x 10(8) cpm/micrograms). We tested the stability of newly synthesized [3H]-uridine-labeled RNA in tissue sections subjected to epoxy plastic solvents and found that no detectable loss of label occurred during preparation of semi-thin (1-2 micron) plastic sections for situ hybridization. In addition, it was possible to detect alpha-casein mRNA in deplasticized sections of mammary gland tissue taken from normal, pregnant, or lactating mice, pre-neoplastic mammary alveolar hyperplasias, explant cultures, and mammary tumors. A positive hybridization signal was consistently obtained in sections of mammary tissues where the estimated average copy number for total casein mRNA was greater than or equal to 250/cell. In mammary tumors, where the estimated casein mRNA content was much lower (less than 5/cell), our positive hybridization signal occurred in regions of the tumor that, in consecutive sections, stained positive for casein by immunoperoxidase. After formaldehyde-glutaraldehyde fixation, loss of hybridizable RNA from epoxy-embedded tissues and sections appears to be minimal. Image resolution was greatly enhanced over frozen or paraffin sections of mammary tissue. Non-specific binding of the radioactive probes was very low. Protease treatment of the sections was not necessary for detection of hybridizable signal.

Animals↗
PubMed1994

Effects of cytokines on gingival fibroblasts in vitro are modulated by the extracellular matrix.

Fibroblast function in gingival tissue is thought to be regulated by the local cellular environment--both the extracellular matrix and soluble factors. In an attempt to artificially re-create this situation fibroblasts have been cultured within 3-dimensional collagen gels in an environment more physiologically comparable to connective tissue. Using such a model we investigated the effects of the extracellular matrix on gingival fibroblast growth and synthetic activity and on the cellular responsiveness to 4 soluble factors--epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta 1) and interleukin-1 beta (IL-1 beta). Fibroblasts cultured within collagen gels showed similar growth rates, an increased production of collagen but reduced levels of hyaluronan synthesis in comparison to cells in monolayer culture. Cellular responsiveness to soluble mediators was also modulated by the collagen matrix, with a generalised reduction in response by cells embedded within the matrix. The stimulatory effects of EGF and PDGF on cell growth in monolayer over a 14-day period were only found during the initial stages of culture within gels. Similarly the stimulation of matrix production by cells induced by TGF-beta 1 on plastic was reduced or even negated when cells were cultured in collagen gels. On plastic IL-1 beta significantly stimulated cell growth but had no effect on either collagen or hyaluronan production by fibroblasts. In gel cultures, this cytokine had no effect on cell proliferation, but significantly inhibited both collagen and hyaluronan synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2003

Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants.

A method for preparing thin fresh-frozen sections from large samples and hard tissues is described and the applications are shown. A new adhesive film is introduced to produce the frozen sections. The sample is frozen in a cooled hexane or liquid nitrogen, and then freeze-embedded with 4-5% carboxymethyl cellulose (CMC) in the coolant. A specially prepared adhesive film is fastened to the cut surface of the sample in order to support the section and cut slowly with a disposable tungsten carbide blade. The adhesive film is made of a thin plastic film and an adhesive before use. This method produces 2-microm thick fresh-frozen sections from a large sample, bone or tooth. The "film-section" i.e. the section attached to the adhesive film, can be used for many types of studies such as histology, general histochemistry, enzyme histochemistry, immunohistochemistry, in situ hybridization, elemental analysis, and autoradiography for water-soluble materials. Immunohistochemistry and in situ hybridization can be carried out with nonfixed and undecalcified sections. The section on the adhesive film can be transferred to a glass slide and mounted under a cover slip, and stained sections can be examined with an optical microscope at high magnification. This method is also useful for preparing frozen sections from samples of fish, insects, and plants. Furthermore, samples of particular areas can be collected from the film-section by means of a laser microdissection technique. The multiple possible applications of the adhesive film render it highly useful for studies in biological and medico-dental fields.

Adhesives↗
PubMed1988

Increase in the calcium content of cardiac tissue after postfixation with osmium tetroxide.

