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PubMed2002

The use of microcomputed tomography to study microvasculature in small rodents.

Appropriate nephron function is dependent on the intrarenal arrangement of blood vessels. The preferred and primary means to study the architecture of intrarenal circulation has been by filling it with opaque substances such as india ink, radio-opaque contrast material, or various polymers for study by light or scanning electron microscopy. With such methodologies, superficial vessels may obscure deep vessels and little quantitative information may be obtained. Serial-section microtomy has not been practical because of problems relating to alignment and registration of adjacent sections, lost sections, and preparation time and effort. Microcomputed tomography (micro-CT) overcomes such limitations and provides a means to study the three-dimensional architecture of filled vessels within an intact rodent kidney and to obtain more quantitative information. As an example of micro-CT's capabilities, we review the use of micro-CT to study the alterations in renal microvasculature caused by the development of liver cirrhosis after chronic bile duct ligation. In this example, micro-CT evidence shows a selective decrease in cortical vascular filling in the kidney, with a maintenance of medullary vascular filling. These changes may contribute to the salt and water retention that accompanies cirrhosis. These results indicate that micro-CT is a promising method to evaluate renal vascular architecture in the intact rodent kidney relative to physiological and pathological function.

Angiography↗
PubMed1998

Methylmercury intoxication and histochemical demonstration of NADPH-diaphorase activity in the striate cortex of adult cats.

The effects of methylmercury (MeHg) on histochemical demonstration of the NADPH-diaphorase (NADPH-d) activity in the striate cortex were studied in 4 adult cats. Two animals were used as control. The contaminated animals received 50 ml milk containing 0.42 microgram MeHg and 100 g fish containing 0.03 microgram MeHg daily for 2 months. The level of MeHg in area 17 of intoxicated animals was 3.2 micrograms/g wet weight brain tissue. Two cats were perfused 24 h after the last dose (group 1) and the other animals were perfused 6 months later (group 2). After microtomy, sections were processed for NADPHd histochemistry procedures using the malic enzyme method. Dendritic branch counts were performed from camera lucida drawings for control and intoxicated animals (N = 80). Average, standard deviation and Student t-test were calculated for each data group. The concentrations of mercury (Hg) in milk, fish and brain tissue were measured by acid digestion of samples, followed by reduction of total Hg in the digested sample to metallic Hg using stannous chloride followed by atomic fluorescence analysis. Only group 2 revealed a reduction of the neuropil enzyme activity and morphometric analysis showed a reduction in dendritic field area and in the number of distal dendrite branches of the NADPHd neurons in the white matter (P < 0.05). These results suggest that NADPHd neurons in the white matter are more vulnerable to the long-term effects of MeHg than NADPHd neurons in the gray matter.

Animals↗
PubMed2002

Histomorphometric analysis of rat alveolar wound healing with hydroxyapatite alone or associated to BMPs.

Several materials and techniques have been proposed to improve alveolar wound healing and decrease loss of bone height and thickness that normally follow dental extraction. The objective of this research was the histologic analysis of bone morphogenetic proteins implanted into dental alveoli of rats after extraction. A total of 45 adult male Wistar rats were divided into three groups of 15 animals each: control (no treatment), implanted with pure hydroxyapatite (HA, 3 mg) and implanted with hydroxyapatite plus bone morphogenetic proteins (HA/BMPs, 3 mg). Five animals from each group were sacrificed at 7, 21 and 42 days after extraction for the histometric analyses of the osteoconductive potential of hydroxyapatite associated or not with BMPs. After dissection, fixation, decalcification and serial microtomy of 6-micron thick sections, the samples were stained with hematoxylin-eosin for histologic and histometric analyses. Both HA and HA/BMPs caused a delay in wound healing compared to control animals, evaluated by the percentage of bone tissue in the alveoli. The treatment with HA/BMPs had the greatest delay at 21 days, even though it produced values similar to the control group at 42 days. The materials did not improve alveolar repair in the normal period of wound healing and the association of HA/BMPs did not have osteoconductive properties with granulated hydroxyapatite as the vehicle.

Alveolar Bone Loss↗
PubMed2006

Effects of Tissucol and epsilon aminocaproic acid in the healing process following dental extraction in dehydrated rats.

