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PubMed1985

Embryonic development of the rat cerebellum. II. Translocation and regional distribution of the deep neurons.

In thymidine radiograms and plastic-embedded sections, the migration of cerebellar deep neurons was traced from their germinal source to their final settling sites. The route proved to be roundabout and three developmental events could be distinguished during the process. First, between days E14 and E16, transversely oriented cells of the nuclear transitory zone move in an arc from the ventrolateral neuroepithelium of the lateral cerebellar primordium in a medial direction. Second, between days E16 and E18, the cells of the rostral component of the nuclear transitory zone assume a longitudinal orientation. We postulated that this is the period of axonogenesis, the longitudinally oriented cells issuing efferents that join the superior cerebellar peduncle ipsilaterally and the transversely oriented cells (representing the neurons of the caudal fastigial nucleus) sending decussating fibers to the uncinate fasciculus (the hook bundle of Russell). Third, between days E18 and E21, the earlier-produced superficial cells of the nuclear transitory zone and the later-produced deep cells of the cortical transitory zone (the young Purkinje cells) exchange positions. The descent of the deep neurons is in the direction of the fibers of the inferior cerebellar peduncle, which becomes distributed throughout the cerebellum on day E17. The ascent of the Purkinje cells is in the direction of the external germinal layer, which begins to spread from caudal to rostral on day E17. The three deep nuclei, the lateral (dentate), interpositus, and medial (fastigial), can be distinguished before their descent into the depth of the cerebellum, and by day E22 a small-celled and a large-celled subdivision is identifiable in each nucleus.

Animals↗
PubMed1985

Embryonic development of the rat cerebellum. III. Regional differences in the time of origin, migration, and settling of Purkinje cells.

The time of origin, site of origin, migratory path and settling pattern of the Purkinje cells of the cerebellar hemispheres, anterior vermis, and posterior vermis were investigated in thymidine radiograms and plastic-embedded materials from rat embryos ranging in age from 15 to 22 days. In the hemispheres there is a rostral-to-caudal cytogenetic gradient: the Purkinje cells of lobulus simplex, crus I, and crus II are produced earlier than the Purkinje cells of the paramedian lobule and paraflocculus, followed by the Purkinje cells of the flocculus. The Purkinje cells of the vermis, in general, are generated later than those of the hemispheres, and with a reverse gradient from caudal to rostral: the Purkinje cells of the posterior vermis (lobules X-VI) being produced ahead of the Purkinje cells of the anterior posteriorly directed wedge of early-produced Purkinje cells through the vermis. Evidence was obtained that the Purkinje cells of the hemispheres derive from the lateral cerebellar primordium capping the lateral recess of the fourth ventricle anteriorly. The Purkinje cells of the anterior vermis originate from the subisthmal cerebellar primordium medially lining the isthmal canal. The Purkinje cells of the posterior vermis originate in the postisthmal cerebellar primordium overlying the tela choroidea caudally. The young Purkinje cells migrate from the neuroepithelium to the surface of the cerebellum in a strictly caudal-to-rostral order, paralleling the spread of the EGL superficially from posteroventral to anterodorsal. This pattern is independent of the time of origin of Purkinje cells. In the posterior vermis the earliest-settling Purkinje cells of the uvula follow a short radial course, and a discrete Purkinje layer is formed 3 days after they are generated. In the anterior vermis the Purkinje cells of lobulus centralis, which follow an anterodorsal migratory course, are still settling on day E22, 7 days after their production, presumably awaiting the fusion of the cerebellar base anteriorly. The fissura prima forms medially at the interface region of Purkinje cells derived from the postisthmal and subisthmal cerebellar primordia. For 1-2 days after their settling, the Purkinje cells of the newly forming lobules can be distinguished by certain cytological criteria from the Purkinje cells in the more caudally-situated, earlier-settled lobules.

Animals↗
PubMed1986

Different neuron populations in the feline lateral cervical nucleus: a light and electron microscopic study with the retrograde axonal transport technique.

