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PubMed1994

Quantitative analyses of neuroepithelial cell shapes during bending of the mouse neural plate.

Despite a wealth of information about cell behaviors contributing to neurulation in chick embryos, similar behaviors in mouse embryos have yet to be well characterized. This study examines cell behaviors occurring during bending of the mouse neural plate, in particular, qualitative and quantitative changes in neuroepithelial cell shape. Our current results demonstrate that in mouse embryos 1) the median hinge point (MHP), a localized region of neural plate that becomes anchored to the underlying prechordal plate mesoderm or notochord/notochordal plate and forms a midline longitudinal furrow around which folding of the remaining neural plate (i.e., the part of the neural plate not involved in MHP formation) occurs, develops during stages of neural fold elevation; 2) the MHP is enriched with wedge-shaped neuroepithelial cells but has significantly fewer spindle-shaped, inverted wedge-shaped, and globular neuroepithelial cells than do the adjacent paired lateral areas of the neuroepithelium (L); and 3) each L is enriched with spindle-shaped, inverted wedge-shaped, and globular neuroepithelial cells but has significantly fewer wedge-shaped neuroepithelial cells than does the MHP. Thus wedging of neuroepithelial cells occurs during bending of the mouse neural plate and is localized to the MHP during neural fold elevation. Similarly, previous studies in the chick have shown that neuroepithelial cells become wedge shaped during bending of the neural plate and that such cell wedging is localized to the MHP during neural fold elevation. Such studies also have shed light on the roles of MHP formation and localized wedging of neuroepithelial cells within the MHP in the chick; however, such roles have yet to be elucidated in the mouse.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Programmed cell death during the earliest stages of spinal cord development in the chick embryo: a possible means of early phenotypic selection.

The spatiotemporal distribution of cell death in the chick embryo neural tube and spinal cord (brachial region) was examined between stage (St.) 12 and 22, in plastic semithin sections. Between St. 12 and 16, the total number of pycnotic cells per segment was low, whereas after St. 16 the number of pycnotic cells was substantially increased. Between St. 17 and 19 three cell death foci or regions could be recognized. One region, the dorsal pycnotic zone, was located in the most dorsal part of the spinal cord, including the neural crest, with the highest number of pycnotic cells observed at St. 18. The second region, or ventral pycnotic zone, was located between motoneurons and the floor plate and had the highest number of dying cells at St. 17. The third region, the floor plate pycnotic zone, was located in the midportion of the floor plate and had the greatest amount of cell death at St. 19. Although low numbers of pycnotic cells were also observed in other regions between St. 17 and 19, no pycnotic cells were found in the ventrolateral region that gives rise to motoneurons. Ultrastructural observations as well as data from in situ nick end labeling indicate that the pycnotic cells observed in the neural tube die by apoptosis and that the debris from the dead cells is phagocytized primarily by adjacent healthy neuroepithelial cells. Although the spatiotemporal distribution of pycnotic cells suggests that cell death at these early stages could play a role in establishing the pioneer axonal pathway for spinal commissural neurons, preliminary observations following perturbations of cell death do not support this notion. Alternatively, early cell death may be involved in the regulation of cellular patterning along the dorsoventral axis of the neural tube by a kind of negative selection of specific progenitor cells.

Animals↗
PubMed1995

Substance P-immunoreactive neurons in the human retina.

Substance P (SP) is a neuropeptide that acts as a neurotransmitter or a neuromodulator in the retina. The aim of this study was to identify the type(s) and the distribution of the SP-immunoreactive (SP-IR) cells in the human retina. We have used an antiserum to SP to immunostain neurons in postmortem human retinae. Immunostained retinae were processed with the avidin-biotin complex (ABC) to visualize the cells either whole mounted in glycerol or embedded in plastic. Some retinae were also sectioned at 20 microns in order to obtain radial views of stained cells. SP-IR amacrine cells stain intensely and appear to be of a single type in the human retina. They are large-field cells with large cell bodies (16 microns diameter) lying in normal or displaced positions on either side of the inner plexiform layer (IPL). Their sturdy, spiny, and appendage-bearing dendrites stratify in stratum 3 (S3) of the IPL, where many overlapping, fine dendrites intermingle to form a plexus of stained processes. Either cell bodies or primary dendrites emit an "axon-like" process that, typically, divides into two long, fine processes, which run in opposite directions for hundreds of micrometers in S5 and S3 before disappearing as distinct entities in the stained plexus in S3. Long, fine dendrites also pass from the dendritic plexus to run in S5 and down to the nerve fiber layer to end as large varicosities at blood vessel walls. In addition, fine processes are emitted from the dendritic plexus that runs in S1, and some pass up to the outer plexiform layer (OPL) to run therein for short distances. The SP-IR amacrine cell has many similarities to the thorny, type 2 amacrine cells described from Golgi studies. In addition to the SP-IR amacrine cells, a presumed ganglion cell type is faintly immunoreactive. Its 20-22 microns cell body gives rise to a radiate, sparsely branched, wide-spreading dendritic tree running in S3. Its dendrites and cell body become enveloped by the more intensely SP-IR processes and boutons from the SP-IR amacrine cell type. The SP-IR ganglion cell type most resembles G21 from a Golgi study.

