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PubMed1998

Three-dimensional microcomputed tomography of renal vasculature in rats.

Current microscopic methods to view renal microvasculature reveal only a very limited portion of the total renal volume. Identification of connectivity for postglomerular vessels in the cortex and the medulla during functional states related to changes in sodium excretion will help better to understand the coupling of renal vasculature to tubular function. The purpose of this study was to investigate the possibility of visualizing the continuity of pre- and postglomerular vasculature using three-dimensional micro-computed tomography (micro-CT). Kidneys from normal rats were perfusion fixed in situ at physiological pressure, filled with latex microfil containing lead chromate, and embedded in plastic. The micro-CT scans of the intact kidneys were carried out on a rotating stage illuminated either by a synchrotron x-ray source or a conventional x-ray spectroscopy tube. Images were reconstructed by a filtered backprojection algorithm and volume-rendering techniques were utilized to display the vasculature. The reconstructed images clearly showed the large distribution vessels and the venous drainage of the kidneys, while pre- and postglomerular vessels and their vascular connections throughout the kidney were displayed in great detail. Efferent arterioles showed the characteristics of their peritubular capillary beds in the cortical and medullary regions. The vascular volume of the cortex was 27%, the outer stripe of the outer medulla 18%, the inner stripe of the outer medulla 30%, and the inner medulla 18%. In conclusion, micro-CT is a promising method to evaluate renal vascular architecture relative to physiological and pathological alterations.

Algorithms↗
PubMed1992

Prediction of cerebral ischemia by ophthalmoscopy after carotid occlusion in gerbils.

BACKGROUND AND PURPOSE: The Mongolian gerbil provides a unique model of unilateral focal cerebral ischemia because of the lack of posterior communicating arteries in all gerbils as well as an absence of an anterior communicating artery in approximately 20% of the gerbil population. It is unclear how to identify unequivocably the subpopulation of animals that would suffer a severe focal cerebral ischemia after unilateral carotid occlusion. METHODS: Ninety-three male gerbils were exposed to unilateral occlusion of the right common carotid artery. The severity of neuronal loss was evaluated histologically in gerbils selected as having significant focal ischemia based on either behavioral criteria (i.e., the demonstration of stereotypical behavior within 1 hour after occlusion) or ophthalmoscopic criteria (i.e., interruption of the retinal arterial perfusion within 10 minutes of carotid ligation as assessed with an ophthalmoscope). After 3 hours of unilateral carotid occlusion, cerebral blood flow was reinstated for 24 hours before fixation for histological analysis. The viability of the CA1 region of the hippocampus, lateral cortex, and medial cortex was scored on a scale of 0-4 based on the percentage of apparent neuronal loss (e.g., 0, no damage; 4, > 75% damage (the Viability Index). RESULTS: Twenty-eight percent of the gerbils met the behavioral selection criteria, and 17% met the ophthalmoscopic criteria. In the specimens selected by behavioral criteria (n = 7), 30% demonstrated no evidence of postischemic neuronal loss; the mean +/- SEM Viability Index scores for CA1, lateral cortex, and medial cortex were 1.6 +/- 0.6, 1.0 +/- 0.3, and 0.3 +/- 0.2, respectively. Of the animals selected by ophthalmoscopic criteria (n = 12), 100% had severe ischemic tissue damage to the ipsilateral hemisphere; the Viability Index scores for CA1, lateral cortex, and medial cortex were 3.5 +/- 0.1, 3.1 +/- 0.2, and 1.2 +/- 0.2, respectively; all scores were significantly larger than those observed in the behaviorally selected group. CONCLUSIONS: Selection of animals by ophthalmoscopic criteria provides a reliable, consistent method to predict animals with severe focal cerebral ischemia.

Animals↗
PubMed2001

Re-evaluation of epoxy resin sections for light and electron microscopic immunostaining.

