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PubMed1987

Staining methods for morphometric studies of parietal and gastrin cells in the rat stomach.

In order to select the most suitable procedures for quantitative microscopy of both parietal and gastrin cell populations in the rat stomach, various staining methods were compared. For parietal cell identification in particular, the following procedures were tested: i) the modification of the haematoxylin-eosin method proposed by Marks and Drysdale, ii) the haematoxylin-eosin-saffron fluorochrome stain on paraffin sections, iii) the haematoxylin-azophloxin-saffron fluorochrome stain on paraffin sections, and iv) the May-Grunwald-Giemsa stain on thin sections from plastic-embedded specimens. This last provided the best results in parietal cell individualization and seemed to be the most suitable method for an accurate image analysis. Immunohistochemistry was the only unequivocal way to identify gastrin cells. Two variant procedures were examined; a) the agar-paraffin embedding technique, and b) the combination of a mucin staining with the immunoperoxidase reaction. The first technique provided an easier procedure for handling seriate strips of gastric mucosa for proper enumeration of immunostained cells. The second was presented as a promising variant procedure for a combined investigation of both G-cell population and mucin secretion patterns under differnt experimental conditions in the same specimen.

Animals↗
PubMed1988

Malignant lymphomas in the acquired immunodeficiency syndrome. Additional evidence for a B-cell origin.

An increased incidence of high-grade malignant non-Hodgkin's lymphomas has been reported in homosexual men. This phenomenon appears to represent another facet of the acquired immunodeficiency syndrome (AIDS). Histologically, the majority of these lymphomas have been small noncleaved cell lymphomas or immunoblastic lymphomas, subtypes most commonly associated with a B-cell phenotype, but immunologic data supporting this have been limited. Using a plastic embedding technique, we have examined a series of 31 malignant lymphomas, including nine from the central nervous system (CNS), in patients with AIDS or at high risk for AIDS. All 31 of the lymphomas were positive with one or more of the following B-cell markers: HLA-DR/la, Pan B, Leu 12, Leu 14, and IgM. All 31 were negative for the pan-T reagent Leu 4 and myeloid-macrophage markers (Leu M1, nonspecific esterase). In addition, seven of the nine CNS lymphomas showed strong plasma membrane staining for adenosine triphosphatase, a B-associated marker. These findings provide strong immunologic evidence for a B-cell origin in the lymphomas of AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Immunocytochemical localization of the major glutathione S-transferases in adult Schistosoma mansoni.

Indirect immunofluorescence was used to investigate the tissue distribution of the major isoenzymes of Schistosoma mansoni glutathione S-transferase (GSH S-transferase). When polyclonal rabbit antisera against GSH S-transferase isoenzymes SmGST-1, -02, and -3 were applied to cryostat or plastic-embedded sections of fixed adult worms, a punctate pattern of enzyme distribution was observed that was restricted to the parenchyma. Labeling was much more pronounced in males than females, consistent with the biochemically determined distribution of these enzymes between the sexes. Intense immunolabeling was noted within the subectocytoplasmic core tissue of the tubercles of the male that appeared to be connected to deep parenchymal cells by immunoreactive cell processes. Immunofluorescence could be blocked completely by prior incubation of antisera with affinity-purified enzyme. Although schistosome GSH S-transferases have been reported to be protective antigens, no immunoreactivity was detected within or on the tegument, including the dorsal spines of the male. The lack of tegumental immunoreactivity was confirmed by immunoblotting of tegumental membrane preparations following SDS-PAGE. Muscle fibers, vitelline cells, and cecal epithelium also failed to react. The fact that the GSH S-transferases were not uniformly distributed among all parenchymal cells suggests the existence of subpopulations of parenchymal cells that are preferentially involved in the conjugation of electrophiles with glutathione.

Animals↗
PubMed1988

Progressive lung cell reactions and extracellular matrix production after a brief exposure to asbestos.