Cell Count↗
PubMed1994

The acoustic properties of normal and imbedded bovine bone as measured by acoustic microscopy.

The acoustic impedance of bovine femoral cortical bone was measured with a scanning acoustic microscope used in the reflection mode. The bone was measured in the unimbedded state and after plastic imbedding. The acoustic impedance of the unimbedded specimens was also measured with a standard transmission ultrasonic technique. For the unimbedded bovine specimens there was a good correlation between the bulk transmission impedance and the reflection surface impedance (r2 = 0.976) and the values were 9.32 and 9.29 MRayls, respectively. Plastic imbedding produced a consistent and statistically significant increase in the acoustic impedance of the bone (9.71 MRayls). This experiment verified the use of acoustic microscopy as a quantitative materials analysis technique and it demonstrated the potential for material property analysis of imbedded bone.

Acoustic Impedance Tests↗
PubMed1992

Medium temperature epoxy resin for immunocytochemistry: Quetol 651 with water.

The addition of 1% water to the epoxy resin Quetol increased the labeling intensity of the sample. The significant decrease of the curing temperature of the epoxy resin may assist in preservation of antigens. Water may also reduce the cross-linkage of the resin allowing more antigen to be available to the antibodies. The modified Quetol resin is an option for use in immunocytochemistry studies.

Basidiomycota↗
PubMed1992

Ultrastructural morphology and allergen detection in birch pollen after aqueous, anhydrous-liquid, and vapor fixation techniques.

In order to find a good compromise between preservation of ultrastructural morphology and retention of antigenicity, birch pollen grains were chemically fixed in aqueous p-formaldehyde or glutaraldehyde, in p-formaldehyde or glutaraldehyde dissolved in anhydrous glycerol, and in p-formaldehyde or glutaraldehyde vapor. Representative cytoplasmic areas were inspected for the preservation of ultrastructural morphology and for their capacity to bind a monoclonal antibody against Bet v I, the major birch pollen allergen. The experiments showed that cytoplasmic morphology was best preserved after vapor fixation in p-formaldehyde. This fixation also led to the highest degree of specific antibody binding.

Allergens↗
PubMed1995

Ultrastructural characterization of neurons recorded intracellularly in vivo and injected with lucifer yellow: advantages of immunogold-silver vs. immunoperoxidase labeling.

Immunoperoxidase labeling of lucifer yellow provides a sensitive method for morphological characterization of neurons recorded intracellularly in vitro or in vivo. However, the reaction product is often so dense that it obscures ultrastructural details necessary for the analysis of synaptic contacts onto individually filled neurons. In the present study, we describe a silver intensification procedure using 1 nm gold labeling of lucifer yellow as an optimal means for immunocytochemically identifying single physiologically characterized neurons at the ultrastructural level. Single neurons in the frontal cortex of anesthetized rats were impaled in vivo and filled with lucifer yellow. The brains were then perfused with an acrolein fixative. Single vibratome sections through the recording site were reacted with a rabbit antibody directed against lucifer yellow followed by goat anti-rabbit 1 nm gold-labeled IgG and silver intensified. For comparison, additional sections were processed for immunoperoxidase detection of lucifer yellow. Labeled sections were processed for light microscopy or embedded in plastic for electron microscopy. The immunogold-silver label as well as peroxidase reaction product of lucifer yellow was readily detected in cell bodies, proximal and distal dendrites, and spines. However, in contrast to immunoperoxidase, the immunogold-silver reaction did not obscure subcellular organelles. Most importantly, the synaptic junctions formed by afferents to the filled neuron were more easily identifiable following the immunogold-silver procedure. This clear visualization of postsynaptic densities is essential for examining synaptic circuitry between afferents and physiologically characterized neurons.

Animals↗
PubMed2007

Novel procedure for high-fidelity tendon histology.