Epoxy resins provide optimal tissue morphology at both the light and the electron microscopic level and therefore enable correlative studies on semithin and thin sections from the same tissue block. Here we report on an approach to retain these advantages for immunolabeling studies by adapting and combining well-known techniques, i.e., surface etching with sodium ethoxide and heat-mediated antigen retrieval. We propose a simple procedure for immunostaining semithin and thin epoxy resin sections. To check its applicability, well characterized, commercially available antibodies (against E-cadherin, alpha-catenin, and beta-catenin) were used on sections of human small intestine. By light microscopy, the immunostaining efficiency was compared on cryo-, paraffin, and epoxy semithin sections processed in parallel. The most detailed results were obtained on semithin sections, where the labeling precisely delineated the lateral plasma membrane of the enterocytes. At the electron microscopic level the procedure did not damage the structures and allowed an efficient, reproducible immunogold labeling extending homogeneously over exceptionally wide tissue areas. The three antibodies specifically labeled the zonula adherens of the junctional complex between epithelial cells and, in agreement with light microscopic observations, the lateral plasma membrane.

Cadherins↗
PubMed2002

NFkappaB and caspase-3 activity in apoptotic hepatocytes of galactosamine-sensitized mice treated with TNFalpha.

Tumor necrosis factor-alpha (TNFalpha) induces apoptosis in hepatocytes only under transcriptional arrest induced by galactosamine (GalN). In this study we demonstrated the shuttle of the transcription factor NFkappaB (nuclear factor-kappa B) in the liver tissue of mice within 30 min-4.5 hr hours after GalN/TNFalpha treatment. NFkappaB translocation from cytoplasm to the nucleus is initiated by its separation from the inhibitory IkappaB proteins which include IkappaBalpha, IkappaBbeta, and IkappaB. Thirty minutes after GalN/TNFalpha administration, NFkappaBp65 in hepatocellular nuclei becomes increasingly detectable and reaches its highest level after 2.5 hr. Then export back into cytoplasm begins but, surprisingly, approximately 30% of NFkappaB remains in the nuclear fraction and appears as an immunoprecipitate in the nuclei of apoptotic hepatocytes. Non-apoptotic hepatocytes do not show any reaction product in the nuclei 4.5 hr after treatment. Correspondingly, the amount of dissociated IkappaBbeta decreases in the cytoplasm up to 2.5 hr and increases again afterwards, although it does not reach the level of the control samples. No evidence of IkappaBbeta in the nuclei was found either immunocytochemically or biochemically. Caspase-3 activity, which is responsible for apoptosis, increases significantly after 3.5 hr. At that time, apoptotic hepatocytes can occasionally be observed and, 4.5 hr after GalN/TNFalpha treatment, constitute approximately 30% of the hepatocytes.

Animals↗
PubMed2006

Harder and stiffer bone osseointegrated to roughened titanium.

Mechanisms underlying the beneficial anchorage of roughened titanium implants have not been identified. We hypothesized that the implant surface roughness alters intrinsic biomechanical properties of bone integrated to titanium. Nano-indentation performed on two- and four-week post-implantation bone specimens of rats revealed that bone integrated to acid-etched titanium was approximately 3 times harder than that integrated to the machined titanium, both at the osseointegration interface and at the inner area of the peri-implant bone. The hardness of the acid-etched surface-associated bone was equivalent to that of untreated cortical bone at week 4, while the bone hardness around the machined surface was equivalent to that of the untreated trabecular bone. The elastic modulus of the integrated bone was 1.5 to 2.5 times greater around the acid-etched surface than around the machined surface. Analysis of the data suggests that the implant surface roughness affects the biomechanical quality of osseo-integrated bone, and that the bone integrated to the acid-etched surface is harder and stiffer than the bone integrated to the machined surface.

Acid Etching, Dental↗
PubMed1975

Light microscopic localization of cytochemical reactions in epoxy-embedded material for electron microscopy.