Inhaled chrysotile asbestos fibers have been shown to deposit initially on the first alveolar duct bifurcations. In brief accidental exposure to asbestos, this would be the most likely site of a significant cellular or fibrotic reaction. The characteristics and progression of tissue reactions occurring at first alveolar duct bifurcations after a single brief asbestos exposure was defined using morphometric techniques. Seven-week-old rats were exposed, nose only, for 1 hour to chrysotile asbestos fibers. After the exposure, the animals were kept in air for 2 days or 1 month, and then their lungs were fixed by vascular perfusion or by intratracheal instillation of 2% glutaraldehyde. The first bifurcations of seven alveolar ducts in each animal were isolated from plastic-embedded tissue and thin-sectioned for electron-microscopic analysis. Two days after exposure, the volume of epithelium and interstitium in the duct bifurcations had increased by 78% and 28%, respectively (P less than 0.05). The total number and volume of alveolar macrophages on the bifurcations increased about 10 times (P less than 0.05), whereas the number and volume of interstitial macrophages increased threefold (P less than 0.05). Statistically significant increases in the numbers of Type I (82%) and Type II (29%) epithelial cells also occurred. One month after the 1-hour exposure, the volume of epithelium and the number of Type I and Type II cells were still greater than control values, but these differences no longer achieved statistical significance. The volume of the interstitium, on the other hand, increased 67% (P less than 0.05), and this was accompanied by a persistently high number of interstitial macrophages, accumulation of myofibroblasts/smooth muscle cells, and an increased volume of interstitial matrix. These results demonstrate that a brief exposure to chrysotile asbestos causes a rapid response that involves an influx of macrophages to the first alveolar duct bifurcations and alterations in the alveolar epithelium. These acute structural changes are followed by a progressive response manifested by increased numbers of interstitial cells and localized interstitial fibrosis.

Aerosols↗
PubMedDate not supplied

Electron microscopy of fine needle aspiration biopsies of mediastinal and paramediastinal lesions.

The ultrastructural cytologic study of fine needle aspiration (FNA) biopsies from eight cases with mediastinal and paramediastinal lesions is reported. In these cases, electron microscopy (EM) was essential in cytologically determining the correct type of the cancer cells. The results in these cases suggest that portions of FNA biopsies from deep sites, where aspiration is difficult or requires computed tomographic scanning, should be routinely processed for plastic embedding, so that EM examination can be performed if the cells are undifferentiated, scanty or poorly preserved by light microscopic examination. The proper cytologic identification of the cell might, in fact, have a major bearing on the therapeutic choices and on the prognosis.

Adenocarcinoma↗
PubMed1988

The structure of the epididymis of the giant rat (Cricetomys gambianus, Waterhouse): histological, histochemical and microstereological studies.

The structure of the ductus epididymidis of the African giant rat (Cricetomys gambianus, Waterhouse) was studied by means of histological (paraffin and plastic-embedded tissues) and histochemical techniques. Five distinct zones (I-V) were detected along the epididymal duct on the basis of epithelial height and cytological composition and structure. The so-called apical cells, which have been observed in some species of animals, were not seen in the initial segment of the duct. Clear or light cells were present in the epithelium of the terminal segment, thus making the giant rat the third species in which this cell has been found, after the laboratory rat and the hamster. A large number of intra-epithelial lymphocytes and macrophage-like cells was commonly observed in the epithelium throughout the duct; the latter were strongly PAS-positive while the former tended to increase in number caudally. A microstereological study of the epididymis showed that Zones I (initial segment) and II contained a significantly (P less than 0.001) higher proportion of epithelium and a relatively greater (P less than 0.001) proportion of blood vessels in the interductal connective tissue than the more caudal segments of the epididymis which possessed a significantly (P less than 0.01) higher proportion of interductal connective tissue.

Acid Phosphatase↗
PubMed1985

Burkitt's lymphoma. Distinction of subgroups by morphometric analysis of the characteristics of 55 cell lines.

In an attempt to clarify the controversy about the distinction between Burkitt's and non-Burkitt's small noncleaved lymphomas, 55 cell lines derived from 48 Burkitt's lymphoma patients were characterized by morphometry on plastic-embedded sections. The results of the measurements permitted the identification of five main cytologic types, with regard to nuclear size, nuclear area dispersion and irregularity of nuclear profiles. The presence of the Epstein-Barr virus (EBV) and the geographic origin of the tumors seemed to play essential roles in the determination of nuclear size, with a significantly larger size seen in EBV-positive cell lines, and especially in the African lines among these. Immunoglobulin profile and monoclonal antibody expression also correlated with the nuclear size. Two conclusions may be drawn from this analysis. An in vivo transformation of the cells of Burkitt's lymphoma can be postulated to explain the wide morphologic spectrum of lymphomas presenting a rearrangement of chromosome 8. The fact that typical and atypical Burkitt's lymphomas cannot be differentiated by study of their derived cell lines raises the question as to the validity of the distinction between the two subtypes of small noncleaved lymphomas.

