Silver Grains Research Articles

Muscarinic cholinergic receptor subtypes in cerebral cortex of Fisher 344 rats: A light microscope autoradiography study of age-related changes

The density and localization of muscarinic cholinergic M1–M5 receptor subtypes was investigated in frontal and occipital cortex of male Fisher 344 rats aged 6 months (young-adult), 15 months (mature) and 22 months (senescent) by combined kinetic and equilibrium binding and light microscope autoradiography. In 6-month-old rats, the rank order density of muscarinic cholinergic receptor subtypes was M1 > M2 > M4 > M3 > M5 both in frontal and occipital cortex. A not homogeneous distribution of different receptor subtypes throughout cerebrocortical layers of frontal or occipital cortex was found. In frontal cortex silver grains corresponding to the M1 and M2 receptor subtypes were decreased in 15- and 22-month-old groups. The M3 receptor density was remarkably and moderately decreased in layers II/III and V, respectively, of rats aged 15 and 22 months. A reduced M4 receptor density was observed in layer I and to a lesser extent in layer V of mature and senescent rats, whereas no age-related changes of M5 receptor were found. In occipital cortex a diminution of M1 receptor was observed in layers II/III and V of mature and senescent rats. The M2 receptor expression decreased in layer I of 15- and 22-month-old senescent rats, whereas M3–M5 receptors were unchanged with exception of a slight decrease of the M4 receptor in layer IV and of M5 receptor in layers II/III. These findings indicate a different sensitivity to aging of muscarinic receptor subtypes located in various cerebrocortical layers. This may account for the difficulty in obtaining relevant results in manipulating cholinoceptors to counter age-related impairment of cholinergic system.

Effects of morphology on phonons in nanoscopic silver grains

The morphology of nanoscopic Ag grains significantly affects the phonons. Atomistic simulations show that realistic nanograin models display complex vibrational properties. (1) Single-crystalline grains. Nearly pure torsional and radial phonons appear at low frequencies. For low-energy, faceted models, the breathing mode and acoustic gap (lowest frequency) are about 10% lower than predicted by elasticity theory (ET) for a continuum sphere of the same volume. The sharp edges and the atomic lattice split the ET-acoustic-gap quintet into a doublet and triplet. The surface protrusions associated with nearly spherical, high-energy models produce a smaller acoustic gap and a higher vibrational density of states (DOS) at frequencies $\ensuremath{\nu}<2\phantom{\rule{0.3em}{0ex}}\mathrm{THz}$. (2) Twined icosahedra. In contrast to the single-crystal case, the inherent strain produce a larger acoustic gap, while the core atoms yield a DOS tail extending beyond the highest frequency of single-crystalline grains. (3) Mark's decahedra, in contrast to (1) and (2), do not have a breathing mode; although twined and strained, do not exhibit a high-frequency tail in the DOS. (4) Irregular nanograins. Grain boundaries and surface disorder yield nondegenerate phonon frequencies, and significantly smaller acoustic gap. Only these nanograins exhibit a low-frequency ${\ensuremath{\nu}\phantom{\rule{0.1em}{0ex}}}^{2}$ DOS in the interval 1--2 THz.

Effect of resin hydrophilicity on water‐vapour permeability of dental adhesive films

This study examined the water-vapour permeability of thin polymerized resin films fabricated from five co-monomer blends of increasing degrees of hydrophilicity, as measured by their Hoy's solubility parameters. Neat resin films were prepared from five experimental light-curable resins (n = 10). Each film was mounted in a Fisher permeability cup with 8 g of water placed inside the cup. The experiments were conducted in a modified twin-outlet desiccator connected to a vacuum pump in one outlet to permit a continuous airflow to encourage water evaporation. Weight losses by water evaporation were measured at 3, 6, 9, 24, 30, and 48 h by using an analytical balance. Additional resin films were examined by using transmission electron microscopy (TEM) after immersion in ammoniacal silver nitrate. A significant correlation was observed between the cumulative water loss at 48 h and the Hoy's total cohesive energy density (delta(t)). Transmission electron microscopy revealed silver-filled channels along film peripheries and silver grains of decreasing dimensions toward the film centres in co-monomer blends 3, 4, and 5 of increasing hydrophilicity. Hydrophilic dentin adhesives polymerized in thin films are prone to water loss by evaporation. This probably accounts for the water droplets seen on the surface of vital-bonded dentin after the application of simplified dentin adhesives.