Tissues from mice were fixed in 1.5% glutaraldehyde, treated for the ultrastructural localization of alkaline phosphatase or Mg++-dependent adenosine triphosphatase, post-fixed in osmium tetroxide, dehydrated and embedded in plastic for electron microscopy. The sites of reaction were visualized in 1-mu plastic sections counterstained with toluidine blue, using a phase contrast microscope. The data show a close correlation between the sites of reaction observed with the phase contrast microscope and the sites studied with the electron microscope. The use of this technique for the study of these phosphatases in normal and pathologic tissues is recommended in order to achieve a high degree of accuracy in selecting a portion of the tissue sample for electron microscopy and to obtain greater resolution in the localization of these enzymes with the light microscope.

Adenosine Triphosphatases↗
PubMed1978

Masking of protein antigen by modification of amino groups with carbobenzoxychloride (benzyl chloroformate) and demasking by treatment with nonspecific protease.

Cryostat sections of various substrates were treated with carbobenzoxychloride in acetone to modify antigens. By applying specific fluorescent antibodies, it could be shown that the antigenic determinants of rabbit gamma-globulin and bovine insulin were totally masked. The antigenicity of ACTH was markedly reduced, whereas the polysaccharide antigens of Salmonella typhimurium were only partially masked. After masking, antigenicity could be restored by treatment with nonspecific protease. The reversible protection of amino groups by carbobenzoxychloride may be a way to preserve protein antigens during embedding in plastics, as such materials also bind to amino groups, blocking the antigenicity of proteins.

Acetone↗
PubMed1982

Immunocytochemical and autoradiographic demonstration of prolactin binding to human breast cancer cells in tissue culture.

Specific binding of human prolactin to human breast cancer cells in culture was demonstrated for the first time by immunocytochemistry ad autoradiography. Using the former technique, it was found that prolactin binding to the cell surface was heterogeneous: some cells showing intense to moderate peroxidase reaction product and some cells showing absence of reaction product. After the cells were incubated with 125I-labeled prolactin and then embedded in plastic, autoradiography of sections revealed both surface-localized and intracellular silver grains, the latter suggesting internalization of prolactin. These two cytological techniques are useful for visualizing prolactin binding to human breast cancer cells and are applicable to studies on prolactin binding in human breast cancer.

Autoradiography↗
PubMed1983

Cobalt uptake and localization of putative calcium binding sites in striated muscle.

Frog sartorius muscles were soaked overnight in saline containing 50 mM cobalt and subsequently fixed with or without precipitation of intracellular cobalt by exposure to ammonium sulfide. Treated fibers and appropriate controls were embedded in plastic in the usual manner. Thick sections (3-4 micrometers) of each sample were treated with silver in order to intensify precipitated cobalt. The samples were then resectioned for ultrastructural examination. Fibers exposed to cobalt with or without precipitation had normal ultrastructure; indeed, the transverse lines of the A-zone and M-band appeared to be more obvious after exposure to cobalt than in control samples. Exposure to silver produced numerous silver grains only in those samples that contained precipitated cobalt. The distribution of silver grains after intensification matched expected calcium binding sites. Silver grains were abundant over membranes of the sarcoplasmic reticulum, sarcolemma, mitochondria, and nucleus and rare or absent from filaments and sarcoplasm. This technique appears to be generally useful for locating intracellular calcium binding sites.

Animals↗
PubMed1988

Chick osteoblasts contain fluoride-sensitive acid phosphatase activity.