The concentration of osmium has been measured by destructive chemical analysis in glutaraldehyde fixed heart tissue postfixed with osmium tetroxide and embedded in epoxy resin. After such treatment, the mean atomic number of the specimen (Z) is close to 10, which permits a quantitative analysis of calcium (Ca) by the continuum method, using Z2/A as a correcting factor (A: atomic weight). Wavelength-dispersive X-ray microanalysis has been used to determine the Ca concentration of frog cardiac tissue fixed in glutaraldehyde and embedded in resin. These measurements have been repeated on tissue postfixed in osmium tetroxide; contrary to expectations, the apparent Ca concentration is much higher in osmium treated than in nontreated tissue. However, this result is observed with OsO4 solutions prepared in glass, not with solutions prepared in plastic. It is shown by energy dispersive X-ray analysis of droplets that OsO4 solutions prepared in glass contain large amounts of calcium, potassium and silicon. Care must be taken in preparing OsO4 fixatives when the fixed tissues are to be subjected to X-ray microanalysis of such elements as Ca or Si.

Animals↗
PubMed1986

Large liposome agglutination technique for the serological detection of syphilis.

In response to tissue invasion by Treponema pallidum, an antibody complex, called reagin, appears in the serum of the syphilitic patients. Reagin has the ability to combine with cardiolipin, therefore, cardiolipin has traditionally been used as the antigen in different test configurations for the serological detection of syphilis: the VDRL (Venereal Disease Research Laboratory) flocculation test and the RPR (rapid plasma reagin) test. Here we introduce a liposome preparation composed of cardiolipin, lecithin and cholesterol and containing a colored dye which upon mixing with syphilitic serum on a slide or a plastic card results in clearly visible liposome agglutination. We show that the liposomes in the size range of 1-10 micron are essential for proper sensitivity. The liposomes are colored with either a water-soluble dye entrapped in the aqueous space of liposomes or a lipophilic dye embedded in the membrane. By virtue of the liposome size and coloration the agglutination pattern is easily visible within 3-5 min without the use of a mechanical rotator. The approach described here can be extended to the detection of other antibodies and polyvalent antigens.

Agglutination Tests↗
PubMed1981

Post-embedding immunoperoxidase staining of glial fibrillary acidic protein for light and electron microscopy.

Post-embedding peroxidase-antiperoxidase methods to stain glial fibrillary acidic (GFA) protein in Araldite-embedded sections for light and electron microscopy were developed. The Jimpy mouse spinal cord was used because it is gliotic and contains abundant glial filaments and GFA protein. For light microscopy, specific staining was obtained in thick and in ultrathin sections mounted on glass following removal of the plastic with sodium ethoxide. Consistent specific staining for GFA protein in ultrathin sections mounted on nickel grids required partial removal of the plastic with 1% sodium ethoxide and further treatment with 2% sodium dodecyl sulfate (SDS).

Animals↗
PubMed1995

Clustering and co-localization of immunogold double labelled neural cell adhesion molecule isoforms in chick forebrain.

A quantitative immunoelectron microscopic study was carried out in the intermediate portion of hyperstriatum ventrale of chick forebrain (a region exhibiting learning-related morphological plasticity) in order to examine the fine distribution of the neural cell adhesion molecule (N-CAM). Antibodies against alpha 2,8 polysialic acid (PSA), characteristic for embryonic N-CAM, and those against the protein backbones of all N-CAM isoforms were sequentially labelled (post-embedding) with 5- and 15-nm gold particles, and binding to their epitopes was analyzed using the statistics of point processes. The results showed that: (1) PSA tends to form clusters (up to 200 nm in size), in contrast to the protein backbones of all N-CAM isoforms, and (2) the two tissue bound antibodies are co-localized spatially in clusters of sizes up to 100 nm. The data suggest that there is a spatial sub-cellular domain enriched in both PSA and protein isoforms of N-CAM.

Animals↗
PubMed1998

Working times and dimensional accuracy of the one-step putty/wash impression technique.