The existence of a linear system consisting of sympathetic endings in rat skin

In a previous pilot study we suggested the novel notion that the catecholaminergic sympathetic nerve endings are non-homogeneously distributed in the rat skin. In the present study we have utilized several independent methods to determine the in vivo distribution of catecholamine-containing fibers in rat skin. Using whole body macro-autoradiography with an iodine125-labeled tyrosine, we localized the distribution of iodine-125-catecholamine in rat skin. The images on the film showed various pairs of symmetrical linear arrays running from the head through the back and to the hind limbs of the animal that we arbitrarily termed sympathetic substance lines (SSLs). The distribution of catecholamine in rat skin was also visualized by light microscopy autoradiography with tritiated tyrosine. The majority of silver grains in the sections were located among hair follicles along a band or zone. Furthermore, a modified sucrose-phosphate-glyoxilic acid (SPG) method was adapted to observe sympathetic fibers in the skin sections. Dense clusters of fluorescent nerve fibers in correspondence of arrector pili muscles (AP muscles) were located along lengthwise lines of the body, in a pattern coinciding with the linear arrays identified by macro-autoradiography. We concluded that concentrated clusters of noradrenergic nerve fibers innervate AP muscles and form a longitudinal linear system in the whole skin. These results are discussed in terms of physiological functions associated with hair follicles, sensory signal pathways and Meridians in Chinese traditional medicine.

Resident–Intruder Trait Aggression is Associated with Differences in Lysine Vasopressin and Serotonin Receptor 1A (5‐HT1A) mRNA Expression in the Brain of Pre‐Pubertal Female Domestic Pigs (Sus scrofa)

Aggressive behaviour exhibited by domestic pigs following encounters with unfamiliar individuals is a serious welfare and economical problem. Aggression resulting in skin lesions is similarly prevalent in prepubertal pigs of either sex. Little is known about the neural circuits and neuropeptides that control aggression in the pig. Because there is evidence for the involvement of the vasopressin and serotonergic systems in the regulation of aggressive behaviour in male mammals, we sought differences using quantitative in situ hybridisation of vasopressin and serotonin 1A receptor (5-HT1A) mRNA expression within specific brain regions of aggressive and nonaggressive prepubertal female pigs. The number of cells expressing vasopressin mRNA was significantly higher in aggressive pigs in the medial amygdala, lateral septum (LS) and showed a similar trend in the bed nucleus of the stria terminalis (BnST) but not the paraventricular nucleus (PVN) or supraoptic nucleus. The 5-HT1A receptor was widely expressed through the porcine brain and a significantly lower intensity (silver grain density) of 5-HT1A mRNA expression was observed in the BnST. In the medial amygdala and LS fewer cells expressed 5-HT1A mRNA in aggressive pigs but no differences were found in the PVN. In the absence of inbred strains or selection lines, these findings have shown that prior identification of phenotypic behavioural extremes in a population in advance of neural studies is a useful technique. Moreover, these findings support a central role for vasopressin and serotonin in the mediation of high trait aggression in prepubertal female pigs.

Coincidence Planar Imaging for Dynamic [ 18F]FDG Uptake in Nude Mice with Tumors and Inflammation: Correlated With Histopathology and Micro-autoradiography

The Institute of Nuclear Energy Research of Taiwan has developed a dynamic coincidence detection device for positron emitted radiotracer pharmacodynamic study in small mice models. In this study, we set up an experimental paradigm by determining [fluorine-18]-2-deoxy-2-fluoro-D-glucose ([18F]FDG) dynamic uptake in tumors and inflammations in nude mice as the foundation for future applications in therapy development. Histopathology and micro-autoradiography of these tumors and inflammations were obtained for confirmation. Dynamic coincidence planar images of six tumors and two inflammations in nude mice were acquired over 4 hours immediately after injection of 25.9 MBq of [18F]FDG into the right thigh of each animal. After image reconstruction, the lesion-to-background ratios were calculated in regions of interest over the lesion and contralateral thigh to determine the equilibrium status of the radiotracer. All mice were sacrificed for histopathologic examination and six of the mice were examined with micro-autoradiography. [18F]FDG uptake in tumors and inflammations both reached equilibrium about 3 hours after injection. At equilibrium, [18F]FDG uptake into tumors was two to four times higher than the background. Uptake into the 4-day and 8-day inflammations was 2.3 and 5.5 times higher than the background, respectively. Histopathology showed macrophage and neutrophil infiltration around the tumors and in the inflammations. Micro-autoradiography showed dense silver grains in the granulation tissue surrounding the tumors and inflammations. The preliminary results suggested that dynamic [18F]FDG coincidence planar imaging can help in determining the suitable time for static [18F]FDG imaging in nude mice models. The optimal time for static [18F]FDG positron emission tomography imaging was around 3 hours after injection. The paradigm for determining a dynamic [18F]FDG uptake pattern was demonstrated for future new therapeutic drug experimental use.