Africa↗
PubMed1988

Cortical bone repair. The relationship of the lacunar-canalicular system and intercellular gap junctions to the repair process.

Repair of cortical bone was studied in 2.4-millimeter-diameter mid-diaphyseal femoral and tibial defects in young New Zealand White rabbits using light microscopy, transmission electron microscopy, and histomorphometry. The initial source of repair tissue is the marrow. Vessels grow into the defect, accompanied by undifferentiated mesenchymal cells. Woven bone is synthesized initially at the periphery of the defect on pre-existing cortex. Differentiating mesenchymal osteoblasts surround themselves with osteoid in a woven conformation. Once a scaffold has formed, surface osteoblasts align themselves in a regular array on the woven matrix surface and synthesize osteoid in a lamellar conformation. The long axes of the repair vessels, lamellae, and osteocyte lacunae are perpendicular to the long axis of the bone. Polarized-light microscopy showed maintenance of this pattern at six, eight, and twelve weeks, even when the defect was filled with lamellar bone. Remodeling is performed slowly by osteoclast cutting cones over a period of several months. The lacunar-canalicular system is clearly demonstrated in plastic-embedded, toluidine blue-stained sections. A canaliculus passes into or away from a lacuna every 1.9 micrometers over the entire osteocyte perimeter. Undifferentiated mesenchymal cells have no processes, as seen by transmission electron microscopy, but soon sprout a florid array of processes as differentiation to early mesenchymal osteoblasts proceeds. Osteoblast and osteocyte cell processes are packed with intermediate filaments that are continuous with those in the cell bodies. Intercellular gap junctions are seen between surface osteoblasts, between osteoblasts and underlying osteocytes, and between osteocyte cell processes in the canaliculi.

Animals↗
PubMed1988

Morphometric analysis of follicular center cells: a new approach.

Accurate and reproducible categorization of follicular center cells (FCC) on the basis of their nuclear size and shape is difficult. In addition, the relationship of cleaved to noncleaved FCC has been questioned. For these reasons, 2126 FCC (and 947 mantle cells) from five plastic embedded reactive lymph nodes were studied using a new morphometric approach. Using an image analyzer, the following nuclear features were studied: area (NA); our previously described shape factors which measure ellipticity (NCIe) and irregularity (NCIni); and a new objective measurement of relative chromatin dispersal (chromatin dispersal index, CDI). Definite clefts and nucleoli were visually identified and recorded. Compared to the mantle cells, FCC nuclei were significantly larger, more elliptical, more irregularly shaped, and had more dispersed chromatin. Among the FCC the only correlation that could be identified between the above parameters was that the larger FCC tended to have more dispersed chromatin (r = 0.46). Significantly more dispersed chromatin was, however, associated with nonclefted cells, less irregularly shaped cells having a NCIni less than the median NCIni, cells with nucleoli, and larger cells having a NA greater than the median NA. Clefted FCC had significantly greater nuclear irregularlity compared to nonclefted FCC but a similar degree of ellipticity. Distribution curves for the NA, NCIe, NCIni, and CDI revealed a continuous range of results rather than a limited number of distinct cell types. The addition of an objective quantitative measurement of chromatin dispersal permits a more complete morphometric description of FCC.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Nucleus↗
PubMed1987

Rapid freezing techniques and cryoprotection of biomedical specimens.

It has been realized almost from the start of biological electron microscopy that the use of low temperature in specimen preparation might alleviate the problems associated with conventional chemical fixation and plastic embedding. In practice it has been very difficult to realize any benefits, owing to the problems encountered in trying to prepare samples with the water transformed to ice, yet without the sample itself hopelessly distorted by ice crystals. The difficulty is caused by the poor diffusibility of heat through water, the main constituent of almost all biological samples. Even with a maximal temperature gradient between the sample surface and the coolant, and a perfect contact between the two, heat diffusibility through the sample quickly becomes the limiting parameter affecting cooling rate. Unless the cooling rate is as high as 10(2) or even 10(3) degrees C.msec.-1, the ice formed will be crystalline. Such high cooling rates can only be attained in the first few micrometers of even very small samples: therefore, for bulk samples one must either accept the use of a 'suitable' cryoprotectant, or else accept the presence of ice crystals. Theoretical and practical considerations for freezing samples using liquid and solid coolants will be reviewed. Special consideration will be given to the present status of cryoprotection and the indications and contra-indications for its use.

Animals↗
PubMed1987

Improved nuclear contour indices for lymphoid morphometry.

