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PubMed1998

[Diagnosis of bone tumors].

The correct diagnosis of bone tumors involves considerable methodological efforts. Because of the rarity of primary bone tumors Uehlinger initiated a centralisation in the form of bone tumor registries. A sufficient number of these registries exist in the German-speaking countries (for example, Münster, Madgeburg, Giessen, Freiburg, Basel, Wien). Our experience with the organisation of interdisciplinary collaboration, methodology and the value of special examination techniques (immunohistochemistry, DNA analysis, cytogenetics) are reported. Since 1975, 7,576 primary bone tumors have been recorded in the Hamburg Bone Tumor Registry. A total of 9,895 biopsy/resection specimens have been examined. Frozen section diagnosis in combination with imprint cytology can be performed as for other tumors. Routine techniques are paraffin embedding, if necessary following EDTA decalcification, and plastic embedding. By means of contact x-rays good insights can be gained into the extent of bone matrix formation or the osteolytic activity of bone tumors. These findings correlate well with the results of clinical imaging. In special cases DNA analysis and various immunohistological reactions can contribute substantially to a correct diagnosis. So far cytogenetics and molecular biology are of only limited use in routine diagnostics, apart from Ewing's sarcoma. In spite of sufficient experience of many registries there are still a number of inaccurately defined primary bone tumors with uncertain biology, for example low grade malignant osteosarcoma. A clear characterization of these tumors using new techniques is a task for the future.

Bone Neoplasms↗
PubMed1995

EM-tomography of section collapse, a non-linear phenomenon.

Using back projection for reconstruction and tilt series of Epon or Lowicryl embedded and sectioned material, we demonstrated: (1) a reduction in thickness of 50% for Epon and 80% for Lowicryl sections, and (2) a non-uniform density distribution along the electron-optical axis in sections. The highest density was found at the vacuum exposed side of the section. The formvar side of the section showed a similar increase in density, but not to the same extent. Minimalization of electron exposure, even without pre-exposure, did not affect the reconstructed thickness, nor did it affect the non-uniform density distribution. However, parallax measurements showed that at 150K, collapse of Epon sections does not take place. For EM-tomography of plastic embedded material our findings imply that at the top and bottom portion of the sections the dimensions of the reconstructed structures are distorted, but that in the middle portion the dimensions are reliably retained.

Acrylic Resins↗
PubMed1999

A quantitative histochemical procedure for measurement of starch in apple fruits.

Measurements of starch (e.g. amyloplasts in stomatal guard cells, sieve elements, root tips or the starch sheath) is often very difficult using most analytical methods. An evaluation was made of interactive computer image analysis of starch measurements in apple fruits. The results obtained indicate that quantitative histochemistry can be an appropriate method to quantify starch. Correlations for starch values between the image analysis system and a colorimetric system were quantified. The thickness of plastic-embedded slices had no influence on the accuracy of the area occupied by image-quantified starch (starch/slice) or on its variance. The magnification of the objective also had no effect on measured starch-occupied areas (starch/slice), but there were big differences in variance. The number of replications required to establish statistically significant differences were calculated.

Histocytochemistry↗
PubMed1995

The ras-like rab3A protein is present in pinealocytes of the gerbil pineal gland.

As part of our molecular and functional characterization of the compartment of synaptic-like microvesicles (SLMVs) in mammalian pinealocytes, we have now analyzed the pineal gland of the Mongolian gerbil for the presence of rab3 proteins. Members of this subfamily of small G proteins are thought to regulate the intracellular trafficking and/or membrane fusion of secretory vesicles. Immunostaining of serial semi-thin sections of plastic-embedded pineals with monoclonal antibodies which recognize rab3A revealed the occurrence of rab3A in pinealocytes throughout the gland. Rab3A immunoreactivity was markedly enriched in dilated pinealocyte process terminals known to contain accumulations of SLMVs. The latter could be labeled with the rab3 antibodies at the ultrastructural level when immunogold staining was performed. Our results lend further support to the hypothesis that mammalian pinealocytes are endowed with a population of SLMVs that serve secretory functions.

Animals↗
PubMed1993

Combined organ of Corti/modiolus technique for preparing mammalian cochleas for quantitative microscopy.

