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The human gonocytes in the early phases of gonadal differentiation. Observations of one embryo at Carnegie stage 11.

With the aim of emphasize the characteristics of the migration of human primordial gonocytes through the mesenchyme of the dorsal mesentery towards the genital ridge, a very early human embryo, corresponding to Carnegie stage 11 (23-24 days of development) was studied together with other embryos from the 4th to the 10th week of pregnancy. The early embryo was embedded in resin, serially cut and the observations were made on semithin coronal sections, de-plasticized and stained with toluidine blue. Migrating primordial germ cells were identified in the thickness of the splanchnopleure, where they appeared as pale and round elements with a nucleolated nucleus and granular cytoplasm. The clusters of primordial gonocytes were constantly surrounded and in close relationship with smaller, polyhedral and intensely stained elements. These features suggest the hypothesis that gonocytes and dark cells join together during the process of migration and that the last can participate in the formation of the gonadal bud. The hypothesis is confirmed by the observation of dark cells, surrounding the gonocytes, in the primitive ovaries or testes, in later stages of development. Perhaps they do not participate directly in the formation of the sexual cords, but reach their periphery where they assume an elongate shape.

Cell Movement↗

A method for collecting semithin epoxy serial sections for light microscopy and 3-D reconstruction.

In the three-dimensional reconstruction of neuronal structure, it is imperative that ribbons of semithin or ultrathin sections be obtained. Resin-embedded semithin sections display better structural details than paraffin-embedded sections. The cutting and collecting of long ribbons of resin-embedded semithin sections using a microtome, requires the use of large troughs on glass knives. A simple plastic trough has been described which facilitates the collection of ribbons directly onto a coverslip. As the ribbons are formed, they are floated on a coverslip. The water in the trough is slowly drained through a tubing which is attached to a syringe. The ribbons settle on the coverslip, which is easily removed and placed on a hotplate to dry the sections.

Animals↗

A selective optical sensor based on [9]mercuracarborand-3, a new type of ionophore with a chloride complexing cavity.

A highly selective optical sensor for chloride, based on the multidentate Lewis acid ionophore [9]mercuracarborand-3, is described herein. This sensor is constructed by embedding the mercuracarborand ionophore, a suitable pH-sensitive lipophilic dye, and lipophilic cationic sites in a plasticized polymeric membrane. The multiple complementary interactions offered by the preorganized complexing cavity of [9]mercuracarborand-3 is shown to control the anion selectivity pattern of the optical film. The film exhibits a significantly enhanced selectivity for chloride over a variety of lipophilic anions such as perchlorate, nitrate, salicylate, and thiocyanate. Furthermore, the optical selectivity coefficients obtained for chloride over other biologically relevant anions are shown to meet the selectivity requirements for the determination of chloride in physiological fluids, unlike previously reported chloride optical sensors. In addition, the optical film responds to chloride reversibly over a wide dynamic range (16 microM-136 mM) with fast response and recovery times.

Algorithms↗

Consecutive light microscopy, scanning-transmission electron microscopy and transmission electron microscopy of traumatic human brain oedema and ischaemic brain damage.

Cortical biopsies of 11 patients with traumatic brain oedema were consecutively studied by light microscopy (LM) using thick plastic sections, scanning-transmission electron microscopy ((S)TEM) using semithin plastic sections and transmission electron microscopy (TEM) using ultrathin sections. Samples were glutaraldehyde-osmium fixed and embedded in Araldite or Epon. Thick sections were stained with toluidine-blue for light microscopy. Semithin sections were examined unstained and uncoated for (S)TEM. Ultrathin sections were stained with uranyl and lead. Perivascular haemorrhages and perivascular extravasation of proteinaceous oedema fluid were observed in both moderate and severe oedema. Ischaemic pyramidal and non-pyramidal nerve cells appeared shrunken, electron dense and with enlargement of intracytoplasmic membrane compartment. Notably swollen astrocytes were observed in all samples examined. Glycogen-rich and glycogen-depleted astrocytes were identified in anoxic-ischaemic regions. Dark and hydropic satellite, interfascicular and perivascular oligodendrocytes were also found. The status spongiosus of severely oedematous brain parenchyma observed by LM and (S)TEM was correlated with the enlarged extracellular space and disrupted neuropil observed by TEM. The (S)TEM is recommended as a suitable technique for studying pathological processes in the central nervous system and as an informative adjunct to LM and TEM.