We used histological and biochemical methods to determine the cellular origin of bone matrix fluoride-sensitive acid phosphatase in chicken bone. Embryonic chicken calvariae were embedded in plastic and sections stained for acid phosphatase at various concentrations of substrate and fluoride. Acid phosphatase activity was observed in osteoblasts and osteoclasts but not in fibroblasts. Striking inhibition of osteoblastic acid phosphatase occurred at 100 microM fluoride, a concentration that had no apparent effect on osteoclastic acid phosphatase. Inhibition of osteoblastic and osteoclastic acid phosphatase by fluoride was also examined using extracts of embryonic chicken calvarial cells, mouse osteoblasts (MC3T3-El cell line), and purified chick osteoclasts, respectively. Fluoride is a partial competitive inhibitor of both chicken and mouse osteoblastic acid phosphatases, with apparent inhibition constants of 10-100 microM. These concentrations of fluoride correspond to those that increase bone formation in vitro and in vivo. In contrast, the apparent inhibition constant for fluoride of osteoclastic acid phosphatase was much higher (i.e., 0.5 mM). In summary, this study demonstrates that chicken osteoblasts contain an acid phosphatase that is sensitive to inhibition by low concentrations (i.e., microM) of fluoride.

Acid Phosphatase↗
PubMed1991

Simultaneous immunohistochemical detection of IUdR and BrdU infused intravenously to cancer patients.

Cell cycle kinetics of solid tumors in the past have been restricted to an in vitro labeling index (LI) measurement. Two thymidine analogues, bromodeoxyuridine (BrdU) and iododeoxyuridine (IUdR), can be used to label S-phase cells in vivo because they can be detected in situ by use of monoclonal antibodies (MAb) against BrdU (Br-3) or IUdR (3D9). Patients with a variety of solid tumors (lymphoma, brain, colon cancers) received sequential intravenous IUdR and BrdU. Tumor tissue removed at the end of infusion was embedded in plastic and treated with MAb Br-3 and 3D9 sequentially, using a modification of a previously described method. Clearly single and double labeled cells were visible, which enabled us to determine the duration of S-phase (Ts) and the total cell cycle time (Tc), in addition to the LI in these tumors. Detailed control experiments using tissue culture cell lines as well as bone marrow cells from leukemic patients are described, including the comparison of this double label technique with our previously described BrdU-tritiated thymidine technique. We conclude that the two methods are comparable and that the IUdR/BrdU method permits rapid and reliable cell cycle measurements in solid tumors.

Antibodies, Monoclonal↗
PubMed1996

Altered aggrecan synthesis correlates with cell and nucleus structure in statically compressed cartilage.

Previous studies have shown that static equilibrium compression of cartilage tissue in vivo and in vitro decreases chondrocyte synthesis of aggrecan molecules. In order to identify mechanisms of cellular response to loading, we have investigated alterations in cell and nucleus structure and the accompanying changes in the synthesis of aggrecan in statically compressed cartilage explants. Using glutaraldehyde fixation and quantitative autoradiography of compressed and radiolabeled cartilage disks we spatially localized newly synthesized aggrecan. Using stereological tools to analyze these same specimens we estimated the cell and nucleus volume, surface area and directional radii. We found that aggrecan synthesis was reduced overall in compressed tissue disks. However, the compression induced a spatial (radial) inhomogeneity in aggrecan synthesis which was not present in uncompressed disks. This spatial inhomogeneity appeared to be directly related to mechanical boundary conditions and the manner in which the load was applied and, therefore, may represent a spatially specific functional adaptation to mechanical loading. Coincident with reduced aggrecan synthesis, we observed reductions in cell and nucleus volume and radii in the direction of compression which were in approximate proportion to the reduction in tissue thickness. Cell and nucleus dimensions perpendicular to the direction of compression did not change significantly. Therefore the observed deformation of the cell and nucleus in statically compressed cartilage approximately followed the dimensional changes imposed on external specimen surfaces. The strong correlation observed between local changes in aggrecan synthesis and alterations in cell and nucleus structure also lend support to certain hypotheses regarding the intracellular signal transduction pathways that may be important in the biosynthetic response of chondrocytes to mechanical loading.

Aggrecans↗
PubMed1992

Measurements of tooth length in panoramic radiographs. 1. The use of indicators.