A comparative light and electron microscopic study was made of the two major neuron populations in the feline lateral cervical nucleus (LCN) that have an ascending projection--namely, the cervicothalamic and cervicomesencephalic neurons. The small population of neurons in the LCN that remained unlabelled after extensive injections into all known areas of LCN termination was also investigated. The intra-axonal retrograde tracing technique was employed and three different tracers were used: native horseradish peroxidase, horseradish peroxidase conjugated to wheat germ agglutinin, and cholera toxin subunit B (CTb). The diaminobenzidine and tetramethylbenzidine methods were used for the peroxidase reaction, and an immunocytochemical staining method with monoclonal antibodies was employed to localize CTb. The light microscopic cell count was performed on plastic-embedded semithin (2 micron) sections, and a section-embedding technique was used for sampling regions of interest for electron microscopy. The total number of neurons in the LCN was calculated to be 8,300, which is higher than numbers reported from other studies on frozen sections. It was concluded from control experiments that many neurons fail to counterstain in frozen sections. The average proportion of labelled cervicothalamic neurons was 94%, which accords with earlier reports. The frequency of labelled neurons projecting to the midbrain was 25-49%, depending on the method used. The highest number of labelled neurons was found with the immunocytochemical method with use of CTb as a tracer. In the electron microscopic study, the cell area, form factor, nuclear area, bouton covering ratio, mean appositional length of boutons, and bouton density were measured. There were no significant differences in these respects between the cervicothalamic and cervicomesencephalic neurons, but the unlabelled neurons differed significantly from the labelled ones, with a smaller somal area, a lower bouton covering ratio, and a larger relative nuclear area. The ascending neurons were scattered over the entire LCN, but the neurons that remained unlabelled after the extensive injections was found in its ventromedial part. It is suggested that these are interneurons. In the light microscopic study of the animals injected with CTb, the ventromedial part of the LCN displayed labelled structures resembling terminals. Electron microscopic examination of the sections revealed boutons synapsing on unlabelled neurons and dendrites. Occasionally labelled neurons and dendrites were also contacted.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗
PubMed1988

Anatomy of glutamic acid decarboxylase immunoreactive neurons and axons in the rat medial geniculate body.

This is a study of the form, density, and distribution of glutamic acid decarboxylase (GAD) immunoreactive neurons and puncta (axon terminals) in the adult rat medial geniculate complex. GAD-positive elements were stained by either the peroxidase-antiperoxidase or avidin-biotin procedures. Thalamic architectonic subdivisions were defined independently in Golgi, Nissl, plastic-embedded semi-thin, and fiber-stained preparations, and from investigations of medial geniculate connectivity. GAD-positive neurons represent only approximately 1% of medial geniculate neurons. They occur in the three major medial geniculate subdivisions (ventral, dorsal, and medial). There is variability between subdivisions in the form and number of such neurons, and among the puncta. In the ventral division, immunopositive somata may have sparsely branched dendrites as long as 300-400 microns and capped with varicose expansions or bouton-like sprays of appendages. These closely appose the somata or primary dendrites of other cells; the axons of these GAD-positive neurons are also immunostained. In the dorsal division there are fewer GAD-positive neurons and their structure is different. Their dendrites are rarely immunoreactive for more than 100-150 microns; nor can their immunostained axons be traced very far. In the medial division the number of GAD-positive neurons, considering the relatively small size of this division, was high. These neurons rarely have immunostained dendrites, and more than one type of neuron is immunoreactive. The average somatic diameter of GAD-positive neurons is about 60% of that of non-immunostained cells in semi-thin material; however, the range of somatic area and the dendritic variability of these neurons suggest that cells representing more than one population are immunopositive and include all but the largest neurons. The puncta also show regional differences. Small (0.5-2 microns in diameter), medium (2-3 microns), or large (greater than 3 microns) puncta occur. In the ventral division, the predominantly medium-sized puncta are about four times as numerous on a unit/area basis than in the dorsal division, where they are far smaller and more delicate; medial division puncta are as numerous as those in the ventral division, but are much larger and coarser, and may form perisomatic arrangements. Controls were devoid of specific immunostaining.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

New high-resolution 2-deoxyglucose method featuring double labeling and automated data collection.