Experimental radioimmunoguided surgery for peritoneal metastases of gastric cancer using anticarcinoembryonic antigen-specific T84.66 F(ab’)2

In gastric cancer, peritoneal dissemination is the most frequent cause of the noncurative resection and recurrence after curative resection. We therefore evaluated the feasibility of radioimmunoguided surgery (RIGS) in the treatment of peritoneal metastases of gastric cancer and the use of anti-CEA-specific T84.66 F(ab')2 as an efficient immune agent. Two human gastric cancer cell lines, MKN45 and RF48, were intraperitoneally xenografted into nude mice, which were later injected with 125I-labeled T84.66 F(ab')2. Peritoneal tumors were localized by RIGS 5 days after antibody injection. The minimum number of cells detected by a gamma probe was assayed by in vitro tumor cell localization. We observed 37 peritoneal metastases: 8 invisible (long diameter, <1 mm), 6 small (1- < 5 mm), and 23 large (> or =5 mm) tumors. The accuracy, sensitivity and specificity of RIGS in detecting peritoneal metastasis were 82% (69/84), 76% (28/37), and 87% (41/47), respectively. RIGS accuracy did not differ with respect to tumor diameter. Mean labeling indices over minimal and maximal normal counts were 6.1+/-1.2 (mean +/- SEM) and 4.7+/-1, respectively. Mean scores of CEA immunostaining and silver grains in tumors were significantly higher than those in the nontumor-bearing peritoneum (P < 0.001). There was a close correlation among radioactivity, immunostaining and microautoradiography (P < 0.001-0.005). We observed six false-positive and nine false-negatives which may have been due to high blood background and negative radioimmune reactivity, respectively. 125I-labeled T84.66 F(ab')2 efficiently targeted peritoneally disseminated gastric cancer cells, suggesting that RIGS using this immune agent may accurately detect occult peritoneal metastases in patients with gastric cancer.

Synthesis of alveolar bone Sharpey's fibers during experimental tooth movement in the rat

There is little information concerning the effects of tooth movement on the relative synthesis of bone matrix and Sharpey's fiber collagenous proteins. The purpose of this study was to investigate this situation using radioautographic techniques. The maxillary first molar tooth in rats was tipped toward the midline using an appliance and the animals were injected with 3H-proline after 3 days and sacrificed 24 hr later. Maxillae were sectioned and silver grain proportional areas (grain density/5,000 microm2) evaluated over Sharpey's fibers and adjacent alveolar bone matrix using computerized densitometry and histomorphometric techniques. These data were compared to a group of untreated animals by Fisher's exact test. At depository surfaces of experimental tissues, the silver grain proportional area over bone matrix was significantly greater than over Sharpey's fibers (P<0.05) and control bone matrix (P<0.01). The silver grain proportional area over Sharpey's fibers was not different between the groups. At resorptive surfaces, the silver grain proportional area over both bone matrix and Sharpey's fibers was significantly greater in experimental tissues compared to controls (P<0.01). Thus, movements of adjacent teeth affect both the quantity and ratios of collagenous protein incorporation into Sharpey's fibers and adjacent alveolar bone, which is dependent on the intensity and characteristics of the force.