Adolescent↗

Effect of normal endometrial stroma on growth and differentiation in Ishikawa endometrial adenocarcinoma cells.

Endometrial cancer is characterized by alterations in the stromal cells and the supporting extracellular matrix in addition to the intrinsic alterations of the malignant epithelial cells. We have developed a cell culture model that demonstrates the role of stromal cells in the regulation of proliferation, hormone responsiveness, and differentiation of an endometrial adenocarcinoma cell line (Ishikawa). Conditioned medium (CM) was collected from normal primary human endometrial stromal cells grown on plastic or within the basement membrane extract, Matrigel. The CM produced by stromal cells cultured in contact with Matrigel markedly inhibited Ishikawa cell proliferation compared with CM from stromal cells cultured on plastic. Ishikawa cell proliferation varied with steroid hormone treatment in the presence of CM from stromal cells embedded in Matrigel. When the Ishikawa cells were placed in coculture in contact with stromal cells in Matrigel, production of a differentiated epithelial secretory product, glycodelin, was induced. Gene expression of stromal cell hormone receptors, growth factors, and integrins was analyzed by reverse transcription-PCR in the presence of Matrigel to determine the potential factors involved in stromal regulatory function. These combined studies imply that the phenotype of the Ishikawa cells can be induced to differentiate to more closely resemble normal endometrial epithelium by reintroduction of stromal factors and appropriate extracellular matrix. Additionally, the study shows that basement membrane proteins influence the regulatory function of stromal cells as they mediate epithelial cell growth.

Adenocarcinoma↗

Cerium as amplifying agent--an improved cerium-perhydroxide-DAB-nickel (Ce/Ce-H2O2-DAB-Ni) method for the visualization of cerium phosphate in resin sections.

A new visualization (Ce/Ce-H2O2-DAB-Ni) procedure for cerium (Ce III) phosphate in semithin and ultrathin plastic sections (Epon 812, Lowicryl K4M, glycol methacrylate) of rat kidney tissues that had been incubated before embedding for the demonstration of phosphatases (alkaline and acid phosphatase, 5(1)-nucleotidase, Mg-dependent ATPase) is described. For this purpose the hydrophobic Epon resin was removed in NaOH-ethanol solution, whereas the hydrophilic Lowicryl and methacrylate sections did not required any etching. The primary reaction product Ce III-phosphate was amplified in a Ce III-citrate solution, subsequently oxidized with H2O2 and then visualized in a H2O2 containing DAB-nickel medium (Ce IV-perhydroxy induced DAB polymerization principle). The method yielded a very clear localization of enzyme activity. The final reaction product (DAB-nickel polymers) in 0.5 - 2.0 microns semithin sections is blue-black; the background staining is completely prevented. An increase of the staining contrast was obtained by posttreatment with OsO4 (osmium black formation). Furthermore, the enzyme reaction product could be demonstrated in 40 nm thick ultrathin sections by silver intensification, which utilized the high argyrophilia of the polymerized DAB-nickel complexes. This procedure replaces the earlier published technique.

3,3'-Diaminobenzidine↗

The influence of different cryopreparations on the distribution of ions in bullfrog myocard cells.

Bullfrog heart muscle trabecula are shock-frozen in liquid propane cooled by liquid nitrogen and then processed for X-ray microanalysis in two different ways: 1. Freeze-drying followed by vacuum embedding. 2. Cryoultramicrotomy and freeze-drying. Stained sections of freeze-dried, embedded tissue exhibit detailed ultrastructure, but are useless for X-ray microanalysis. Unstained, dry cut plastic-sections are suitable for X-ray microanalysis, but the ultrastructure appears faint. Higher electron optical contrast and peak-to-background ratio of X-ray spectra are generally obtained in freeze-dried cryosections. Both preparation methods show that the X-ray spectra are influenced by the quality of cryofixation. The phosphorus/potassium ratio in nuclei increases with increasing ice crystal size.