A new approach to high-resolution 2-deoxy-D-glucose (2DG) emulsion-autoradiography which combines improved retention of 2DG labeling, staining with immunohistochemical and other specific markers, and automated data collection and analysis of local silver grain and stain densities is described. The Durham et al. (J. Neurosci. 1:519-526, '81) procedure for fixation of 2DG with periodate-lysine-paraformaldehyde (PLP, McLean and Nakane: J. Histochem. Cytochem. 22:1077-1083, '74) was adapted to increase retained label roughly tenfold. Phenobarbital anesthesia is induced 45 minutes after 2DG injection. Barbiturate anesthesia increases brain glycogen (Nelson et al.: J. Neurochem. 15:1271-1279, '68) and presumably increases the incorporation of intracellular 2DG from 2DG-6P into brain glycogen and other molecules (Nelson et al.: J. Neurochem. 43:949-956, '84; Pentreath et al.: Neuroscience 7:759-767, '82). Iodoacetate is added to cold fixative to prevent glycogen breakdown (Cammermeyer and Fenton: Histochemistry 76:339-356, '82). This high-resolution 2DG protocol is directly compatible with many other neuroanatomical techniques. We demonstrate 2DG emulsion autoradiography combined with cytochrome oxidase (CO) histochemistry, markers for axonal pathway tracing, plastic embedding for semithin sections, and immunohistochemical staining for glutamate decarboxylase (GAD). The method should be compatible with antibodies for other antigens and with other neuroanatomical stains. To collect the data directly from microscope slides, a computer-controlled microscope was integrated with image-processing software to eliminate the need for manual counting and scoring of autoradiograms. Regions of interest are scanned automatically at high resolution to map regional labeling and/or stain density. There is excellent correspondence between computer-enhanced two-dimensional maps of the data and the original autoradiograms. Automated counts for five specimens were compared to counts of labeled cells by trained observer. The correlation between the two sets of measurements is high (r = .93). Automated data collection has been generalized to measure regional stain densities on the autoradiographed sections for direct comparison with silver grain density. The method is extremely flexible, especially since new image-processing strategies can be developed in software to extract the desired information from materials labeled by other methods (e.g., HRP).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Cytoarchitectonic organization of the spinal cord in the rat: II. The cervical and upper thoracic cord.

A laminar cytoarchitectonic scheme of the cervical and upper thoracic segments of the rat spinal cord is presented in which Rexed's principles for the cat are applied. The material examined in the current investigation consists of 50-80 microns-thick unstained or Nissl-stained sections, and 2 microns-thick plastic-embedded sections stained with paraphenylenediamine. The cytoarchitectonic organization was found to be basically similar to that of the cat. As in our previous study of the cytoarchitectonic organization of the lower thoracic and lumbosacral spinal cord (Molander et al.; J. Comp. Neurol. 230:133-141, '84), the borderlines between the laminae were often found to be ambiguous, suggestive of zones of transition rather than sharp borders. In addition to the laminar scheme, the distribution of certain important cell groups, including the column of Clarke, the central cervical nucleus, the lateral cervical nucleus, the lateral spinal nucleus, and the internal basilar nucleus, is described.

Animals↗
PubMed1993

Ultrastructure of the extraocular photoreceptor in the genitalia of a butterfly, Papilio xuthus.

This paper describes the ultrastructure of a sensory neuron found in the extraocular photoreceptive site on the butterfly genitalia. Our previous studies have shown that there are two pairs of the photoreceptive sites in a butterfly (four per individual). Each photoreceptive site is recognizable by a transparent area in the pigmented cuticle of the genitalia. From the nerve that extends from the transparent cuticle to the last abdominal ganglion, a sustained train of single unit spikes can be recorded in response to a light flash. The single unit spikes disappear when the transparent cuticle is covered, thus indicating that a single photoreceptor exists close to it. Here, we examined complete serial semithin sections of plastic-embedded photoreceptive sites of both male and female, and observed an ovoid structure close to the transparent cuticle. The structure contained the cell body of a sensory neuron whose axon was in the nerve branch from which the photoresponse had been recorded. Further electron microscopy revealed that the cell body extended a few distal processes that protrude tubular membrane from the tip, forming a structure resembling the phaosome (from Greek; phaos = light, some = body) which was first described in the earthworm dermal photoreceptors. The sensory neuron was also found in a surgically isolated nerve-photoreceptor preparation that responded to the light. We therefore propose that the phaosome-containing sensory neuron is the butterfly genital photoreceptor.

Animals↗
PubMed1994

Laminar distribution and neuronal targets of GABAergic axon terminals in cat primary auditory cortex (AI).