The morphometric analysis of benign and neoplastic lymphoid proliferations relies heavily on nuclear shape factors to identify clefted, cerebriform or convoluted nuclei. Most studies employ the size-independent nuclear contour index (NCI = perimeter/square root area) or the closely related (nuclear) form factor to evaluate the degree of nuclear irregularity. These indices, however, cannot distinguish a truly irregular shape from a perfectly smooth elliptical one. A variation of the standard NCI is therefore proposed in which the NCI of the ellipse (NCIe) that best approximates the nuclear shape is determined to indicate the degree of nuclear elongation or ellipticity and the separate NCI of the nuclear irregularity (NCIni) is determined to indicate the true irregularity of the nuclear perimeter, independent of nuclear elongation. These two new shape factors were tested on a series of shapes and on mantle and follicular-center cells present in 3-micron plastic-embedded sections from three tonsils with reactive lymphoid hyperplasia. Whereas the standard NCI increased with increasing ellipticity or nuclear irregularity, the NCIe increased only with increasing ellipticity and NCIni with increasing nuclear irregularity. Mantle cell and, to a greater extent, follicular-center-cell nuclei showed mean NCIe and NCIni greater than what would be expected from a perfectly round nucleus. These nuclei, therefore, were both more elliptical and more irregular in outline than a perfect circle. The NCIe and NCIni were shown to vary independently in both mantle and follicular-center cells. These new and relatively simple indices should lead to a more accurate morphometric description of lymphoid cells.

Cell Nucleus↗
PubMed1988

Short-lasting accumulation in osteoid bone seams of radioactive iron injected as citrate into mice.

The possible role in vivo of osseous structures in binding radioactive iron injected as a low-molecular-weight complex was studied in mice, using combined autoradiography and histomorphometry on sections of undecalcified, plastic-embedded femur epiphyses/metaphyses. A single intraperitoneal injection of 10 microCi 59Fe (1.2 micrograms Fe) per animal as citrate within 3 hours led to a preferential accumulation of this metal in the osteoid mineralized tissue interphase (osteoid seams) of bone. Within the next 2 days the labeling intensity in this localization diminished markedly to approximate levels of the bone marrow and calcified bone. The bulk of the injected radioiron was utilized according to known erythrokinetics. Findings suggest a direct entry of "free," ie, not transferrin-bound, iron into osteoid seams and its consecutive rapid removal from this site.

Animals↗
PubMed1987

[Effects of systemic administration of strychnine, L-allylglycine, bicuculline and picrotoxin on the transsynaptic neural destruction in the medullary dorsal horn following transection of the rat inferior alveolar nerve].

Adult rats underwent unilateral transection of the inferior alveolar nerve and subsequent intraperitoneal injection of strychnine (1 mg/kg, 3-23 days) at various posttransectional intervals. When they were sacrificed at 18-30 days posttransectionally, many pyknotic neuronal cell bodies were observed in plastic embedded toluidine blue-stained 1 micron-thick sections of the medullary dorsal horn. They were mostly found in the dorsal part of the dorsal horn ipsilateral to the neurotomy and were more abundant in laminae I/II than in laminae III/IV. Similar pyknotic neurons were found after 1 or 2 days of L-allylglycine administration (55.7 mg/kg/day) at a posttransectional interval of 20 days. Unlike those observed after strychnine treatment, the pyknotic neurons after L-allylglycine treatment were evenly distributed throughout laminae I-IV of the dorsal half of the medullary dorsal horn. Twenty-three days of bicuculline (2 mg/kg/day) or picrotoxin (0.5 mg/kg/day) treatment at an interval of 7 days did not yield pyknotic neurons. The results are discussed in the light of intrinsic synaptic circuitry of the dorsal horn.

Allylglycine↗
PubMed1986

Does sympathectomy or antihypertensive treatment affect the morphometry of basal cerebral arteries in spontaneously hypertensive rats?

Spontaneously hypertensive rats (SHR) and stroke-prone spontaneously hypertensive rats (SHRSP) were treated with a combination of a beta 1-blocker (Metoprolol) and a calcium antagonist (Felodipine) from 1 to 4 or from 4 to 6 months of age. Cross sections of plastic embedded basal cerebral arteries were measured with a digitizer. The ratio between media thickness and luminal radius (m/r ratio) could then be calculated for a standardized condition assuming a smooth, circular internal elastic membrane. The treatment caused a significant decrease of the m/r ratio in various basal cerebral arteries of young and adult SHR and SHRSP, i.e. the therapy may prevent as well as reverse hypertensive structural changes. Unilateral superior cervical ganglionectomy or preganglionic denervation did not affect the structure of basal cerebral arteries in young SHRSP or normotensive controls. A slight decrease of m/r ratio was indicated in the smallest pial arteries on the sympathectomized side, but the results did not allow any conclusion as to differences in effect between preganglionic denervation and ganglionectomy.

