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PubMed1978

Revision of light-microscopic kidney biopsy diagnosis in glomerular disease.

Paraffin-embedded kidney biopsies sent to our laboratory in 1967 were re-embedded in Epon and studied with electron and light microscopy (1-micrometer thick sections). Of 28 biopsies with enough material 18 were cases with clinical suspicion and symptoms of glomerular disease. In electron microscopy all of these were shown to be of glomerular origin by demonstrating either dark deposits in or at the basement membrane, or amyloid fibrils, or areas with retraction of the foot processes of the epithelial cells. Original light microscopy in 1967 had given proof of glomerular origin in 9 of these 18 cases. Light microscopy of plastic sections of these originally paraffin-embedded biopsies was also helpful in diagnosis and showed deposits in 9 out of 14 cases with ultrastructural deposits. The study shows that considerable benefits can be gained through re-embedding biopsies in plastic, especially in cases with minor alterations.

Adult↗
PubMed1995

Backscattered electron imaging of the undersurface of resin-embedded cells by field-emission scanning electron microscopy.

In this study backscattered electron (BSE) imaging was used to display cellular structures stained with heavy metals within an unstained resin by atomic number contrast in successively deeper layers. Balb/c 3T3 fibroblasts were cultured on either 13-mm discs of plastic Thermanox, commercially pure titanium or steel. The cells were fixed, stained and embedded in resin and the disc removed. The resin block containing the cells was sputter coated and examined in a field-emission scanning electron microscope. The technique allowed for the direct visualization of the cell undersurface and immediately overlying areas of cytoplasm through the surrounding embedding resin, with good resolution and contrast to a significant depth of about 2 microm, without the requirement for cutting sections. The fixation protocol was optimized in order to increase heavy metal staining for maximal backscattered electron production. The operation of the microscope was optimized to maximize the number of backscattered electrons produced and to minimize the spot size. BSE images were collected over a wide range of accelerating voltages (keV), from low values to high values to give 'sections' of information from increasing depths within the sample. At 3-4 keV only structures a very short distance into the material were observed, essentially the areas of cell attachment to the removed substrate. At higher accelerating voltages information on cell morphology, including in particular stress fibres and cell nuclei, where heavy metals were intensely bound became more evident. The technique allowed stepwise 'sectional' information to be acquired. The technique should be useful for studies on cell morphology, cycle and adhesion with greater resolution than can be obtained with any light-microscope-based system.

3T3 Cells↗
PubMed1996

A simple technique for flat osmicating and flat embedding of immunolabelled vibratome sections of the rat spinal cord for light and electron microscopy.

We describe here a simple technique for flat-osmicating and flat-embedding of immunolabelled vibratome sections. The technique is particularly useful for large specimens such as whole cross-sections of rat spinal cord. After the vibratome section has been flat-osmicated on a flat surface under a glass coverslip, it is dehydrated and then embedded by placing it in a small drop of epoxy resin on a flat base of polypropylene plastic and overlaying a small square piece of cellulose acetate cut from heat-resistant overhead projector transparency film for photocopying. The thin layer of resin containing the flat-embedded vibratome section is then separated from the base and glued onto the flat end of a pre-polymerised blank block of resin. The method produces flat-embedded vibratome sections and thus allows serial large uniformly labelled semithin and ultrathin sections to be obtained of the whole cross-section of the rat spinal cord. This facilitates the observation and quantification of labelled cells in the specimen. Because of its simplicity the technique also allows one worker to process more than 100 vibratome sections at the one time.

Animals↗
PubMedDate not supplied

Microwave stabilization versus chemical fixation. A morphometric study in glycolmethacrylate- and paraffin-embedded tissue.