A histological study was conducted of the alveolar bone healing process following tooth extraction of dehydrated rats after the implantation of fibrin adhesive (TISSUCOL) associated to previous irrigation of the wound with a 5% epsilon aminocaproic acid solution (EACA). Seventy two rats were used, divided into three groups receiving different treatments after the surgical procedure. In group I, the gingival mucosa was sutured after extraction of the right upper incisor. In groups II and III, chronic dehydration was produced by water deprivation for 9 days (3 days in the preoperative period and 6 days in the postoperative period). In the animals of Group II, after tooth extraction, the gingival mucosa was sutured in the same way as performed in group I. In group III, after extraction, the dental socket was irrigated with 5% EACA, followed by implantation of the fibrin adhesive (TISSUCOL). The mucosa was sutured in the same way as performed in the other groups. At 3, 7, 15 and 21 postoperative days, the animals were sacrificed in number of 6 for each group. Specimens containing the dental socket were removed and fixed in 10% formalin and decalcified in an equal part formic acid and sodium citrate solution. After routine processing, the specimens were embedded in paraffin for microtomy. We obtained 6 microm semi-serial slices that were stained with hematoxylin and eosin for histological evaluation. The results showed that the water deprivation in the pre- and postoperative periods caused a delay in the alveolar bone healing process. The use of the fibrin adhesive (TISSUCOL) produced an improvement in the fibrinolytic picture caused by dehydration.

Aminocaproic Acid↗
PubMed2004

[Onco-surgical significance of anatomo-morphological specificity of glottic region of the larynx].

Glottis is a medium level of the larynx, involving the vocal cords, vocal process of arytenoid cartilage, and the anterior and posterior commissures. This region of heterogeneous histomorphological structure has specific characteristics: the internal striated muscles, the outer and inner perichondrium, atypical intra-cartilaginous areas of ossification, and unusual muscular insertion to cartilage. Microtomy of 3 thick successive sections was performed at the level of the upper surface of vocal cords. Standard histological staining methods were used. Microphotographs were taken by light microscope under different magnification along with histomorphometric measurements. Cancer spread is partially restricted by anatomic barriers: vocal ligament and tendon of the anterior commissure, and subsequently by the elastic cone. Easier tumor extension may be due to absence of the internal perichondrium and ossification of thyroid cartilage. Microvascularization of the anterior commissure is significant for tumor spread in glottis and paraglottic space. The role of commissural lymph network in local spread of the cancer is completely vague. It is certain that there are causes, still unrecognized, which have an effect on the pathways and direction of malignant tumor spread.

Adult↗
PubMed1980

Methods for the study of leaf anatomy in palms.

Large size, hardness, combinations of thick-walled fibers and sclereids with thin-walled parenchyma cells, and the occurrence of silica, calcium oxalate, and tannins make anatomical preparations of palm leaves difficult. Samples for anatomical study should encompass one-half a pinna or a comparable portion from palmate and entire leaves including the midrib, all large ribs, and the margin. Similar pieces from herbarium specimens are reconstituted in glycerin alcohol, aerosol OT and distilled water (10:3:90). All samples are fixed in formol-acetic-alcohol (FAA) but stored in glycerin alcohol to minimize hardening. Transverse and longitudinal sections 15 microns thick, epidermal macerations, and pieces for clearing and for scanning electron microscopy are prepared from the FAA fixed material. Samples for electroscanning are gradually changed to 100% acetone, critical point dried with CO2, and coated with 100-300 angstroms of gold. Leaf material for microtomy is treated with hydrofluoric acid, embedded in Paraplast, and sectioned at 15 microns at a temperature of 7.2 C. Paraplast sections are floated directly on a modification of Sass' Adhesive III, mounted unstained or stained in safranin and fast gaee, and observed in polarized light. Epidermal peels are prepared by soaking pieces 5 mm square for 12-24 hours in undiluted bleach. Pieces for observation of transverse veins are cleared by treatment in 5% sodium hydroxide in a 60 C oven, washed rapidly in three changes of distilled water, and placed in one-third strength commercial bleach until clear. The same procedures can be used to prepare reproductive material for anatomical observations, but time schedules must be increased for larger specimens.

Botany↗
PubMed1988

Identification of normal or neoplastic murine mesenchymal cells in chimeric tissues or heterotransplants.

An improved method for identifying murine mesenchymal cells in chimeric tissues or heterotransplants using Hoechst dye 33258 is described. Following fixation in formalin-saline, tissues are embedded in JB-4 plastic. Sections 3 micron thick are then stained in a 10 microgram/ml solution of Hoechst 33258 in Hanks' balanced salt solution for 5-10 min at 4 C. After rising, the sections are coverslipped using a modified polyvinyl alcohol mounting medium. This approach offers several advantages over existing techniques: 1) uniform section thickness is more easily obtained than with paraffin or cryostat microtomy, thereby allowing improved resolution and more reliable identification of mesenchymal cells with small nuclei such as skeletal muscle myocytes or fibroblasts, 2) the preparations are stable over long periods and can be repeatedly viewed or photographed, and 3) calcified tissues can be examined without prior decalcification. An example is shown of species identification using rat chondrosarcoma cells grown in nude mice.