The form, density, and neuronal targets of presumptive axon terminals (puncta) that were immunoreactive for gamma-aminobutyric acid (GABA) or its synthesizing enzyme, glutamic acid decarboxylase (GAD), were studied in cat primary auditory cortex (AI) in the light microscope. High-resolution, plastic-embedded material and frozen sections were used. The chief results were: 1) There was a three-tiered numerical distribution of puncta, with the highest density in layer Ia, an intermediate number in layers Ib-IVb, and the lowest concentration in layers V and VI, respectively. 2) Each layer had a particular arrangement: layer I puncta were fine and granular (less than 1 micron in diameter), endings in layers II-IV were coarser and more globular (larger than 1 micron), and layer V and VI puncta were mixed in size and predominantly small. 3) The form and density of puncta in every layer were distinctive. 4) Immunonegative neurons received, in general, many more axosomatic puncta than immunopositive cells, with the exception of the large multipolar (presumptive basket) cells, which invariably had many puncta in layers II-VI. 5) The number of puncta on the perikarya of GABAergic neurons was sometimes related to the number of puncta in the layer, and in other instances it was independent of the layer. Thus, while layer V had a proportion of GABAergic neurons similar to layer IV, it had only a fraction of the number of puncta; perhaps the intrinsic projections of supragranular GABAergic cells are directed toward layer IV, as those of infragranular GABAergic neurons may be. Since puncta are believed to be the light microscopic correlate of synaptic terminals, they can suggest how inhibitory circuits are organized. Even within an area, the laminar puncta patterns may reflect different inhibitory arrangements. Thus, in layer I the fine, granular endings could contact preferentially the distal dendrites of pyramidal cells in deeper layers. The remoteness of such terminals from the spike initiation zone contrasts with the many puncta on all pyramidal cell perikarya and the large globular endings on basket cell somata. Basket cells might receive feed-forward disinhibition, pyramidal cells feed-forward inhibition, and GABAergic non-basket cells would be the target of only sparse inhibitory axosomatic input. Such arrangements imply that the actions of GABA on AI neurons are neither singular nor simple and that the architectonic locus, laminar position, and morphological identity of a particular neuron must be integrated for a more refined view of its role in cortical circuitry.

Animals↗
PubMed1993

Germinal center analysis with the tools of mathematical morphology on graphs.

Only few studies devoted to quantitative analysis of tissue architecture have been performed. The analysis of neighborhood relationships between cells, using graphs and mathematical morphology (MM), constitutes one approach. We propose to analyse quantitatively the architecture of a tissue with the tools of MM on graphs. The use of graphs seems best suited to take into account the neighborhood relationships between cells, independently from their mutual distances: two cells are considered as neighbors if no interfering cell is placed in a given sense between them. Such neighboring cells are linked by an arc in a graph. On such a graph, all tools of MM may be applied. We investigate two of them in the present work: (1) the distance transforms permits to analyse the repartition of a cellular population A relative to population B, (2) the size distribution permits to analyse the tendancy of a cell population to form clusters. We have applied this method using Gabriel's graph, derived from the Voronoï diagram, to determine the "zone of influence" of a cell. We have analysed the neighborhood relationships between cells in germinal centers (GC) from lymph nodes. Twelve hyperplastic GCs from follicular hyperplasia (FH) and 5 neoplastic GCs from small cleaved cell follicular lymphoma (++SCCFL) have been studied; 2 microns hematoxylin-eosin plastic embedded sections have been analysed. Cell nuclei have been identified manually by the observer by giving a numerical and image color code. Gabriel's graphs have been constructed with all centrofollicular cells. Subgraphs with only lymphoid cells, large cells or small cleaved cells have also been studied. The application of MM transformations on the graphs using software package Morphograph has allowed a quantitative description of cell distribution in the tissue. The distances of centrofollicular lymphoid cells from the periphery of the GCs (mantle zone), from nonlymphoid cells as macrophages located within the GCs and between large lymphoid cells and small lymphoid cells have been determined in FH and SCCFL. Using iterative closings and openings, we have detected aggregates of small and large cells and characterized their size and distribution in the GCs. The application of this method on lymph node biopsy has allowed a quantitative description and comparison of GCs in different pathological conditions.

Biopsy↗
PubMed1994

A new saw technique improves preparation of bone sections for light and electron microscopy.