An improved cochlear preparation technique is described with the following key features: 1) Preservation of the organ of Corti (OC) and the spiral ganglion cells (SGCs) in the same cochlea for quantitative, high-resolution microscopic evaluation; 2) Dissection of the plastic-embedded cochlea so that the entire OC can be prepared as whole mounts for quantitative study while the modiolus remains in a single block; 3) Decalcification and serial sectioning of the modiolus so that all SGC bodies are available for microscopic examination. This technique will be valuable for correlating the condition of the OC, nerve terminals, nerve fibers and the SGC bodies in normal and damaged cochleas.

Animals↗
PubMed1997

Human neutrophil lipocalin (HNL) is a specific granule constituent of the neutrophil granulocyte. Studies in bronchial and lung parenchymal tissue and peripheral blood cells.

The neutrophilic granulocyte is a cytotoxic and potentially tissue-injuring cell participating in the destructive processes and symptoms seen in a variety of inflammatory diseases. Sensitive immunoassays have been introduced to measure the levels of specific secretory proteins of various inflammatory cells in blood and other body fluids. The aim has been to develop highly specific markers for each cell type. The results obtained by immunoassay have indicated that human neutrophil lipocalin (HNL) is a protein unique to the neutrophil. The present study investigated the specificity of HNL as a neutrophil marker in peripheral blood and lung tissue by using flow cytometry and immunocytochemistry. Flow cytometry and immunocytochemistry on peripheral blood showed that monoclonal antibodies to HNL only react with neutrophils and not with other types of leukocytes. Immunocytochemistry on plastic-embedded sections and on frozen sections of lung tissue showed that a cocktail of six monoclonal antibodies to HNL specifically reacts with neutrophils and not with epithelial cells or macrophages. By immunoelectron microscopical studies performed on healthy human neutrophils after low temperature embedding in Lowicryl K4M following aldehyde fixation and partial dehydration, it could be shown that HNL colocalized with lactoferrin (a known marker for secondary or specific granules) and that myeloperoxidase was localized in the primary or azurophil granules. The results confirm that HNL is a unique component of the secondary granules of the neutrophil granulocyte.

Acute-Phase Proteins↗
PubMed2002

Er:YAG laser scaling of diseased root surfaces.

BACKGROUND: The removal of calculus and plaque is an essential component of a therapeutic approach to control periodontal disease. Er:YAG laser scaling was recently introduced as an alternative to conventional scaling procedures. In this histological study, the effects of laser instrumentation of diseased root surfaces are compared to mechanical removal of plaque and calculus with ultrasonic instruments and scalers. METHODS: Areas of subgingival calculus were identified on 40 freshly extracted human teeth. Each of these areas was randomly divided into 2 equal parts. The control site was treated either with scaling and root planing or with an ultrasonic instrument. The test site was cleaned using an Er:YAG laser according to the manufacturer's instructions. The end point of debridement was the inability to mechanically or visibly detect any remaining calculus. After pre- and postsurgical photographs and impressions for scanning electronic microscopic investigation, a plastic embedding technique was used to cut the undecalcified roots into 15 microm thick sections. RESULTS: Clinically and histologically, scaling resulted in complete debridement at all samples, producing a smooth root surface. At the test sites, laser scaling was accompanied by an increased removal of tissue and roughened surfaces. CONCLUSIONS: Laser scaling results in an increased loss of cementum and dentin, which should be taken into account in clinical situations.

Aluminum Silicates↗
PubMed1977

Immunofluorescence on resin-embedded material.

Some antigenic determinants can be preserved in tissues after plastic embedding. In the present study liver tissue was fixed with glutaraldehyde or paraformaldehyde, dehydrated with ethanol and toluene and embedded in araldite which was then polymerized at 37 degrees C. Immunofluorescence was performed on semi-thin sections etched with hydrogen peroxide. This procedure allows correlation with light microscopy (on the same stained semi-thin section) and with electron microscopy (on adjacent ultra-thin section). Good results were obtained with anti-nuclear, anti-mitochondrial, anti-microsomal and anti-alpha1-antitrypsin sera.

Antibodies, Antinuclear↗
PubMed1994

Staining and histomorphometry of microcracks in the human femoral head.