Divergent glial fibrillary acidic protein and its mRNA in the activated supraoptic nucleus

Previous studies have shown decreased immunoreactive glial fibrillary acidic protein (GFAP) in the supraoptic nucleus (SON) when magnocellular neuroendocrine cells (MNCs) are activated by lactation or dehydration. This is thought to underlie structural plasticity of glial processes that occurs during these times. Here, we investigated how this apparent reduction in protein relates to GFAP mRNA expression in the dehydrated rat as visualized by in situ hybridization. Densitometry of silver grains in the SON revealed low levels of mRNA expression in control, 2-day dehydrated and 21-day rehydrated (R21) animals. Conversely, the SON from 7-day dehydrated (D7) subjects displayed significantly more silver grains. Thus, the pattern of GFAP mRNA expression is the inverse of what we previously observed for GFAP immunoreactivity in tissue sections of the SON. No differences in mRNA levels due to hydration state were seen in the lateral hypothalamic area, suggesting that increases in GFAP mRNA at D7 were specifically related to MNC activation. These data indicate a divergence in GFAP mRNA and protein expression in the SON.

Nanoleakage within the hybrid layer: A correlative FEISEM/TEM investigation

The aim of this study was to compare the nanoleakage patterns of the resin-dentin interfaces of three dentin bonding systems at both TEM and field emission in lens SEM (FEI-SEM) levels. A standardized smear layer was created with 180-grit silicon carbide paper (SiC) on dentin disks obtained from 18 noncarious human third molars. Specimens were randomly divided into three groups and bonded with a two-step total etching adhesive (Single Bond, SB), a two-step, self-etching adhesive (Clearfil SE BOND, SEB), and a one-step, self-etching adhesive (XENO III, XEIII). Nanoleakage was evaluated by using an ammoniacal silver-nitrate solution. Specimens were processed for TEM and FEI-SEM observation. The TEM of SB revealed silver deposits in adhesive and hybrid layers (HL). High-magnification FEI-SEM micrographs clearly identified these deposits as spherical clusters mainly associated with nonembedded collagen fibrils. TEM and FEI-SEM examination of SEB revealed some clusters of silver deposits within porosities and small channels of the HL. Additional silver deposits were observed between the peritubular dentin walls and the resin tags. XEIII revealed very fine and diffuse silver grains throughout the entire HL. SEM visualization of nanoleakage at a high level of resolution has not been previously described. FEI-SEM technology supported the TEM visualization with three-dimensional morphological data of the relations between the HL constituents and nanoleakage. The results of the present study confirm the hypothesis that both total- and self-etch adhesives are not able to fully infiltrate the dentin substrate.

Distribution of L1cam mRNA in the adult mouse brain: In situ hybridization and Northern blot analyses

Previous immunohistochemical analysis revealed a wide distribution of L1cam-positive neural and nonneural structures in adult mouse brain. Although there were numerous punctate immunoreactive nerve terminals, only a few immunoreactive neuronal cell somata were present (Munakata et al. [2003] BMC Neurosci. 4:7). To explore the distribution of L1cam mRNA-containing cells, which are interpreted to be L1cam-producing cells, we performed in situ hybridization histochemistry with an antisense L1cam cRNA probe. L1cam mRNA was distributed widely from the olfactory bulb to the upper cervical cord with an uneven localization pattern in adult brain. All positive cell somata with silver grains after emulsion autoradiography were neuronal, and no grains were detected on nonneural cells in the present study. A high density of signals for neuronal L1cam mRNA was found in the thalamus, mammillary body, and hippocampus. In addition, strong hybridization signals were localized in various nuclei: main and accessory olfactory bulb, compact part of the substantia nigra, pontine gray matter, tegmental reticular nucleus, Edinger-Westphal nucleus, trigeminal motor nucleus, locus coeruleus, mesencephalic trigeminal nucleus, raphe nuclei, facial nucleus, ambiguus nucleus, dorsal motor vagal nucleus, and inferior olivary nucleus. Some long projection neurons such as the pyramidal, mitral, principal neurons of several cranial nuclei, and presumably monoaminergic cells containing noradrenalin, dopamine, and serotonin, expressed high levels of L1cam.