A newly developed saw technique was developed to produce sections circa 10 microns or thicker from fresh bone or dentine and from plastic embedded undecalcified bone tissue with or without implant materials. The method comprises only one step because grinding or polishing to make the sections thinner is not necessary. The bone slices can be decalcified rapidly without using aggressive solvents and used for making ultrathin sections for electron microscopy. Sections of fresh dentine of 15 to 30 microns are transparent which makes it possible to study osteoclastic resorption in vitro. Sections, 10 microns thick, with an intact interface of bone and implant material can be observed for biocompatibility studies.

Animals↗
PubMed1975

Reaction associated with a silicone rubber gel: an experimental study.

A blend of Silastic 382 (Room Temperature Vulcanizing, RTV) Medical Grade silicone oil and a catalyst was prepared in vitro, in both the catalyzed and noncatalyzed state, and injected subcutaneously in mice, rats, and rabbits. When properly blended, this catalyzed material, referred to as "silicone gel," formed a soft rubbery mass that remained at the site of injection. Properly catalyzed silicone rubber gel produces no macroscopic inflammatory reaction, attracts few polymorphonuclear leucocytes, and after 5-6 days a thin fibrous capsule begins to form aroung the gel. No degeneration of the silicone gel was observed during the 62 days of this experiment. Additional rats with this silicone gel have been under observation for 8 months and clinically have shown no changes in the local mass of silicone. If the catalyst is partly oxidized when added to the silicone fluid, the degree of gelling is much less. A local mass usually forms at the site of injection with some of the fluid diffusing into the tissue, forming minute cysts. The inflammatory reaciton is characterized by polymorphonuclear leucocytes, associated with many macrophages and giant cells phagocytizing oil droplets and particles of the diatomaceous earth. The pathogenesis of the inflammatory reaction is discussed, referring to the ionic change and the emigration of polymorphonuclear leucocytes to particles of plastics embedded in tissue.

Animals↗
PubMed2003

Flow perfusion culture of marrow stromal osteoblasts in titanium fiber mesh.

The objective of this study was to evaluate the effect of two cell culture techniques, static and flow perfusion, on the osteogenic expression of rat bone marrow cells seeded into titanium fiber mesh for a period up to 16 days. A cell suspension of rat bone marrow stromal osteoblasts (5 x 10(5) cells/300 microL) was seeded into the mesh material. Thereafter, the constructs were cultured under static conditions or in a flow perfusion system for 4, 8, and 16 days. To evaluate cellular proliferation and differentiation, constructs were examined for DNA, calcium content, and alkaline phosphatase activity. Samples were also examined with scanning electron microscopy (SEM) and plastic-embedded histological sections. Results showed an increase in DNA from day 4 to day 8 for the flow perfusion system. At day 8, a significant enhancement in DNA content was observed for flow perfusion culture compared with static culture conditions, but similar cell numbers were found for each culture system at 16 days. Calcium measurements showed a large increase in calcium content of the meshes subjected to flow perfusion at day 16. The SEM examination revealed that the 16-day samples subjected to flow perfusion culture were completely covered with layers of cells and mineralized matrix. In addition, this matrix extended deep into the scaffolds. In contrast, meshes cultured under static conditions had only a thin sheet of matrix present on the upper surface of the meshes. Evaluation of the light microscopy sections confirmed the SEM observations. On the basis of our results, we conclude that a flow perfusion system can enhance the early proliferation, differentiation, and mineralized matrix production of bone marrow stromal osteoblasts seeded in titanium fiber mesh.

Alkaline Phosphatase↗
PubMed1996

Mineral anisotropy in mineralized tissues is similar among species and mineral growth occurs independently of collagen orientation in rats: results from acoustic velocity measurements.