A morphological and morphometrical study was performed on testicular cells after microwave stabilization of the tissue while immersed in phosphate buffered saline (PBS), 0.9 NaCl or Tris-HCl. Fixation in Carnoy's fluid without irradiation was chosen as a control chemical fixation method. After microwave stabilization or chemical fixation, the testes were embedded in paraffin or in plastic (glycolmethacrylate). An excellent morphology, comparable to that after chemical fixation in Carnoy's fluid, was observed in the plastic sections of tissue irradiated in PBS or NaCl, even when the sections were subsequently treated with an aggressive reagent at high temperature, required for the Feulgen reaction. The nuclear area of the microwave-stabilized Sertoli cells was 37-46% smaller in haematoxylin-eosin stained, paraffin sections in comparison with that in the glycolmethacrylate sections. The microwave-stabilized, paraffin-embedded tissue was much more vulnerable to the hot HCl treatment of the Feulgen staining than the chemically fixed tissue, resulting in an additional 10-20% decrease in nuclear size. The latter finding is particularly important for quantitative microscopy, where the Feulgen staining method is often employed.

Animals↗
PubMed1991

Ultrasound for the detection of foreign bodies.

STUDY OBJECTIVE: To determine the ability of an emergency physician to detect a variety of foreign bodies in an experimental model using a portable ultrasound device. DESIGN: Ten pieces of beef were sliced into cubes approximately 6 cm on each side. Six different groups of foreign bodies were examined: gravel, cactus spine, glass, metal, wood, and plastic. An independent observer placed the objects in a random fashion into the beef cubes. One hundred twenty observations were made using sets of ten beef cubes at a time. Five foreign bodies were placed into each set of ten beef cubes. INTERVENTIONS: A blinded emergency physician used a portable ultrasound with a 7.5-MHz transducer to determine the presence or absence of a foreign body in each cube. MEASUREMENTS AND MAIN RESULTS: Ultrasound detected 59 of 60 foreign bodies, including all cubes of meat embedded with gravel, cactus spine, plastic, metal, and wood. Glass was detected nine of ten times. Of the 60 cubes of meat with no foreign bodies, one false-positive was recorded. This yielded sensitivity, specificity, positive predictive value, and negative predictive value of 98%. Positive determinations by ultrasound were significantly greater in the meat cubes with foreign bodies compared with the control group with no foreign bodies (P less than .001 by chi 2). Although the subset of glass foreign bodies had one false-positive and one false-negative, it was not significantly different in comparison with the other groups (P greater than .05 by chi 2). CONCLUSION: Ultrasound has promise as a diagnostic tool for the detection of a variety of foreign bodies. Further clinical studies using ultrasound for the detection of foreign bodies are warranted.

Emergencies↗
PubMed1996

A new method of acrylic cranioplasty.

BACKGROUND: Previous acrylic cranioplasty techniques have relied on wire or suture fixation of the acrylic to the skull. A new methyl methacrylate cranioplasty technique, using acrylic with titanium plating, is described. METHODS: Titanium plates were bent into a "Z" shape and attached to the skull at the perimeter of the skull defect, extending into the defect. The acrylic was then poured into the defect and held in place during setting with a sheet of plastic. The plates are thereby embedded in the acrylic. RESULTS: Standard methyl methacrylate and titanium plates and screws were used to perform a new method of cranioplasty. CONCLUSIONS: A rigid, form-fitting, aesthetic construct can be easily and quickly created. This technique also offers the benefit of allowing secondary craniotomy, if necessary.

Humans↗
PubMed1987

Retention, localization and staining of lipids in tissues embedded in water-miscible methacrylates: using the solubility parameter to analyse the influences of resins, plasticizers, and other reagents.

The influences of a wide variety of technical factors (the nature of the monomer and plasticizer, the duration and temperatures of the processing steps, etc.) on retention, localization and staining of lipid in tissues embedded in water-miscible methacrylates were assessed experimentally. This analysis was aided by the use of the solubility parameters of the substances as quantitative indicators of miscibility. The technical requirements for, and limits of, lipid histochemistry using resin sections have therefore been established on a quantitative basis.

Acrylates↗
PubMed2000

Widefield deconvolution epifluorescence microscopy combined with fluorescence in situ hybridization reveals the spatial arrangement of bacteria in sponge tissue.