We developed staining techniques that permit identification and histomorphometric analysis of microcracks in the human femoral head 1) from thick, ground bone sections (100 microns) by prestaining with the Villanueva mineralized bone stain (MIBS), and 2) from plastic embedded, undecalcified thin bone sections (5-15 microns) by staining in gallocyanin chrome alum-Villanueva blood stain methods. Both methods represent a significant improvement in the stainability of the microcracks, cellular and tissue elements, and the simultaneous assessment of osteoid seams and tetracycline markers by histomorphometry. Shrinkage and other artifacts were minimized, which helped to clarify some of the uncertainties arising from artifacts resulting from some bone staining methods. Histomorphometric analyses of microcracks were conducted on thick, ground sections of subchondral and trabecular bone. Microcracks were more prevalent in the subchondral bone and osteochondral junction than in the more distant trabeculae. We have consistently localized microcrack areas in bone tissues prepared in these ways.

Aged↗
PubMed1995

Gap junctions revealed by freeze-fracture electron microscopy.

Gap junctions provide the basis for the formation of elaborate networks of communication between cells in animal tissues. Electron microscopic examination of thin sections of plastic embedded gap junctions has provided valuable information on the anatomy and function of these remarkable structures. Freeze-fracture electron microscopy, however, has made available unique vistas of gap junction-bearing intramembrane surface--surface previously inaccessible to the researcher's eyes. Data on population density, distribution, size, geometry of intramembrane particle packing, and structural responses of gap junction components to experimental manipulation are simply and easily obtained with freeze fracture. Recent developments of sophisticated protocols of immunocytochemistry as applied to freeze-fracture replicas further serve to reinforce the notion that freeze-fracture is a powerful tool for study of gap junctions. Molecular techniques of gap junction gene transfection promise to add a truly unique dimension to investigations of the broad spectrum of functional roles of gap junctions.

Animals↗
PubMed2000

Apical inflammatory root resorption: a correlative radiographic and histological assessment.

AIM: To assess the reliability of routine single radiographs in the diagnosis of inflammatory apical root resorption by correlating the radiographic and histological findings. METHODOLOGY: The material comprised serial and step serial sections of plastic-embedded root-apices with attached apical periodontitis lesions that were prepared for a previous study and the diagnostic radiographs. The histological sections of 114 specimens were analysed by light microscopy and categorized into three groups: (i) those without any resorption (0); (ii) those with moderate resorption (+); and (iii) those with severe resorption (+ +). The radiographs were examined by a separate examiner and graded with a similar categorization of no resorption (0); moderate (+); and severe (+ +) apical resorption. RESULTS: Radiographically, 19% of the teeth were diagnosed as having apical inflammatory root resorption, whereas histologically, 81% of the teeth revealed apical inflammatory root resorption. A correlative radiographic and histological assessment (n = 104) revealed a coincidence of diagnosis in 7% of the specimens and noncoincidence of diagnosis in 76% of the specimens. CONCLUSIONS: The results indicate that routine single radiographs are not sufficiently accurate or sensitive to consistently diagnose apical root resorptive defects developing as a consequence of apical periodontitis.

Dental Cementum↗
PubMed1997

Mast cells and atopic dermatitis. Stereological quantification of mast cells in atopic dermatitis and normal human skin.

Stereological quantification of mast cell numbers was applied to sections of punch biopsies from lesional and nonlesional skin of atopic dermatitis patients and skin of healthy volunteers. We also investigated whether the method of staining and/or the fixative influenced the results of the determination of the mast cell profile numbers. The punch biopsies were taken from the same four locations in both atopic dermatitis patients and normal individuals. The locations were the scalp, neck and flexure of the elbow (lesional skin), and nates (nonlesional skin). Clinical scoring was carried out at the site of each biopsy. After fixation and plastic embedding, the biopsies were cut into 2 microns serial sections. Ten sections, 30 microns apart, from each biopsy were examined and stained alternately with either toluidine blue or Giemsa stain and mast cell profile numbers were determined. The study yielded the following results: (1) in atopic dermatitis lesional skin an increased number of mast cell profiles was found as compared with nonlesional skin, (2) comparing atopic dermatitis skin with normal skin, a significantly increased number of mast cell profiles per millimetre squared was found in specimens from the neck, (3) staining with toluidine blue yielded a lower number of mast cell profiles than Giemsa staining, (4) the use of Carnoy's fixative resulted in a lower mast cell profile count than the use of formaldehyde, and (5) there was no statistically significant correlation between the clinical score and the number of mast cell profiles per millimetre squared. Using stereological techniques, this study indicated that mast cells might participate in the inflammatory process in skin leading to atopic dermatitis.