It has been reported that the mineral crystals in long bones have their c-axis aligned with the bone axis, presumably because collagen fibrils in bone also align with the bone axis. However, the predominant collagen orientation in bone often does not appear to be aligned with the mineral crystals, especially in rat primary bone. We hypothesized that mineral orientation in bone is not necessarily related to collagen orientation. An acoustic microscope was used to measure elastic constants of mineralized tissues from rat, cow, monkey, and human bone, and mineralized turkey leg tendon (MTLT). Measurements were made before and after demineralization with 10% ethylenediaminetetraacetic acid (EDTA) or decollagenization with 7% sodium hypochlorite. The elastic anisotropy ratio (AR) was defined as the ratio of the elastic coefficient in the longitudinal direction to the elastic coefficient in the transverse direction. Anisotropy ratios of mineralized tissues were not affected by formalin fixation or plastic embedding. An evaluation of tissues from the different species showed that the AR after decollagenization was not significantly different (p > 0.4, analysis of variance) among the groups, while AR after demineralization varied from 1.04 (rat bone) to 1.51 (MTLT). There was no correlation between AR after demineralization and AR after decollagenization (r = 0.13, p = 0.5). This showed that the elastic anisotropy of collagen is more variable than mineral anisotropy in bone and MTLT. Another experiment showed that mineralization of turkey leg tendon changes the elasticity of the collagen matrix, making it less anisotropic. A final, prospective experiment was performed in which tibiae of rats were subjected to mechanical loading for 16 weeks. After 12 days, new periosteal woven bone was observed on the tibiae and, after 16 weeks, this new bone was consolidated and mineralized. Mineral in the newly formed woven bone was virtually isotropic (AR = 1.07) after 12 days of loading, then became more anisotropic (AR = 1.52) after 16 weeks of mechanical loading, as the mineral density of the new bone increased. This increase in anisotropy of bone mineral occurred even though the collagen matrix was woven and had no measureable fibril orientation. We conclude that (1) collagen anisotropy and mineral anisotropy are not necessarily correlated in mineralized tissues, (2) mineralization can affect the collagen matrix elasticity of mineralized tissues, and (3) an organized mineral structure can form in the absence of an organized collagen matrix.

Acoustics↗
PubMed1987

Postreplication labeling of E-leaflet molecules: membrane immunoglobulins localized in sectioned, labeled replicas examined by TEM and HVEM.

Conventional freeze-fracture techniques were combined with immunogold labeling and with plastic embedding and sectioning to analyze the distribution of membrane immunoglobulins (mIgs) and their associated intramembrane particles (IMPs) in E-face replicas of murine B-lymphocyte plasma membranes. Immunogold labels were applied to cells after the process of freeze-fracture and replication. Conventional stereoscopic transmission electron microscopic examination of sectioned, labeled replicas (SLRs) revealed that the gold-labeled mIgs were bound to and localized on the outer leaflets of split and replicated membranes. The gold labels were attached to the external determinants of the mIg molecules, which were retained beneath and contiguous with the replicated E-faces. The mIgs were also localized on the external surface of unreplicated microvilli. In addition, thick sections examined by high-voltage transmission electron microscopy (HVEM) revealed large expanses of replica with well-resolved IMPs. mIgs colocalized with small-diameter (less than 60 A) IMPs in E-face replicas of B-lymphocytes whose mIgs were patched by anti-immunoglobulin. Thus, postreplication E-surface labeling of split and replicated membranes is a high-resolution technique that is suitable for the study of membrane protein distribution in E-face replicas and contiguous nonreplicated tissue.

Animals↗
PubMed1990

Autoradiographic studies of selective amino acid uptake by neural and nonneural elements in the gerbil cochlea.

The cochlea is well suited for studies of the uptake properties of auditory neurons and nonneuronal supporting cells. Probe concentrations of radioisotopically labeled amino acids, including putative neurotransmitters and their precursors, breakdown products, and blockers, can be introduced via the natural, fluid-filled channels of the inner ear. Uptake patterns can be mapped at cellular and intracellular levels using light and electron microscopic autoradiographic methods. The procedures for introduction of label, fixation, plastic embedment, and light and electron microscopic autoradiography are described with special reference to the cochlea. Labeling patterns observed with over 20 amino acids are summarized for hair cells, spiral ganglion neurons, efferents, and nonneural elements of the stria vascularis, limbus, and modiolus. Limitations on the interpretation of results and their implications for the general usefulness of the methods are discussed.

Amino Acids↗
PubMed1976

Morphology and kinetics of spermatogenesis in Xenopus laevis.