PURPOSE: Using the one-step polyvinyl siloxane impression technique, this study compared the effect of putty material working time on the dimensional accuracy of recovered improved stone casts. MATERIALS AND METHODS: An acrylic resin master cast was fabricated with embedded reference points to enable both horizontal and vertical measurements. Four commercially available polyvinyl siloxane impression putties with light body washes were evaluated: 1) Extrude (Kerr), 2) Cutter (Coltere), 3) Express (3M), and 4) Reprosil (Caulk). Each putty was mixed by hand for 30 seconds, placed in a plastic stock tray, and seated on the master cast at 0, 30, 60, 90, and 120 seconds after mixing (N = 100, n = 5). The impressions were poured in improved stone, and vertical and horizontal measurements were made to 0.001 mm between reference points on recovered casts using an optical traveling microscope. ANOVA (p < or = .05) was used to compare vertical and horizontal measurements to master cast dimensions. RESULTS: No statistically significant difference was found among casts for all materials and time periods tested. When horizontal measurement ranges were evaluated, Extrude casts exhibited the widest range of measurement, especially when the trays were seated at 90 and 120 seconds. When vertical measurement ranges were evaluated, Express casts at 120 seconds exhibited the widest range. CONCLUSIONS: A high range of variability in vertical and horizontal dimensions occurred at the later time periods, especially for Extrude and Express casts. As a result of rapid polymerization and poor flow properties of putty materials, the impression tray should be seated within 60 seconds after putty mixing to reduce dimensional variability of recovered casts.

Analysis of Variance↗
PubMed2006

Formation and subdivision of deformation structures during plastic deformation.

During plastic deformation of metals and alloys, dislocations arrange in ordered patterns. How and when these self-organization processes take place have remained elusive, because in situ observations have not been feasible. We present an x-ray diffraction method that provided data on the dynamics of individual, deeply embedded dislocation structures. During tensile deformation of pure copper, dislocation-free regions were identified. They showed an unexpected intermittent dynamics, for example, appearing and disappearing with proceeding deformation and even displaying transient splitting behavior. Insight into these processes is relevant for an understanding of the strength and work-hardening of deformed materials.

Journal Article↗
PubMed1994

The preparation of native livers for morphological studies.

We describe experiences with a perfusion fixation apparatus that was used for studies on approximately 500 native livers. Immediately after excision of the specimen, small samples from needle biopsy specimens are obtained for snap-freezing and the remaining portions of the specimens are fixed and embedded in paraffin. In the laboratory, the portal vein and, if possible, the hepatic artery or common hepatic duct are then cannulated and the livers are perfused for 3 days with Kaiserling's solution. An electric pump drives the perfusion apparatus and allows the formalin to cascade through stacked plastic containers, with the specimens attached to the inflow nozzles for the fixative. Eight or more livers (or other organs and specimens) can be accommodated simultaneously. Angiograms or cholangiograms can be prepared before or after fixation; we prefer the latter. The livers are then sliced with an extra-long knife, which minimizes cutting marks. Most preparations are thoroughly fixed and yield excellent specimens, not only for routine microscopic study but also for special methods such as scanning electron microscopy and trace metal analysis. The liver slices can be stored indefinitely, which allows long-range collection for routine review or research purposes. In approximately 5% of the cases, specimens cannot be perfused properly and thus are unsuitable for this type of preparation. With autopsy specimens this percentage is higher, probably because of postmortem clotting. Gravity perfusion of the livers before placement into the apparatus generally enables identification of specimens with incomplete filling of the vasculature.

Angiography↗
PubMed2005

The development of a dialogue between cortex and midbrain to integrate multisensory information.

The anterior ectosylvian (AES) and rostral lateral suprasylvian (rLS) sulci send critical signals to multisensory superior colliculus (SC) neurons that enable them to integrate information from different senses. When either of these areas is temporarily deactivated in adult animals, the ability of SC neurons to integrate multisensory information and, thereby, enhance their responses to cross-modal stimuli is temporarily compromised. As a consequence, the ability to use cross-modal stimuli to enhance SC-mediated behavioral performance is also compromised. In contrast, removal of either one of these areas during early life has little effect on the development of multisensory processes in the SC or on SC-mediated multisensory behaviors and these animals seem very similar to normal controls. These observations suggest that there is considerable plasticity in these cortico-collicular systems during early life, with each area able to compensate for the early loss of the other. However, when both AES and rLS are removed early in life, there appears to be no compensation. The SC neurons now deal with sensory stimuli, even those embedded in multisensory complexes, as if they were there alone, precluding any SC-mediated behavioral benefit to cross-modal stimuli.

Animals↗
PubMed1988

Biliary stent blockage with bacterial biofilm. A light and electron microscopy study.