A novel method to obtain high-quality histologic sections of rat tendon for light microscopic study is presented. This approach utilizes non-deleterious dehydrating and clearing solvents and a hydrophobic acrylic (methyl methacrylate) resin. This methodology avoids processing and microtomy artifacts common with routine paraffin wax techniques and overcomes specimen size limitations associated with hydrophilic (e.g., glycol methacrylate) resin histologic methods. These novel histologic processing techniques facilitate the reliable assessment of tendons' cellular and matrix components and can be readily adapted to morphologic studies of damage, healing, and repair.

Acrylic Resins↗
PubMed2002

Quadriplegic areflexic ICU illness: selective thick filament loss and normal nerve histology.

Areflexic quadriplegia that occurs in the intensive care unit (ICU) is commonly ascribed to critical illness polyneuropathy based upon electrophysiology or muscle light microscopy. However, electron microscopy often documents a selective thick filament loss myopathy. Eight ICU patients who developed areflexic quadriplegia underwent biopsy. Seven patients had received steroids, and 2 had also received paralytic agents. Electrodiagnostic studies revealed absent or low-amplitude motor responses in 7. Sensory responses were normal in 5 of 6 and absent in 1. Initial electromyography revealed absent (n = 3), small (n = 3), or polyphasic (n = 1) motor unit potentials, and diffuse fibrillation potentials (n = 5). In all 8, light microscopy of muscle revealed numerous atrophic-angulated fibers and corelike lesions, and electron microscopy revealed extensive thick filament loss. Morphology of sural and intramuscular nerves, and, in one autopsied case, of the obturator nerve and multiple nerve roots, was normal. Although clinical, electrodiagnostic, and light microscopic features mimicked denervating disease, muscle electron microscopy revealed thick filament loss, and nerve histology was normal. This suggests that areflexic ICU quadriplegia is a primary myopathy and not an axonal polyneuropathy.

Adult↗
PubMed1993

Different forms of 130 kD connective tissue protein are specific for boundaries in the nervous system and basement membrane of muscle cells in leech.

The nervous system and muscle tissue of the leech express two different organ-specific forms of connective tissue protein. The nervous system-specific form appears in regional boundaries separating cell bodies, axonal tracts and areas of the neuropile during late embryogenesis. In contrast, the muscle-specific form appears earlier during development in the basement membrane of muscle cells. In extraction experiments both forms behave like extracellular matrix proteins and because of their molecular weight, are considered members of a group of cell type-specific 130 kD proteins (leech gp130s). However, the two forms differ in their posttranslational modification. As determined by Con A and lentil lectin affinity chromatography, only the nervous system-specific, but not the muscle-specific form, has fucosylated and high mannose N-linked carbohydrates. These differences in the developmental onset and glycosylation suggest that nervous system-specific and muscle-specific connective tissue proteins are regulated differently and participate in different molecular interactions.

Animals↗
PubMed2004

Prostasomes are secreted from poorly differentiated cells of prostate cancer metastases.

BACKGROUND: Prostasomes are small (40-500 nm), granule-like bodies, found in normal epithelial cells of the prostate and secreted into the prostate duct system. Also poorly differentiated prostate cancer cells are producing prostasomes, since we could isolate and purify prostasomes from vertebral metastases with biochemical methods. To find out whether these prostasomes are secreted into extracellular sites of the metastases, we used electron microscopy. METHODS: Small biopsies from vertebral metastases of prostate cancer, taken directly from the operating field at surgery, were immediately fixated, embedded in plastic and processed for electron microscopy. RESULTS: We found that prostasomes could be identified extracellularly in the interstitial tissues as well as in the cytoplasm of the metastatic cells. CONCLUSION: We conclude that prostasomes produced by the cells of vertebral metastases of prostate cancer are distributed both intracellularly and extracellularly in the interstitial spaces of the tissue. Thus, prostasomes of metastases could perhaps be exploited as targets for immunodiagnosis and/or immunotherapy.

Biopsy↗
PubMed2000

Vascular remnants in the rabbit vitreous body. I. Morphological characteristics and relationship to vitreous embryonic development.