The light microscopic characteristics of spermatogenic stages of the germ cell line in the anuran, Xenopus laevis, have been described as they appear in both nuclear squash preparations and plastic embedded thick sections of intact testes. Tritiated thymidine autoradiography was employed to unequivocally identify stages. Using this methodology, it was determined that the premeiotic DNA synthetic period occurs in a cell which is morphologically indistinguishable from a late secondary spermatogonial cell. In addition, kinetic studies with tritiated thymidine were employed to determine the duration of meiotic prophase and spermiogenesis in Xenopus. Results indicate that at 18 degrees C, the most rapidly matururing cells in the testis spend four days in leptotene, six days in zygotene, twelve days in pachytene, one day in diplotene, one day in meiotic division, and twelve days in spermiogenesis. An estimate for the duration of the premeiotic S stage was indirectly calculated from combined data to yield a value of six to seven days. Pooled spermatocyte counts measuring the frequency of occurrence of individual stages produced results which correlated closely with estimates obtained by tritiated thymidine labelling. Individual counts, however, show wide variations between testes from single animals and between testes from different animals, whether sacrificed together or at different times of the year. Nevertheless, in all cases, both morphology and labelling patterns indicate that spermatogenesis is continuously active. The variation observed in individual testes appears to be the result of waves of non-random entry of spermatogonial stem cells into the population of cells irreversibly committed to differentiation.

Animals↗
PubMed1994

Quantification of neutrophil migration following myocardial ischemia and reperfusion in cats and dogs.

Endothelial cell dysfunction and cardiac myocyte injury resulting from ischemia and reperfusion have been associated with accumulation of neutrophils in the myocardium. To determine whether the accumulation is related primarily to intravascular sequestration or extravascular infiltration of neutrophils during the early period of reperfusion, we morphometrically quantified the tissue distribution of neutrophils in cats and dogs. At the end of the reperfusion period, the base of the heart was cross-clamped to preserve neutrophil location at the moment of death. Point-counting methods were used to determine the distribution of neutrophils inside and outside coronary arterioles and venules (< or = 100 microns in diameter) as well as coronary capillaries 5-10 microns in diameter in 0.5-microns-thick, plastic-embedded sections. Ischemia-reperfusion resulted in a threefold increase in neutrophil number in the lumen of arterioles and venules at 60 min of reperfusion and up to a sevenfold increase at 270 min of reperfusion (P < .05) compared to time-matched control nonischemic hearts. The ratio of intravascular neutrophils in venules to arterioles was 2:1. Intracapillary neutrophils increased, but not significantly, at 60 min of reperfusion. At 270 min of reperfusion, intracapillary neutrophils increased 11-fold (P < .05). The percentage of total neutrophils that accumulated outside arterioles and venules in cat hearts was 8% at 60 min of reperfusion (not significant, NS) and 28% at 270 min of reperfusion (P < .05). In dog hearts, the percentages were 26% (NS) and 44% (P < .05), respectively. The percentage of total neutrophils that accumulated outside capillaries was < 6% in both cat and dog hearts (NS). The combination of rapid intravascular sequestration, delayed extravascular infiltration, and low incidence of neutrophil-cardiac myocyte contact in situ in these two species suggests that neutrophil-mediated cardiac myocyte injury during early reperfusion may initially depend on diffusion of inflammatory mediators and subsequently require direct contact between neutrophils and cardiac myocytes.

Animals↗
PubMed1982

Hamster nasal glands: their structure, sialic acid content, and vulnerability to actinomycin D.

This study examines the structure of mucosal glands in the walls of the hamster maxillary recess, compares the histochemical appearance of nasal glands to their sialic acid content, and determines the vulnerability of nasal glands to actinomycin toxicity. Observations were made on plastic-embedded tissue with light and transmission electron microscopes. Determinations of total sialic acid in mucosal samples were conducted with thiobarbituric acid. Experimental hamsters were administered 0.2 micrograms of actinomycin D (IP)/gm body weight/day for five days. Types of granules present in the later nasal gland (LNG) and glands of the maxillary recess (MRGs) include: 2.0 micrometers lightly basophilic, lightly electron-dense granules and 1.5 micrometers strongly basophilic, electron-dense granules in the same acinar cell type in both the LNG and MRGs; 1.5 micrometers metachromatic granules in some acinar cells of the LNG; 1.0 micrometer moderately electron-dense granules in cells of MRG ducts; and 0.7 micrometers electron-dense granules in cells of LNG intercalated ducts. Acid glycoproteins, demonstrable by histochemistry, are present in the LNG but not in the MRGs. However, the total sialic acid content of tissues from MRG tissue is greater than that of other tissues measured. A minor number of LNG acini, those with metachromatic granules, have branching basal cytoplasmic projections. Many dark cells are present in striated ducts of the LNG. Histological alteration due to actinomycin-D toxicity, conspicuous in parotid salivary parenchyma, is greater in MRG than in LNG tissue.

Animals↗