Adult↗
PubMed1994

Ultrastructural studies of diffuse axonal injury in humans.

Diffuse axonal injury (DAI) is observed commonly in traumatically brain injured humans. However, traditional histologic methods have proven of limited use in identifying reactive axonal change early (< 12 h) in the posttraumatic course. Recently, we have reported, in both humans and animals, that antibodies targeting neurofilament subunits are useful in the light microscopic recognition of early reactive change. In the present study, we extend our previous efforts in humans by analyzing the progression of traumatic brain injury (TBI)-induced axonal change at the ultrastructural level. This effort was initiated to follow the subcellular progression of reactive axonal change in humans and to determine whether this progression parallels that described in animals. Two commercially prepared antibodies were used to recognize reactive axonal change in patients surviving from 6 to 88 h. The NR4 antibody was used to target the light neurofilament subunit (NF-L), and the SMI32 antibody was used to target the heavy neurofilament subunit (NF-H). Plastic-embedded tissue sections were screened for evidence of reactive axonal change, and once identified, this reactive change was analyzed at the ultrastructural level. At 6 h survival, focally enlarged, immunoreactive axons with axolemmal infolding or disordered neurofilaments were seen within fields of axons exhibiting no apparent abnormality. By 12 h, some axons exhibited continued neurofilamentous misalignment, pronounced immunoreactivity, vacuolization, and, occasionally, disconnection. At later stages, specifically 30 and 60 h survival, further accumulation of neurofilaments and organelles had led to the further expansion of the axis cylinder, and clearly disconnected reactive swellings were recognized. These contained a dense core of disordered immunoreactive neurofilaments partially encompassed by a cap of less densely aggregated organelles. At 88 h, the reactive axons were larger and elongated, consistent with the continued delivery of organelles by axoplasmic transport. At the later time points, considerable heterogeneity was observed, with focally enlarged disconnected axons being observed in relation to axons showing less advanced reactive change. Our findings suggest that neurofilamentous disruption is a pivotal event in axonal injury.

Adult↗
PubMed1995

Regenerated nerve fibers in the noise-damaged chinchilla cochlea are not efferent.

Nerve-fiber regeneration in the chinchilla cochlea following a traumatic noise exposure was systematically described by Bohne and Harding (1992). However, their study did not determine the origin of the regenerated nerve fibers (RNFs). In the present study, 23 chinchillas were exposed for 12 h to a 0.5 kHz octave band of noise at 120 dB SPL. After a 3-month or 1-year recovery period, their right cochleas were incubated to demonstrate acetylcholinesterase (AChE) activity and then briefly counterstained with Neutral Red or OsO4. Their left cochleas were fixed with OsO4 and dissected using a combined organ of Corti (OC)/modiolus technique that preserved both structures for high-resolution microscopy. All cochleas were prepared as plastic-embedded flat preparations. Damage was located in the basal two-thirds of the cochlea and generally consisted of multiple lesions in the OC, often involving total degeneration of one or more OC segments (i.e., OC wipeouts). The OC wipeouts were separated from one another by areas which contained some identifiable cells of the OC (i.e., OC remnants). Most RNFs were found in OC wipeouts adjacent to OC remnants. In those animals (83%) with significant OC damage, 13 (100%) 3-month-recovery chinchillas had 1-96 RNFs while 6 (86%) 1-year-recovery chinchillas had 7-62 RNFs. In the AChE-stained cochleas, none of the RNFs were AChE-positive, but normal AChE-positive fibers were found in the undamaged apical turn. A variable number of surviving spiral ganglion cells was present in those regions of Rosenthal's canal that had originally innervated the missing hair cells in the OC wipeouts and remnants. It is concluded that RNFs are not part of the efferent cochlear system and therefore, most likely belong to the afferent system.