Distribution of rDNA in the nucleus of Giardia lamblia: detection by Ag-I silver stain

Previous investigations have proved that diplomonads have primitive cell nuclei and lack a nucleolus. We determined the distribution of ribosomal DNA (rDNA) in diplomonad nuclei that lacked a nucleolus. Giardia lamblia was used as the experimental organism with Euglena gracilis as the control. The distribution of rDNA was demonstrated indirectly by the modified Ag-I silver technique that can indicate specifically the nucleolus organizing region (NOR) by both light and electron microscopy. In ultrathin sections of silver stained Euglena cells, all silver grains were concentrated in the fibrosa of the nucleolus, while no silver grains were found in the cytoplasm, nucleoplasm, condensed chromosomes or pars granulosa of the nucleus. In the silver stained Giardia cells, no nucleolus was found, but a few silver grains were scattered in the nucleus. This suggests that the rDNA of Giardia does not form an NOR-like structure and that its nucleus is in a primitive state.

Cell proliferation in the gastric epithelium of the ulcer rat

Objective. Cell division is brisk in the ulcer margin and many of the new cells will migrate over and cover the ulcer bed. The aim of this study was to determine how agents that promote or delay gastric ulcer healing influence cell proliferation in the gastric epithelium. Material and methods. Acetic acid ulcers were produced in the rat gastric corpus; non-ulcer rats served as controls. All rats were given a continuous infusion of 3H-thymidine. Some rats were also given gastrin or indomethacin, or infected with Helicobacter pylori. The rats were killed after 1, 2, 6 or 13 days, and the ulcer margin and undamaged corpus were excised for determination of labeling index (LI) by autoradiography. Antrum, duodenum and colon were also studied. Silver grain counting was carried out in some groups. Results. LI in the ulcer margin grew exponentially, reaching 84% after 6 days; gastrin increased, and indomethacin decreased LI significantly. In 6-day ulcer rats that were given 3H-thymidine only during the first day LI was 5%, while in those given 3H-thymidine only during the last day LI was 27%. LI and silver grain counting results indicated that during the first 6 days of healing the epithelial cells in the ulcer margin divide twice. In the undamaged epithelium of the 1-day ulcer rats LI was <one-third, and in the antral epithelium one-half of that in the non-ulcer controls. Conclusions. During the first 6 days of healing, the LI of the non-parietal epithelial cells increases exponentially in the ulcer margin; this is enhanced by gastrin. The unexpected finding in the ulcer rats of decreased LI in undamaged oxyntic and antral epithelium may be caused by a block of the G1 restriction point in the cell cycle. The reason for this remains obscure.

Improvement of Digital Print in Preference by Adding Noise

Development of the digital imaging technology is remarkable. Combination of high-resolution digital camera with high performance ink-jet printer results in noiseless prints. On the other hand, with disappearance of noise, we lost impression of space and texture which we get from complicated 3D structure of silver grains or dye clouds. In the preceding paper, we showed that adding white noise to some type of picture like wood, cloud, etc. results in improvement increase of scores of preference. In the present paper, it was attempted to improve digital prints in preference by adding white, blue, or 1/f noise.

High quality silver contacts on ceramic superconductors obtained by electrodeposition fromnon-aqueous solvents

Contacts on high temperature superconductors have been fabricated using an electrodepositionprocess in non-aqueous media. This technique allows obtaining specific resistance values lower than10−9 Ω cm2 in a reproducible way. We have studied the differences in the silver grain morphology andmetallic coating homogeneity depending on different electrodeposition parameters, such asdeposition current density or different shaking techniques. The best results are obtained ifthe deposition process takes place in as-grown samples, before performing therequired annealing to the superconducting material. The comparison betweenthe predictions of the metallic layer thickness and the measured values indicatesthat the process is highly efficient and that the silver coating has a high density.

Low friction silver-DLC coatings prepared by thermionic vacuum arc method

By using a new type of plasma discharge, thermionic vacuum arc (TVA), silver diamond-like carbon (DLC) composite overlays were prepared successfully for tribological applications. Carbon was incorporated into the silver matrix at concentration between 19 and 42 mass% as a function of the distance to the two TVA guns used to evaporate simultaneously silver grains and a graphite rod. It was found that the surface of the obtained overlays was smooth (5–10nm peak-to-valley roughness) as measured by atomic force microscope (AFM) in contact mode. Carbon inclusion as DLC phase in the silver matrix reduced the coefficient of friction in dry sliding up to 2.5 times compared to that of the bronze substrate.

Sex-linkage of the chicken ornithine transcarbamylase gene.