Widefield deconvolution epifluorescence microscopy (WDEM) combined with fluorescence in situ hybridization (FISH) was performed to identify and characterize single bacterial cells within sections of the mediterranean sponge Chondrosia reniformis. Sponges were embedded in paraffin wax or plastic prior to the preparation of thin sections, in situ hybridization and microscopy. Serial digital images generated by widefield epifluorescence microscopy were visualized using an exhaustive photon reassignment deconvolution algorithm and three-dimensional rendering software. Computer processing of series of images taken at different focal planes with the deconvolution technique provided deblurred three-dimensional images with high optical resolution on a submicron scale. Results from the deconvolution enhanced widefield microscopy were compared with conventional epifluorescent microscopical images. By the application of the deconvolution algorithm on digital image data obtained with widefield epifluorescence microscopy after FISH, the occurrence and spatial arrangement of Desulfovibrionaceae closely associated with micropores of Chondrosia reniformis could be visualized.

Algorithms↗
PubMed1994

Post-embedding tem signal-to-noise ratio of S-100.

We assessed the reactivity of purified S-100 antiserum in immuno-electron microscopy by counting the number of gold particles per microns 2 over inner ear tissues embedded in different media. Sections containing predominantly Schwann's cell cytoplasm and nucleus, afferent fiber axoplasm and myelin sheath of chick cochleae were reacted with anti-S-100 IgG, an antibody to a calcium binding protein of neuronal tissues, then labeled with anti-IgG-gold conjugate. This investigation was conducted because previously published procedures, unmodified, did not yield acceptable results. Preparation of all specimens was identical. Only the medium (PolyBed 812, Araldite or Spurr epoxies; and LR White, LR Gold or Lowicryl plastics) was changed. The medium was made the changing variable because antigens available in post-embedding immuno-electron microscopy are decreased by heat, either used and/or released during polymerization of the embedding medium. The results indicate that: (a) none of the embedding media above provided optimal signal-to-noise ratio for all parts of the nerve stained in the same section; (b) aggregation of gold particles over cells was highest in embedding media with high background labeling over areas devoid of tissue (noise); (c) aggregation occurred randomly throughout both cellular and acellular regions; and (d) particles aggregated less and were distributed more evenly in tissues from media yielding good ultrastructural integrity.

Animals↗
PubMed1987

A tissue-specific protein in rat osteogenic tissues.

A tissue-specific protein fraction has been detected in rat osteogenic tissue. Dissociative extraction of adult rat bone matrix with 4 M guanidinium chloride solution was followed sequentially by gel chromatography and polyacrylamide gel electrophoresis. By the latter procedure a prominent protein component of molecular weight 19,000 was isolated from the low molecular weight fraction, and antibodies directed against this protein were raised in rabbits. The antibodies were mainly against antigenic sites on this protein, as shown by protein blotting techniques. By embedding rat tissues in hydrophilic plastic and by using immunohistochemical procedures the presence of this protein was demonstrated specifically in bone matrix in vivo, in osteogenic tissue developing in diffusion chamber culture, and in a malignant osteoblast cell line (UMR 106). Soft tissues (liver, kidney, spleen, gut, skin, thymus, eye) showed no reactivity with the antiserum and in vitro a further malignant osteoblast cell line (ROS 17/2.8) did not synthesize the 19,000 molecular weight protein. This protein appears to be expressed solely by osteogenic tissue and may be used as a biochemical criterion of osteogenic differentiation.

Animals↗
PubMed1994

Comparison of four methods for cross-calibrating dual-energy X-ray absorptiometers to eliminate systematic errors when upgrading equipment.

Dual-energy x-ray absorptiometry (DXA) is a widely employed technique for making noninvasive measurements of bone mineral density (BMD). Advances in DXA technology have resulted in the development of new densitometers that offer increased scan speed, improved spatial resolution, and the ability to make measurements at additional skeletal sites. However, changing from a first to a second-generation DXA system generates two additional potential sources of error. First, if the densitometers produce results that are substantially different, diagnostic errors occur if the results from both instruments are compared to the same normative database. Second, even if the densitometers produce results that are nearly identical, small systematic errors may influence interpretation of serial bone density measurements in individual patients. To assess the impact of changing from a first- to a second-generation DXA scanner, we made measurements using the standard "pencil beam" mode on 133 consecutive patients using both a Hologic QDR-1000 and a QDR-2000 densitometer when the latter instrument was calibrated according to the manufacturer's routine procedure using a single anthropomorphic spine phantom. We then recalculated the results for the QDR-2000 densitometer using cross-calibration factors based on (1) a regression line generated by scanning three anthropomorphic spine phantoms whose BMD ranged from osteoporotic to high normal on each instrument, (2) an adult human lumbar spine embedded in tissue-equivalent plastic, or (3) a regression line derived from scans of the first 83 patients that was then applied to the last 50 patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorptiometry, Photon↗
PubMedDate not supplied

Technique for casting chronic extradural and intracerebral cannula arrays.