Animals↗

A new plastic for morphometric investigation of blood vessels, especially in large organs such as the human liver.

In this paper a technique is described, using Araldite CY 223 and hardener HY 2967 as injection material, for preparing corrosion casts or histological sections. The plastic has a viscosity (at 39-40 degrees C) similar to that of blood, a gelling time of approximately 17 min (at 40 degrees C), and an exothermic transition energy of delta H = 80.28 +/- 3.20 cal/gm. The influence of the plastic on the tissue is discussed. The histological sectioning of fixed tissue containing Araldite-filled blood vessels after embedding in 2-hydroxyethyl-methacrylate (GMA) is described. When using GMA in a modification of the mixtures of Ruddell (1967) and Sims (1974), methylbenzoate is recommended as an intermedium in order to obtain a more uniform infiltration and reproducible section thickness. At the same time methylbenzoate is recommended as a storing fluid. Sections of 2-3 micrometers afford satisfying morphologic and morphometric results. This method allows various arterial wall dimensions to be measured easily, and provides a suitable means to compare histometric values with SEM data derived from corrosion casts.

Blood Vessels↗

The glutamatergic innervation of oxytocin- and vasopressin-secreting neurons in the rat supraoptic nucleus and its contribution to lactation-induced synaptic plasticity.

The present ultrastructural study analysed the distribution of glutamatergic synapses on oxytocin- and vasopressin-secreting neurons in the rat supraoptic nucleus (SON) after post-embedding immunogold labelling for glutamate immunoreactivity, visible over synaptic-like vesicles, mitochondria and synaptic densities. Double labelling for glutamate and GABA showed that putative glutamatergic terminals were distinct from GABAergic terminals. In ultrathin sections stained for glutamate and either oxytocin or vasopressin, the proportion of glutamatergic synapses was similar on oxytocinergic and vasopressinergic somata in virgin rats under basal conditions of peptide release as well as in lactating rats, in which oxytocin secretion is enhanced. Cross-sectional soma areas were significantly increased in lactating rats: oxytocinergic profiles were, on average, approximately 40% larger than in virgin rats. However, the incidence of axo-somatic glutamatergic synapses (assessed as mean number of synapses per 100 microm of plasmalemma or proportion of somatic surface apposed to synaptic active zones) did not diminish, indicating that there was a compensatory increase of synapses during lactation. Also, we found an increase in the number of glutamatergic terminals making synaptic contact simultaneously onto two or more oxytocinergic elements in the same plane of section. Our observations therefore indicate that SON oxytocinergic and vasopressinergic neurons are innervated to a similar extent by a relatively large proportion of glutamatergic synapses. They reveal, moreover, that glutamatergic afferents participate in the lactation-induced synaptic plasticity of the oxytocinergic system.

Animals↗

Improvements of glycol methacrylate. I. Its use as an embedding medium for electron microscopic studies.

The technique for using the water-soluble embedding medium glycol methacrylate has been improved for ultrastructural studies by the simplification of the method of formation of prepolymers used in embedding the tissue, by the addition of a cross-linking agent so that sections are stable in the electron beam, and by improving the softness of the blocks by the addition of a plasticizing agent. The preservation of tissue morphology has been improved by complete dehydration in glycol methacrylate monomer prior to infiltration with the prepolymer. Preservations of tissue morphology is further enhanced by complete dehydration in ethanols and embedding in the improved glycol methacrylate medium.

Acrylates↗

Routine use of backscattered electron imaging to visualize cytochemical and autoradiographic reactions in semi-thin plastic sections.