The aim of this study was to investigate both the actual and the radiographic tooth lengths of the maxillary first molar and second premolar and the mandibular premolars in panoramic radiographs. The actual length of 64 extracted teeth was measured. Steel balls were then attached to the cusp and apex and the teeth embedded in plastic moulds. Each pair of plaster casts with their set of teeth was radiographed with an Orthopantomograph twice at an interval of 1 month. The actual and the radiographic tooth length was measured twice by one observer. The mean tooth length of the molar was shorter than that of the premolars. The mean difference between the repeated measurements of the actual tooth length was small and ranged between 0.47 and 1.16% of the tooth length. The mean difference between repeated measurements of the radiographic tooth length was also small, with a method error of 0.13-0.21 mm. The vertical magnification in panoramic radiography was lower for mandibular premolars (13-15%) than for the maxillary second premolar and first molar (17-28%). The palatal root of the maxillary first molar had the highest vertical magnification (28%). Following the second set of radiographs, the mean difference between the measurements was small, except for the palatal root of the maxillary first molar (P < 0.001). Radiographic measurements of this root should therefore be used with caution. The results for the other roots examined indicate that these could be measured with high reproducibility.

Humans↗
PubMed1990

Morphological aspects of the human vestibular nerve.

A quantitative study was made of the number of fibers, their diameters, and distribution in the nerves innervating individual vestibular receptor organs of three human temporal bones. The specimens were obtained from autopsies conducted within 4 hours postmortem. The temporal bones containing the nerves were fixed in 3% glutaraldehyde and decalcified with EDTA until they were soft enough to allow dissection of the nerve branches to the individual receptors. The nerve branches were processed for osmium staining, embedded in plastic, and cut serially in 1-micron-thick sections for light-microscopic histologic evaluation of their fiber composition. Quantitative evaluation of nerve-fiber characteristics was made with the aid of a laboratory computer and programs for graphic representation and statistical analysis. In the nerves to the individual cristae the number of fibers ranged between 1416 and 2335. Fibers with diameters between 2.5 microns and 3 microns were the most numerous. The number of fibers decreased exponentially with increased size. The thickest fibers had diameters up to 11.5 microns. The distribution of fibers in the nerve of each crista was different for the central and intermediate areas of the crista than it was for the ends. Thin fibers with diameters less than 2.5 microns, which represented 36% of the population, projected to the ends of the receptor area. Thick fibers with diameters greater than 4.5 microns, which represented 8% of the fibers, were distributed relatively uniformly except for the extreme ends of the crista, where they were nearly absent. Fibers with intermediate diameters were distributed throughout the crista, although they were more concentrated at the ends. In the nerves to three maculae, the number of fibers ranged from 3744 to 5538. The percentage of fibers of each size, however, was similar to that in the cristae. The greater portion of fibers had diameters between 3.0 microns and 3.5 microns. Because of the anatomical configuration of the maculae, it was not possible to compare fiber diameters from one area to another. The composition and differential projection of fibers to the crista in the human labyrinth were similar to those found earlier in squirrel monkeys and bullfrogs. On the basis of these similarities in the pattern of innervation, it is suggested that the physiological properties in humans must be similar to those in animals.

Aged↗
PubMed2001

Electron microscopic studies of ion- and H2O-transporting epithelial cells in the horizontal ampulla of the pigeon.

Earlier morphological studies of the epithelial structure in the semicircular canals of mammals have focused on the sensory cells of the crista ampullaris. This report draws attention to the fact that there exist at least seven further cell types in the horizontal ampulla walls of pigeon with various functions; the role of ion- and H2O-transporting epithelial cells is dealt with here in detail. While the dark cells appear to play a decisive role in the regulation of ionic composition, the cells in the planum semilunatum may transport H2O and assist in the regulation of endolymph volume. In addition, protein-secreting structures are located in the apical region of the cells of the planum semilunatum. The question whether the proteins are dispersed in the endolymph or contribute to cupula formation remains unclear. The morphology and possible functions of these two cell types are discussed on the basis of electron microscopic results.

Animals↗