A technique for fixing multiple brain cannulae in uniform arrays by embedding guide cannulae in molded plastic pedestals is described. Methods for producing corresponding threaded caps for protection of the outer ends of the cannulae, and procedures for reducing mortality and morbidity when large number of cannulae are implanted in brain tissue, are presented.

Animals↗
PubMed1978

Morphology of osteosarcoma: new qualitative and quantitative investigations.

Results on seven cases of osteosarcoma are reported, based on new morphologic methods and quantitative procedures. Tumor tissue was embedded without prior decalcification in plastic and sectioned. Imprint cytology preparations were produced from fresh tumor tissue, and cell nuclei were measured with an electronic image analysing computer system. The loss of differentiation seen in osteosarcomas differs among osteoblasts, osteocytes, and osteoclasts. The differentiation of osteoclasts, namely their resorptive characteristics, disappears relatively early. Tumor osteocytes show loss of differentiation in their osteocyte processes. The new formation of tumor bone tissue remains in the near normal range of volume density when nuclear polymorphy is limited. The formation of ground substance and mineralization are apparently closely couplet to one another, since in our cases mostly ordered osteoid seams were observed. The capacity for mineralization of bone tissue is lost with marked polymorphy. The significance of these results for diagnostic statements and therapeutic consequences will be further discussed in long term studies.

Adolescent↗
PubMed1997

Magnetically-enhanced radionuclide therapy (MERiT): in vitro evaluation.

PURPOSE: Radionuclide therapy is a promising method for delivering radiation dose selectively to tumors. In situations where electron -emitters are used and the tumor is small relative to the maximum range of therapeutic electrons, these particles exit the tumor before delivering the maximum amounts of radiation dose. In this study, the method of magnetically constraining electrons to small tumors, known as magnetically -enhanced radionuclide therapy (MERiT), is explored using in vitro experiments. METHODS AND MATERIALS: The potential utility of MERiT was investigated by first measuring the reduction of number of electrons exiting a small sphere containing 90Y embedded in a block of plastic scintillator. Measurements of total energy deposited in the plastic scintillator made inside and outside a 7 Tesla magnetic field were compared. Furthermore, an experiment utilizing lymphoma cells of human origin was performed. Groups of cells were added to wells containing 90Y-labeled bovine serum albumin (and control groups containing no radioactivity) were placed either inside a 7 Tesla magnet or at a position where the magnetic field was minimal (essentially zero) for 18 hr. RESULTS: The presence of a 7 Tesla magnetic field reduced the amount of energy deposited in the scintillator by 16.63 +/- 1.05%. This demonstrates that the magnetic field constrains a large fraction of the emissions to the sphere and implies that normal tissues adjacent to radiotracer-avid tumors can be protected from radiation dose. Results from the cell culture experiment showed that the presence of a 7 Tesla magnetic field significantly (p < 0.005) reduced the number of viable cells remaining after treatment with non-specific 90Y-labeled bovine serum albumin by 11.7% compared to the appropriate control group (90Y treated, not exposed to magnetic field). CONCLUSIONS: These initial physical and biological studies indicate that magnetically-enhanced radionuclide therapy can be effective in increasing radiation absorbed dose to small tumors, consequently reducing radiation dose to surrounding normal structures.