Using light and transmission electron microscopy, we observed novel structures in the rabbit vitreous body. They were found in 18 out of 27 eyes from rabbits 0.5-36 months of age. These structures are scattered throughout the entire vitreous matrix. By light and transmission electron microscopy, they appear to be made up of the same structural components. Based upon their morphological appearance, they can be subdivided into two groups which we provisionally named 'intravitreal structure type 1 and 2' or 'IVS-1' and 'IVS-2'. IVS-1 has a highly variable morphology (e.g. star-shaped, round, oval), whereas IVS-2 is tubular. The dimensions of IVS-1 vary in relation to the mesh diameters of the collagen matrix, while those of IVS-2 do not. In adult rabbit eyes, we observed transitions between IVS-1 and intravitreal ghost vessels (acellular remnants of blood vessels), and between IVS-1 and IVS-2. In very young rabbits (14 days) we observed intravitreal ghost vessels consisting of tightly-packed IVS-1. Therefore, we concluded that IVS-1 and 2 are related structures presumably of vascular origin. It appears that they represent fragmented and non-fragmented acellular remnants of hyaloid blood vessels. The presence of vascular remnants throughout the entire vitreous matrix of adult rabbit eyes is in conflict with existing theories on the embryonic development of the vitreous body, which describe a strict spatial separation between the primary (vascular) and secondary (avascular) vitreous. However, it strongly supports an alternative theory that explains the formation of the secondary vitreous by a process of continuous remodelling of the primary vitreous.

Animals↗
PubMed2002

Thymosin beta 4 promotes corneal wound healing and decreases inflammation in vivo following alkali injury.

Previously, thymosin beta 4 (Tbeta(4)) was found to promote wound healing in full thickness skin wounds and heptanol debrided corneas. Here, the effect of Tbeta(4) was examined treatment on corneal wound healing and inflammation in vivo after alkali injury, a more severe wound of the eye. Corneas from 129 Sv mice were chemically burned with a 2 mm disc soaked in 1 N NaOH for 30 sec. Eyes were irrigated copiously with phosphate buffered saline (PBS) and then treated topically with either Tbeta(4) (5 microg/5 microl PBS) or 5 microl PBS twice daily. Animals were killed, the eyes were enucleated, fixed and embedded in plastic resin or prepared for mRNA analysis. Mouse corneas topically treated with 5 microg of Tbeta(4) twice daily after alkali injury demonstrated accelerated re-epithelialization at all time points and decreased polymorphonuclear leukocyte (PMN) infiltration at 7 days post injury (p.i.) when compared to PBS-treated controls. mRNA transcript levels were decreased several fold for interleukin (IL)-lbeta, and the chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2 and monocyte chemoattractant protein (MCP)-1 from 1 to 7 days after injury in the Tbeta(4)- vs. PBS-treated corneas. Thus, Tbeta(4) may provide a new clinical treatment for severe traumatic corneal wound disorders by promoting rapid corneal wound healing and decreasing both PMN infiltration and inflammatory cytokine and chemokine mRNA levels.

Administration, Topical↗
PubMed1997

Electron tomography of neuronal mitochondria: three-dimensional structure and organization of cristae and membrane contacts.

The structure of neuronal mitochondria from chick and rat was examined using electron microscope tomography of chemically fixed tissue embedded in plastic and sliced in approximately 500 nm-thick sections. Three-dimensional reconstructions of representative mitochondria were made from single-axis tilt series acquired with an intermediate voltage electron microscope (400 kV). The tilt increment was either 1 degree or 2 degrees ranging from -60 degrees to +60 degrees. The mitochondrial ultrastructure was similar across species and neuronal regions. The outer and inner membranes were each approximately 7 nm thick. The inner boundary membrane was found to lie close to the outer membrane, with a total thickness across both membranes of approximately 22 nm. We discovered that the inner membrane invaginates to form cristae only through narrow, tubular openings, which we call crista junctions. Sometimes the cristae remain tubular throughout their length, but often multiple tubular cristae merge to form lamellar compartments. Punctate regions, approximately 14 nm in diameter, were observed in which the inner and outer membranes appeared in contact (total thickness of both membranes approximately 14 nm). These contact sites are known to a play a key role in the transport of proteins into the mitochondrion. It has been hypothesized that contact sites may be proximal to crista junctions to facilitate transport of proteins destined for the cristae. However, our statistical analyses indicated that contact sites are randomly located with respect to these junctions. In addition, a close association was observed between endoplasmic reticulum membranes and the outer mitochondrial membrane, consistent with the reported mechanism of transport of certain lipids into the mitochondrion.

Animals↗
PubMed1993

A single low cadmium dose causes failure of spermiation in the rat.