The scanning electron microscope (SEM) was used to examine cytochemical and autoradiographic reactions in 2-microns semi-thin sections of tissues conventionally fixed and embedded in various resins. The sections were examined using both the secondary and backscatter modes of the SEM at magnifications within the range attainable with the light microscope. Both modes allowed the imaging of phosphatase reaction product using cerium and lead capture, lectin-gold, and immunogold labeling, with and without silver enhancement, and autoradiography. Backscattered electron imaging (BEI), however, provided images with more contrast and structural details. This approach allows examination of large sections, with more contrast and resolution than the light microscope, and visualization of reactions not visible with this instrument. The improved imaging and the simple and conventional preparation of specimens indicate that BEI can be used routinely to examine tissue organization, cell structure, and the content of the various cell compartments with a resolution approaching that of transmission electron microscopy.

Ameloblasts↗

Abeta-polyacrolein aggregates: novel mechanism of plastic formation in senile plaques.

High levels of acrolein (H2C=HC-CH=O) occur in Alzheimer's brain. Amyloid-beta (Abeta) peptide co-localizes with acrolein presumably due to Abeta-induced lipid peroxidation. Focal production of acrolein may yield a transient elevation in the concentration of acrolein that may be susceptible to polymerization via basic latex polymer chemistry. Following incubation of Abeta with acrolein (16-750 mM), we observed the formation of thin plastic fragments that were extensively punctuated. Planar aggregates stained for protein and for cross-beta structures suggesting an Abeta-polyacrolein colloidal mixture. Depending on acrolein concentration and incubation time, we observed uniformly sized planar aggregates (approximately 10 microm2) or monolayers (>100 mm2) of thin polyacrolein films embedded with Abeta oligomers. The ability of Abeta to catalyze the polymerization of acrolein is likely due to Abeta's surfactant and redox properties. These observations suggest that plastic in the form of Abeta-polyacrolein latexes may exist in neural tissue contributing to the pathogenesis of Alzheimer's disease.

Acrolein↗

The influence of grain size on the fatigue behavior of annealed 316 LVM stainless steel.

Experimental tests demonstrate that there is a sharp drop in resistance to fatigue fracture when the grain size is increased from 75 to 150 micrometers. This is at least a partial explanation for fatigue fractures of the stems of total hip-joint prostheses reported in the literature. It is also shown that plastic strains associated with fatigue stressing increase with increasing grain size so that loosening of the bone cement embedment may also be part of the premature failure process.

Hip Joint↗

Terahertz imaging system based on a backward-wave oscillator.

We present an imaging system designed for use in the terahertz range. As the radiation source a backward-wave oscillator was chosen for its special features such as high output power, good wave-front quality, good stability, and wavelength tunability from 520 to 710 GHz. Detection is achieved with a pyroelectric sensor operated at room temperature. The alignment procedure for the optical elements is described, and several methods to reduce the etalon effect that are inherent in monochromatic sources are discussed. The terahertz spot size in the sample plane is 550 microm (nearly the diffraction limit), and the signal-to-noise ratio is 10,000:1; other characteristics were also measured and are presented in detail. A number of preliminary applications are also shown that cover various areas: nondestructive real-time testing for plastic tubes and packaging seals; biological terahertz imaging of fresh, frozen, or freeze-dried samples; paraffin-embedded specimens of cancer tissue; and measurement of the absorption coefficient of water by use of a wedge-shaped cell.

Animals↗

Analytical scanning electron microscopy and x-ray microdiffractometry of renal calculi using etched plastic sections.

We describe the technique and illustrate results of renal calculi analyses utilizing correlative analytical scanning electron microscopy and x-ray microdiffractometry on the same 100 micrometer Spurr embedded sections. Essential to the technique is the employment of an extremely sensitive x-ray microdiffractometer, and for morphological analysis, pretreatment of the sections with a sodium ethoxide etching solution. This technique facilitates more precise identification of crystalline constituents especially calcium apatite and magnesium ammonium phosphate hexahydrate (struvite). Further, the technique permits detection and mapping of both crystalline and amorphous constituents within calculi.

Calcium Oxalate↗