Cell Survival↗
PubMed1994

Laparoscopic Conservative Cystectomy of Ovarian Benign Cystic Teratoma

Laparoscopic surgery has almost totally replaced laparotomy in the management of benign adnexal conditions. Benign cystic teratoma (dermoid cyst) is one of the most common ovarian neoplasms in young women. A prospective non-randomized clinical trial was conducted. To compare the efficacy of two different methods of removal of benign cystic teratoma enucleated laparoscopically. Between January 1991 and June 1993, 37 women, all reproductive-age and desiring future fertility, underwent resection of dermoid cysts between 4 and 8 cm in diameter via laparoscopy using the three-puncture-technique. The removal of the cysts from the abdominal cavity was carried out by traction through an extended incision of one of the puncture sites in the first 17 cases (group A), and the other 20 cases (group B) were managed by embedding the cyst in a plastic bag inserted through one of the trocars, aspirating its contents, and subsequent extraction of the bag without extension of abdominal wall stab wounds. The operating time in both groups was comparable (from 35-130 minutes in group A, and 45-120 minutes in group B. However, the time required for removal of the cyst from the abdominal cavity was significantly shorter in group B (10-15 minutes) compared with group A (20-35 minutes). Spillage of cyst contents occurred in six cases in group A, and in two cases in group B. The average hospital stay in both groups was similar (22.8 hours in group A and 23.4 hours in group B). Postoperative complications in group A included one case of excessive bleeding from the extended incision, and three cases of wound infection. None of the patients in group B had short- or long-term postoperative complications. Operative laparoscopy is a safe and effective method in the management of benign cystic teratoma. We found that removal of the enucleated cyst from the abdominal cavity using a plastic bag is advantageous in terms of operating time and postoperative complication as well as in the cosmetic results compared to extraction of the cyst through an extended abdominal incision.

Journal Article↗
PubMed1996

Interleukin-1-induced growth factor expression in postmitotic and resting fibroblasts.

Tissue homeostasis in skin is regulated by epithelial-mesenchymal interactions, mostly operating via diffusible factors. To study the underlying regulatory mechanisms, in vitro systems have been established to mimic the in vivo situation in skin. In co-cultures, keratinocytes grow either adjacent to irradiated fibroblasts on plastic or on top of collagen gels containing fibroblasts, thus forming 3-dimensional organotypic structures. Keratinocyte growth is supported in part by fibroblast-produced factors induced by keratinocyte mediators such as interleukin-1 (IL-1). To better understand this cellular interaction and its modulation by fibroblast proliferation and extracellular matrix (ECM), we examined the effect of IL-1 on growth factor expression in proliferating and growth-arrested x-irradiated human dermal fibroblasts on plastic and in resting cells embedded in collagen gels. By semiquantitative reverse transcriptase PCR, we demonstrated that IL-1alpha and IL-1beta stimulated the expression of KGF, HGF, IL-1alpha, IL-1beta, IL-1RI, and IL-8 in fibroblasts regardless of their physiologic condition, whereas that of TGF-beta remained unaffected. The constitutive mRNA levels were usually lower in irradiated postmitotic and ECM-embedded cells than in proliferating fibroblasts. Cells responded to stimulation with IL-1 under all three culture conditions, although to different degrees depending on the growth factor. As demonstrated for HGF, IL-8, and IL-1beta, the IL-1alpha-induced mRNA expression was followed by production and secretion of protein in irradiated fibroblasts. Thus, our findings show that resting and growth-inhibited fibroblasts, reflecting more closely the situation in dermis, exhibit lower constitutive growth factor expression levels but characteristically respond to IL-1 stimulation.

Adult↗
PubMed1989

Reversible middle cerebral artery embolization in dogs without intracranial surgery.

Using dogs, we developed an intravascular model for reversible middle cerebral artery occlusion that does not involve intracranial surgery or enucleation. Using silicone plastic plugs with a suture embedded within them, we embolized the middle cerebral artery in 19 dogs via the cervical carotid artery. The free end of the suture remained accessible in the neck, and after variable dwell times traction was placed on the suture and the plug was withdrawn. Placement of the plug in the middle cerebral artery produced ischemia in the basal ganglia. The degree and distribution of cortical ischemia were variable as evidence by the pathologically documented scattered nature of infarcts that resulted when the plug was left permanently in the middle cerebral artery and when it was removed after 1 or 2 hours. Angiography demonstrated occlusion of the middle cerebral artery with the plug in place as well as reperfusion when the plug was withdrawn. This modification of a previously described model of middle cerebral artery occlusion provides an opportunity to study structural, physiologic, and biochemical events occurring in acutely hypoperfused cerebral tissue as well as critical changes leading to irreversible injury without the disadvantages of surgical manipulation required by all previous models of reversible cerebral ischemia.

Animals↗