Clogging of endoscopic stents necessitates their replacement in many patients with malignant obstructive jaundice and limits their use in benign strictures. We studied the basic mechanism of clogging to find ways to prevent it. We did light and electron microscopy studies of blocked and functioning stents, which were prepared so that organic structures would be preserved. The material blocking the lumina was composed of a matrix of bacterial cells and their fibrillar anionic extracellular products. Crystals of calcium bilirubinate, calcium palmitate, and cholesterol were embedded within this matrix. Bacterial cells were attached to the stent surface by a fibrillar matrix, suggesting that the initial event in stent clogging is the development of an adherent bacterial biofilm. Bacterial enzyme activity (beta-glucuronidase and phospholipase) leads to the deposition of crystals. The use of antibacterial plastics in the manufacture of stents may reduce bacterial adhesion and stent clogging.

Bacteria↗
PubMed1994

In vitro torque-deformation characteristics of orthodontic polycarbonate brackets.

The purpose of this study was to investigate the torque-deformation characteristics of the following four types of polycarbonate brackets: (1) pure polycarbonate, PPC (anterior Miura, RMO, Denver, Colo.), (2) ceramic reinforced polycarbonate, CRPC (Silkon bracket, American, Sheboygan, Wis.), (3) metal slot reinforced polycarbonate, MRPC (Plastic bracket, Tella Tech, Miami, Fla.), and (4) metal slot and ceramic reinforced polycarbonate, MCRPC (Spirit, Ormco, Glendora, Calif.). A stainless steel bracket, (Mini Diamond, Ormco, Glendora, Calif.), was used as a control. Ten brackets of each type were tested. Each bracket was bonded to a porcelain tooth and engaged in a torquemeter. The tooth-bracket assembly was made stationary by embedding it in die stone. Torsion was applied to the bracket at 4 degrees per minute and the resultant torque (grams.centimeters) and deformation (degree) were measured. For optimum labiolingual tooth movement for a maxillary incisor at 175 grams . centimeters, the amount of angular deflection necessary for the different polycarbonate brackets was the following: (a) 15 degrees for MRPC, (b) 17 degrees for MRPC, (c) 24 degrees for CRPC, and (d) > 30 degrees for PPC. The amount of deformation at this deflection was the least for MRCP followed by MCRPC, CRCP, and PPC. When compared with the stainless steel bracket, all polycarbonate brackets showed significantly (p < 0.0001) higher deformation and lower torque. Within the polycarbonate group, there was a significant difference (p < 0.0001) between each bracket for both measurements. The MRPC produced the highest torque and lowest deformation values followed by the MCRPC, CRCP, and PPC. It appears that only the metal slot reinforced brackets are clinically capable of torquing teeth sufficiently.

Analysis of Variance↗
PubMed1985

Comparative analysis of casein synthesis during mammary cell differentiation in collagen and mammary gland development in vivo.

Substrata upon which epithelial cells are cultured modulate their morphology,growth, and ability to differentiate. Mouse mammary epithelial cells cannot be induced to synthesize caseins, a marker of cell differentiation, when grown on a plastic surface. An analysis was made of the effect of time within a collagen matrix on the ability of normal mammary epithelial cells to be induced to synthesize caseins and that response was compared to mammary gland development in vivo. Primary cultures of mammary cells from unprimed virgin BALB/c mice were embedded in rat-tail collagen gel mixtures and maintained in growth medium. Induction medium containing lactogenic hormones was added at various times. The cells were monitored every 3-7 days over a period of 8 weeks for cell growth, casein synthesis, and ability to grow in vivo in cleared mammary fat pads. Casein accumulation was assayed quantitatively by an ELISA competition assay and qualitatively by the immunoblot procedure using specific antisera prepared against purified mouse caseins. No marked differences in cell numbers and transplantability potential were observed among cells cultured for various times in collagen. Mammary cells grown in collagen for up to 8 weeks retained the capacity to grow in vivo as normal ductal outgrowths. The duration of culture within collagen prior to hormonal stimulation did influence the kinetics of casein synthesis. Cells cultured for 1 week in growth medium did not accumulate detectable levels of casein until after 3 weeks of induction, whereas cells cultured for 2 or 4 weeks responded by accumulating caseins after 2 weeks and 3 days of induction, respectively. While the levels of total caseins that accumulated under optimal conditions of induction in culture approached levels found during lactation in vivo, the relative proportion of specific casein polypeptides synthesized in culture was altered from alpha casein (43K) in favor of the beta casein (30K) species. These results suggest that a period of culture within collagen is required to permit mammary epithelial cells to become responsive for hormone-induced differentiation. It is possible that during growth within the collagen the cells synthesize and deposit extracellular matrix components important in modulating gene expression.

Animals↗