Age Factors↗
PubMed1986

Cryoultramicrotomy and immunocytochemistry in the analysis of muscle fine structure.

Cryoultramicrotomy, which avoids the use of harsh fixation procedures, deleterious dehydration and plastic embedding can be combined with immunocytochemistry to determine the ultrastructural localization of cellular proteins. Our attempts to use the cryosectioning technique in combination with immunolabelling to bridge the gap between light and electron microscopic analysis of muscle morphology have enabled us to obtain new information on fibre typing at the ultrastructural level. Furthermore, we have obtained a marked improvement in the resolution of myofibrillar structures by using semithin cryosections for fluorescence microscopy. Data are also presented on correlated light and electron microscope immunocytochemistry of myocardial intermediate filaments confirming the presence of longitudinally oriented intermediate filaments of desmin in the region of the intercalated discs of mammalian cardiac myocytes, whereas elsewhere in the myocyte the bulk of intermediate filaments of desmin is concentrated in the intermyofibrillar space at the level of the Z disc.

Animals↗
PubMed1987

Lymphocyte nuclear morphology in diffuse well-differentiated lymphocytic lymphoma. Comparative morphometry of normal lymphoid tissues, non-Hodgkin's lymphoma, and Hodgkin's disease.

Precise morphologic data on the relationship of diffuse well-differentiated lymphocytic lymphoma (DWDLL) to other non-Hodgkin's lymphomas (NHLs) are lacking in current classifications of lymphomas. Morphometry of plastic-embedded tissues describes details of the mean nuclear parameters and distribution of lymphocyte-types in 14 cases of DWDLL in nodal and extranodal sites. The results indicate that the proportion of small (unstimulated or nontransformed) lymphocytes in DWDLL varies from 50% to 86%. The mean nuclear area of small lymphocytes in DWDLL is either within the size range or somewhat smaller, and many examples are more irregular in shape, than the small lymphocytes in germinal centers that are morphologically comparable with the majority of lymphocytes in DWDLL and mantle zones. The morphologic relationship of DWDLL to other NHLs, Hodgkin's disease, and paracortical, mantle, and germinal center lymphocytes in lymphoid-reactive hyperplasia was established by morphometric analysis of the nuclear profiles of small lymphocytes. The small (nontransformed) lymphocytes in those subtypes of NHL other than DWDLL have abnormally small and more irregular nuclear profiles than those in normal small lymphocytes, or the small-lymphocyte population of Hodgkin's disease. Such findings may be of diagnostic significance to pathologists.

Biopsy↗
PubMed1987

Immunogold localization of intra- and extra-cellular proteins and polysaccharides of plant cells.

This paper illustrates post-embedding immunogold labelling of protein and polysaccharide molecules of plant cells. For EM studies, one is restricted (for most plant cells) to the post-embedding approach because the surrounding cell wall prevents access of antibodies (and secondary gold-tagged markers) to internal sites. The large size of many plant cells also does not lend itself to diffusional entry of antibodies. The molecules localized include seed storage proteins that are large and present in major quantities, a smaller less abundant, water soluble albumin, an oxygen-binding protein, components of the photosynthetic electron transport chain, and complex sugars from the cell wall. A range of preparative procedures and embedding plastics are used.

Gold↗
PubMed1986

Hematopoiesis in osteoporosis--preliminary report comparing biopsies of the femoral neck and iliac crest.

Undecalcified plastic-embedded semithin sections of femoral neck and iliac crest biopsies taken at operation in 44 patients with intracapsular femoral neck fractures were examined for hematopoiesis, osseous remodelling and osteopenia. Osteopenia, osseous remodelling and hematopoiesis were present in over half of the iliac crest biopsies. More than half of the femoral neck biopsies showed osteopenia and replacement of the hematopoiesis by fat cells with virtual absence of osteoblasts and osteoclasts. In these patients, osteopenia in the iliac crest appears to be of the "high turnover type" while that in the femoral neck is of the "low turnover type." These results indicate that the iliac crest biopsy is not necessarily representative of osseous remodeling and hematopoiesis at other skeletal sites.

Aged↗