Animals↗
PubMed1999

[Comparative study of the effect on stress distribution of porcelain laminate complex with different elastic moduli].

Porcelain laminate has been a kind of popular treatment modality for its aesthetics and less tooth reduction. The models of 3-D FEA of left upper permanent central incisor were designed by microtomy. Comparative studies of stress distribution were carried out to reveal the stress of three designs of tooth preparation with two kinds of occlusions. The results showed that under loads imitating of central occlusion, the stress level increased a little with the rising of elastic modulus of the porcelain material, while the stress values of type II veneer seemed the highest under loads imitating of protruding occlusion. For the designing of laminate, it implies that porcelain material with low elastic modulus should be used in case of type II veneers.

Dental Porcelain↗
PubMed1975

[Contribution of histochemistry to the classification of neuromuscular diseases].

For a long time after the first muscle biopsy performed on man by Bilroth in 1965, histological study of the muscle has been linked to out of date patterns of interpretation most of them without any importance for clinical diagnosis. The use of histochemical techniques in the study of muscle was introduced quite recently and consequently in the last 15 years it has been possible to collect an enormous amount of very important data for the clinical diagnosis of many neuromuscular disorders. The classification of muscle fibers into different types which was possible first of all using the myofibrillar ATPase reaction by Padykula and Hermann permitted pathological interpretation leading to specific correlations between histological and EMG results. Furthermore the use of different histoenzymological methods in the study of mitochondrial enzymes activity and of the enzymes for the glycogen breakdown and synthesis, promoted a more elaborate fibre typing system. Thank to the development of cryostatic microtomy, the above mentioned methods became easy to be performed in the laboratory routine work. The use of the cryostat, which allows a better preservation of muscular tissue, led to a more accurate diagnostic interpretation particularly in relation to morphology. The study of fiber typing revealed many alterations: single fiber type atrophy, type one or type two predominance, type grouping, hypertrophy of a single fiber type and so on, giving to the clinicians the possibility not only of a more elaborate pathogenetic interpretation, but also of a much more precise diagnosis than in the past. Important results have been achieved using the above mentioned histoenzymological methods in the study of the single muscle fibers. Anglo-Saxon Authors provided us with many significant terms, in order to point out alterations of the enzymes distribution within the single muscle fiber: moth eaten, target fiber, rods, central core, subsarcolemmal blebs. Some of these alterations, seen in specific neuromuscular disorders, still represent the only distinguishing feature of these disorders from the nosographic point of view. It is possible to assert without any doubts that in the last 15 years the use of histochemical methods in the study of human muscular tissue has allowed a nosografic classification of neuromuscular disorders very different from that in use before the histochemical age. Thanks to histochemistry the consequent diagnostic and therapeutic advantages have led to very interesting results in such an important field of Neurology.

Animals↗
PubMed2005

[A comparison of tissue formation and the content of polysaccharide between wild and cultured Dendrobium candidum].

OBJECTIVE: To compare the tissue formation and the content of polysaccharide between the wild Dendrobium candidum and the cultured ones and to find any existed differences. METHOD: Bare-handed microtomy and photomicrography; The content of polysaccharide is determined by phenol-sulphuric acid method. RESULT AND CONCLUSION: There are no marked noticeable differences between the wild D. candidum and the cultured ones in terms of the tissure formation and the content of polysaccharide.

Dendrobium↗
PubMed1979

Origin of artifactual quantitation of electrolytes in microprobe analysis of frozen sections of erythrocytes.

Frozen sections of erythrocytes have been used to validate microprobe X-ray analysis of diffusible elements in biological samples. At this meeting last year we reported that intracellular Na concentrations measured by microprobe were much higher than those measured by bulk chemical methods. It was suggested that this might be due to the movement of extracellular material over the cells by microtomy. We now present evidence that such results are better explained by electron scattering within the sample during analysis. This evidence includes observations that the excess measured Na varies directly with section thickness and inversely with accelerating voltage. At 80 kV accelerating voltage there is excellent agreement between microprobe and chemical analysis. It may be concluded that specimen preparation techniques such as used in our laboratory are satisfactory for reliably localizing diffusible elements in small volumes, but that instrumental factors can seriously affect their quantitation.

Electron Probe Microanalysis↗
PubMed1985

Scanning electron microscopy methodology for study of the pathophysiology of calcification in bioprosthetic heart valves.