Ten-week-old male Sprague-Dawley rats were injected ip with cadmium chloride solution in a single dose of 0, 0.5, or 1.0 mg/kg body wt. At 4, 24, 48, and 72 hr after injection, testes of the animals were perfusion fixed, embedded in plastic, and sectioned. Tissue sections were stained and examined under the light microscope. These amounts of cadmium did not result in visible vascular lesion in the testes. However, at a dose of 1 mg/kg, the cadmium treatment resulted in failure of spermiation from stage IX through later stages of spermatogenesis in the seminiferous epithelium. Detailed statistical analysis revealed failed spermiation 24 hr after dosing. As the exposure time increased, failure of spermiation was observed with increasing frequency within an affected stage, and was seen at later stages of spermatogenesis as well. Testes of the rats treated with a cadmium chloride dose of 0.5 mg/kg showed no change in the frequencies of tubules having unreleased spermatids when compared to the controls. There was no difference in the stage frequencies between all the treatment groups and the controls. These results indicate that a single cadmium chloride dose of 1 mg/kg results in failed spermiation in rat seminiferous tubules, without discernible change to the surrounding endothelium. We conclude that cadmium begins to act during early stage VIII of spermatogenesis to induce failure of spermiation, and the action of cadmium is spermatogenic stage-specific.

Animals↗
PubMed1988

Reactions of surrounding tissue to the cementless hip implant Ti-6Al-4V after an implantation period of several years. Autopsy studies in three cases.

Three femoral endoprosthetic stems implanted without cement, made of Ti-6Al-4V alloy, with implantation periods of between 2 and 4 years were examined histologically in the form of undecalcified thin ground sections after having been split into segments and embedded in plastic. During the implantation period total osseointegration in the metadiaphyseal region takes place, while in the proximal area reaction forms with direct contact between metal and bone on the one hand and with an interposed membrane of connective tissue and adjacent secondary bony shell on the other must be distinguished. The osseointegration of the stem is the result of a primary healing of the bone and is due to the biocompatibility of the metal alloy and to the primary stability obtained by the press-fit method.

Aged↗
PubMed1979

Auditory nerve activity and cochlear morphology after noise exposure.

Four chinchillas were exposed for 5 days to an octave band of noise centered at 4 kHz and having an SPL of 86 dB. After a recovery period of approximately 6 months, behavioral audiograms were obtained and auditory nerve fiber activity was recorded. The animals were killed and the cochleas embedded in plastic to obtain a surface preparation and 1 mu radial sections of the organ of Corti. Behavioral threshold shifts ranged from 5 to 20 dB at frequencies between 4 and 11 kHz. Auditory nerve fiber thresholds were elevated up to 70 dB for units with characteristic frequencies between 4 and 14 kHz. Units with higher and lower characteristic frequencies had normal thresholds. Cochleagrams showed narrow lesions of inner and/or outer hair cells over approximately a 1 mm distance. A comparison of the three realms of data revealed the following: (1) The greatest threshold shifts from the noise exposure were seen in the single nerve fiber thresholds while the smallest shifts were seen in the behavioral thresholds, (2) the greatest behavioral and neural threshold shifts and greatest cochlear damage occurred 1 octave above the center frequency of the noise exposure, and (3) based on the frequency-place map of the chinchilla cochlea, the range of fibers with elevated thresholds exceeded the extent of the OHC lesion. A number of anatomical changes were seen that effectively increased the extent of the damage found in the chochleagram. These changes included: distortions in the surface topography of the organ of Corti affecting the orientation of IHC; missing pillar cells in the presence of normal OHC and/or IHC and protrusion of the IHC cuticular plate into the subtectorial space.

Animals↗
PubMed1987

Autometallography: tissue metals demonstrated by a silver enhancement kit.

In biological tissue, minute accumulations of gold, silver, mercury and zinc can be visualized by a technique whereby metallic silver is precipitated on tiny accumulations of the two noble metals, or on selenites or sulphides of all four metals. In the present study a silver enhancement kit, primarily intended for the amplification of colloidal gold particles, has been used to demonstrate these catalytic tissue metals. Sections from animals exposed intravitally to aurothiomalatate, silver lactate, mercury chloride, sodium selenite or perfused with sodium sulphide were subjected to a commercial silver enhancement kit (IntenSE, Janssen Pharmaceutica). It was found that the kit performs adequately to the silver lactate gum arabic developer and to the photographic emulsion technique. The kit can be used as a silver enhancement medium for the demonstration of zinc by the Neo-Timm and selenium methods and for demonstration of gold, silver, and mercury in tissues from animals intravitally exposed to these metals. It can also be used for counterstaining silver treated osmium fixed tissues embedded in plastic.

Animals↗