Acetylcholinesterase↗
PubMed2002

Ciliated epithelium-lined radicular cysts.

OBJECTIVE: This report describes 3 cases of ciliated epithelium-lined radicular cysts among 256 apical periodontitis lesions and also illustrates the occurrence of an Actinomyces-infected periapical cyst. STUDY DESIGN: Serial and step serial sections of 256 plastic-embedded root apices with attached apical periodontitis lesions that were prepared for a previous investigation were reviewed for the presence of ciliated epithelium-lined radicular cysts. The lesions that were found to have such epithelial lining were examined in a transmission electron microscope to elaborate the fine structure of the ciliated cells. RESULTS: A total of 3 ciliated columnar epithelium-lined cysts was found among the 256 apical periodontitis lesions examined. Two of the lesions also contained stratified squamous epithelium. All 3 lesions affected maxillary premolars. One of the lesions was a true cyst, and the other 2 were periapical pocket cysts. The lumen of 1 of the latter revealed the presence of typical "ray-fungus" actinomycotic colonies. CONCLUSION: Although the stratified squamous component of the epithelia that lined the radicular cysts reported here may be derived from the cell rests of Malassez, the ciliated epithelial cells may be of sinus origin. Microbial agents from diseased root canals can advance into radicular cysts, particularly in pocket cysts, with the possible threat of such infection in upper posterior teeth spreading into the maxillary sinus.

Actinomyces↗
PubMed1981

A hematoxylin and eosin-like stain for glycol methacrylate embedded tissue sections.

A staining procedure is described for use with glycol methacrylate embedded tissue sections which does not stain the plastic embedment or remove the sections from the glass slides. The basic dye is celestine blue B. It is prepared by treating 1 g of the dye with 0.5 ml concentrated sulfuric acid. It is then dissolved with the following solution. Add 14 ml glycerine to 100 ml 2.5% ferric ammonium sulfate and warm the solution to 50 C. Finally adjust the pH to 0.8 to 0.9 The acid staining solution consists of 0.075% ponceau de xylidine and 0.025% acid fuchsin in 10% acetic acid. Slides containing the dried plastic sections are immersed in the celestine blue solution for five minutes and in the ponceau-fuchsin solution for ten minutes with an intervening water rinse. After a final wash, the sections are air dried and coverslipped. This staining procedure colors the tissues nearly the same as hematoxylin and eosin procedures.

Animals↗
PubMed2006

Death pathways in noise-damaged outer hair cells.

Using morphological criteria, death pathways in outer hair cells (OHCs) were determined in chinchilla organs of Corti that had been exposed to a high- or moderate-level octave band of noise (OBN) centered at either 0.5 or 4-kHz. The specimens were part of our large collection of plastic-embedded flat preparations of chinchilla cochleae. Three death pathways were identified: (1) oncotic - swollen, pale-staining cell with a swollen nucleus, (2) apoptotic - shrunken, dark-staining cell with a pyknotic nucleus and (3) a newly defined third pathway - no basolateral plasma membrane but cellular debris arranged in the shape of an intact OHC with a nucleus deficient in nucleoplasm. To minimize the secondary loss of OHCs from the entrance of endolymph into the organ of Corti, the specimens used for quantitative analysis of death pathways had the following characteristics: (1) the level to which they were exposed was less than or equal to 95dB SPL, (2) the exposure duration was 6-216h, (3) fixation for microscopic examination took place in vivo 1-2h post-exposure and (4) there were no focal OHC lesions in the organs of Corti. Fifty-eight noise-exposed cochleae met these criteria. In these specimens, degenerating and missing OHCs were classified as to which death pathway the cells had followed or were following. Nine non-noise-exposed cochleae were also evaluated for OHC death pathways. The number of OHCs following the third death pathway was significantly greater in the noise-exposed cochleae than the non-noise-exposed cochleae for total exposure energies greater than those produced by 75dB SPL for 216h to a 0.5-kHz OBN and 57dB SPL for 48h to a 4-kHz OBN. In cochleae exposed to either octave band, OHCs dying by oncosis or apoptosis were uncommon.

Animals↗