Scanning electron microscope (SEM) morphologic analysis combined with energy dispersive characteristic X-ray (EDX) microprobe analysis provides insight into the mechanisms associated with disease-related crystal formation in biological materials. SEM and EDX were employed in analyzing specimens which were embedded in standard fashion in glycolmethacrylate. The specimen surfaces under electron microscope investigation resulted from microtomy used in the preparation of reference light microscope histological sections; thus histology served as a direct reference for the SEM and EDX analyses. The particular application of these methods was in the study of bioprosthetic heart valve calcification, largely responsible for clinical failure of these heart valve substitutes. To simulate the clinically observed mineralization processes, glutaraldehyde-pretreated porcine heart valve leaflets were implanted subcutaneously in rats and subsequently removed at various time intervals from 1 to 56 days. Also, to address the hypothesis that the calcification process generates crystalline materials analogous to those in bone, EDX data obtained from pure hydroxyapatite were compared with the embedded tissue results. Further, EDX results were compared with data obtained by chemical analysis of the bulk specimens to assess the validity of the electron microscope technique.

Animals↗
PubMed1984

Freeze-fracture of 2-cell mouse embryos. A new method for fracture of very small and scarce biological samples.

Membrane morphology and development of intercellular junctions in 2-cell mouse embryos was studied with freeze-fracture electron microscopy. In order to circumvent the dual problem of relative scarcity of samples and their very small size (about 50 micrometers per blastomere), both of which made conventional microtomy-induced freeze-cleavage impractical, a new protocol for handling 2-cell embryos was derived from freeze-fracture procedures established for monolayer cultures. Freshly-collected 2-cell embryos were placed within a 2 mm2 area on plastic Thermonox or Polystyrene coverslips which had been previously coated with 0.002% poly-l-lysine. The embryos were then fixed in 2.5% glutaraldehyde buffered to pH 7.3 with sodium cacodylate and containing 0.01% ruthenium red. The embryos were washed three times in buffer and then glycerinated. The next day, the 2 mm2 area of coverslip containing the embryos (75-100) was cut out with a fine scalpel, inverted and placed on a drop of Elvanol-glycerol mixture and frozen in a slurry of liquid nitrogen-cooled Freon-22. Fracturing was achieved in a Balzers BAF 301 Freeze-Etch Unit by positioning the microtome blade underneath a corner of the inverted coverslip and raising the knife until the coverslip 'popped off'. This procedure left the surface of the Elvanol droplet covered with fractured embryos. The exposed tissue was shadowed with platinum and coated with carbon. This protocol produces large expanses of blastomere plasma membrane as well as cross-fractures of entire blastomeres and 2-cell embryos. The replicas revealed blastomeres endowed with a rich assortment of pleomorphic microvilli.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1980

[Rational simplified plastic embedding with polymethacrylate for non-decalcified bone sections].

A exact judgment of bone and bone marrow tissue is based on an undecalcified treatment technique by means of plastics embedding and hard section microtomy. All hitherto existing routine techniques of plastics embedding of bone tissue used monomeres of methakrylate with addition of so-called emollient substances for polymerisation. Disadvantages of a stronger shrinking of the polymerisation product and uncontrolled, not always avoidable accelerations of the polymerisation process which have an essentially negative influence on the histological results should thus be avoided. A very exact observance of the treatment steps is here necessary. In the further development of our embedding method we turned to the polymethacrylates. The polymerisation adhesive Kalloplast R used in industry proved as the most suitable initial substance for the new method. The yielded results lie in a considerable shortening and further simplification of the embedding technique. The results are always well reproducible. Undesirable shrinking or polymerisation artefacts with impairment of the histological results do no more appear.

Bone and Bones↗
PubMed1978

A freeze-etching and replication study of wall deposition in elongating plant cells.

The architecture of the expanding wall of mung bean hypocotyl (Phaseolus aureus) and collenchyma of celery (Apium graveolens) was examined using freeze-etching without any cryoprotectant, and surface-replication of frozen-ground and air-dried specimens. The polylamellated organization of the wall was seen. Freeze-etching clearly visualized, within one single fracture plans, the intermediate strata in which the microfibril orientation gradually changes between the main transverse and longitudinal directions. They corresponded to the bow-shaped arcs seen with surface replication and conventional microtomy. The organization of newly-formed microfibrils (periplasmic microfibrils) was seen by their imprints on the plasmalemma. When they were being deposited the microfibrils were loose and sinuous though tightly packed, rigid and parallel on the further layers. Therefore it seems that the fibrillogenesis and the spatial orientation of the microfibrils are two subsequent steps. The role of the periplasm in controlling the three-dimensional arrangement of the wall is emphasized.

Cell Membrane↗