The chicken ornithine transcarbamylase gene has been mapped to the short arm of the Z chromosome, near the centromere. Radioactive in situ hybridization was performed using a human cDNA clone, and chromosomes were scored for silver grain accumulation. This represents the first documented case of a mammalian X-linked gene also being sex-linked in an avian species. The significance of this finding in the context of sex chromosome evolution is briefly discussed. Further, differential sex-related OTC expression, whereby females (ZW) consistently produce higher levels of the enzyme than males (ZZ), must imply a peculiar mode of OTC transcription regulation.

Expression of hyperpolarization-activated and cyclic nucleotide-gated cation channel subunit 2 in axon terminals of peptidergic nociceptive primary sensory neurons in the superficial spinal dorsal horn of rats.

Hyperpolarization-activated cyclic nucleotide-gated cation channel proteins (HCN1-4), which are potentially able to modulate membrane excitability, are abundantly expressed by neurons in spinal dorsal root ganglia (DRG). In the present experiment, we investigated whether HCN2 protein is confined exclusively to the perikarya of DRG neurons or is transported from the somata to the central axons of DRG neurons that terminate in the spinal dorsal horn. Using immunohistochemical methods, we have demonstrated that laminae I-IIo of the superficial spinal dorsal horn of the adult rat spinal cord show a strong punctate immunoreactivity for HCN2. Dorsal rhizotomy resulted in a complete loss of immunostaining in the dorsal horn, suggesting that HCN2 is confined to axon terminals of primary afferents. In double labelling immunohistochemical studies, we have also shown that HCN2 widely co-localizes with calcitonin gene-related peptide, but is almost completely segregated from isolectin-B4 binding, indicating that HCN2 is primarily expressed in peptidergic nociceptive primary afferents. The expression of HCN2 in central terminals of peptidergic primary afferents was also verified with electron microscopy. Utilizing the pre-embedding nanogold method, we found that HCN2 is largely confined to axon terminals with dense-core vesicles. Within these terminals, some of the silver grains marking the accurate location of HCN2 molecules were associated with the cell membrane, and others were scattered in the axoplasm. Within the cell membrane, HCN2 was found almost exclusively in extrasynaptic locations. The results suggest that HCN2 may contribute to the modulation of membrane excitability of nociceptive primary afferent terminals in the spinal dorsal horn.

Electron microscopic radioautographic study on protein synthesis in amitotic hepatocytes of the aging mouse.

Ten groups of aging mice, each consisting of three individuals, from fetal day 19 to postnatal month 24, were injected with (3)H-leucine and killed 1 h later; the livers were processed for light and electron microscopic radioautography. On radioautograms obtained from each animal, amitotic nuclear divisions and resulting binucleate hepatocytes were detected and compared to mononucleate hepatocytes. From the results, it was demonstrated that only a few hepatocytes showing amitotic nuclear divisions were found labeled with the precursor demonstrating protein synthesis. However, the numbers of silver grains showing incorporations of labeled precursors in respective amitotic cells were very few. It was clarified that the amitotic cells did not synthesize such macromolecules as mononucleate hepatocytes did. On the other hand, more binucleate cells were found than amitotic cells. Protein synthesis in karyoplasm and cytoplasm in both mononucleate and binucleate cells increased from the perinatal stage, reaching the maxima at adult stage, then decreased to the senescent stage. Grain counts revealed that synthesized proteins were more abundant in binucleate cells than in mononucleate cells at the respective aging stages.

Raman scattering from nanocrystalline Ag compacted under different pressures

Nanocrystalline silver samples compacted under different pressures have been studied by X-ray diffraction and Raman spectroscopy. X-ray photoelectron spectroscopy results of the polished nanocrystalline silver samples suggest the formation of silver dioxide at the interfaces. Raman spectra of the as-polished nanocrystalline silver samples exhibit a weak band at about 240 cm−1, which can be attributed to surface-enhanced bands originating from the silver dioxide dispersed at the surface of the silver grains. When the compacting pressure is increased, the intensity of the Raman peak at about 240 cm−1 decreases due to decreasing silver dioxide in the interfaces. Also, the peak exhibits a frequency shift upwards which has been attributed to a molecular contraction, i.e. residual stress developed during the pressing of the powders. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)