Biomedical subjects

Meredin Stoltenberg

Publications and source records attributed to Meredin Stoltenberg.

17 recordsLinked to original sources

PubMed2006

Silver enhancement of quantum dots resulting from (1) metabolism of toxic metals in animals and humans, (2) in vivo, in vitro and immersion created zinc-sulphur/zinc-selenium nanocrystals, (3) metal ions liberated from metal implants and particles.

Autometallographic (AMG) silver enhancement is a potent histochemical tool for tracing a variety of metal containing nanocrystals, e.g. pure gold and silver nanoclusters and quantum dots of silver, mercury, bismuth or zinc, with sulphur and/or selenium. These nanocrystals can be created in many different ways, e.g. (1) by manufacturing colloidal gold or silver particles, (2) by treating an organism in vivo with sulphide or selenide ions, (3) as the result of a metabolic decomposition of bismuth-, mercury- or silver-containing macromolecules in cell organelles, or (4) as the end product of histochemical processing of tissue sections. Such nano-sized AMG nanocrystals can then be silver-amplified several times of magnitude by being exposed to an AMG developer, i.e. a normal photographic developer enriched with silver ions. The present monograph attempts to provide a review of the autometallographic silver amplification techniques known today and their use in biology. After achieving a stronghold in histochemistry by Timm's introduction of the "silver-sulphide staining" in 1958, the AMG technique has evolved and expanded into several different areas of research, including immunocytochemistry, tracing of enzymes at LM and EM levels, blot staining, retrograde axonal tracing of zinc-enriched (ZEN) neurons, counterstaining of semithin sections, enhancement of histochemical reaction products, marking of phagocytotic cells, staining of myelin, tracing of gold ions released from gold implants, and visualization of capillaries. General technical comments, protocols for the current AMG methods and a summary of the most significant scientific results obtained by this wide variety of AMG histochemical approaches are included in the present article.

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PubMed2005

Gene expression changes induced by bismuth in a macrophage cell line.

We have investigated the effect of bismuth by autometallography, cell viability, TUNEL assay and microarray analysis of a macrophage cell line. The cells accumulate bismuth in their lysosomes in a time- and dose-dependent manner. Cell viability assays show a significant decrease in the number of viable cells related to both bismuth concentrations and exposure time. TUNEL assays after 12 h and 24 h at a bismuth-citrate concentration of 50 microM revealed the presence of 30% and 70% TUNEL-positive cells, respectively, compared with 8% in the controls. We have analysed gene expression profiles for cells exposed to 50 microM bismuth-citrate and for untreated controls at 12 h and 24 h by microarray analysis, which confirmed that bismuth is a powerful metallothionein inducer. A number of glycolytic enzymes are induced by bismuth, suggesting that bismuth is able to induce "hypoxia-like" stress. BCL2/adenovirus E1B 19-kDa-interacting protein 3 (Bnip3) has been suggested as a regulator of hypoxia-induced cell death independent of caspase-3 activation and cytochrome c release. Bnip3 is up-regulated indicating the involvement of Bnip3 as a possible mechanism for bismuth-induced cell death. Differences have been noticed in cell viability and in the modification of the mRNA expression levels at 12 and 24 h. Only 13 genes are modified at both these times, suggesting a time-dependent molecular cascade in which bismuth-exposed cells enter a dormant stage with mRNA down-regulation being followed by cell death of susceptible cells.

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PubMed2005

Abundant expression of zinc transporters in Bergman glia of mouse cerebellum.

Zinc transporters (ZnTs) are membrane proteins involved in zinc ion transportation in mammalian cells. Seven members of ZnT family, ZnT1-7, have been cloned and characterized. These transporter proteins have different cellular and sub-cellular locations, suggesting that they may play different roles in zinc homeostasis in normal and pathological conditions in different tissues. Cerebellum is one of the most zinc-enriched regions in the central nervous system, but little is known about zinc metabolism in the cerebellum. In the present study, we investigated the detailed distributions of four members (ZnT1, ZnT3, ZnT4 and ZnT6) of the ZnT family, in the mouse cerebellum. Immunostaining and confocal microscopic observations revealed a similar staining pattern of ZnTs in the molecular layer and the Purkinje cell layer. Double labeling with anti-S-100beta or anti-MAP2 and anti-ZnTs clearly showed that the Bergman glial cell bodies in the Purkinje cell layer and their radial processes in the molecular layer exhibited strong immunofluorescence of all the tested ZnTs. However, the somata of the Purkinje cells contained a moderate immunostaining for ZnT1, but virtually lack of other three ZnTs. In the granular layer, ZnTs appeared with different immunostaining patterns. ZnT1 was expressed in a small number of neuronal cell bodies and their primary dendrites, whereas ZnT3 and ZnT4 were present in nerve terminals but not in the neuronal somata. ZnT6 was undetectable in either the cell bodies or processes in the granular layer. The present results indicate that the Bergman glial cells may play an important role in zinc metabolism in the mouse cerebellar cortex.

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PubMed2005

In vivo distribution of bismuth in the mouse brain: influence of long-term survival and intracranial placement on the uptake and transport of bismuth in neuronal tissue.

Bismuth is used for a multitude of industrial purposes and has partly replaced toxic heavy metals such as lead and mercury in e.g. lubricants and shotgun pellets. In medicine, bismuth-compounds have long been used to remedy gastrointestinal disorders; lately in combination with antibiotics to treat Helicobacter pylori associated peptic ulcers. An epidemic episode of bismuth-induced encephalopathy in France in the 1970s revealed the neurotoxic potential of bismuth. This incidence, involving almost 1000 patients, remains unexplained and the contribution of other factors besides bismuth has been postulated. Recently an autometallographic technique made it possible to detect bismuth in morphologically intact tissue. In the present study, autometallographicly detectable bismuth was seen throughout the brain following intraperitoneal and intracranial exposure. The neuronal staining pattern seems highly organized with some areas heavily stained and others with low or no staining. Long-term (8 months) intraperitoneal exposure led to higher bismuth uptake than short-term (2 weeks) exposure. Following both intraperitoneal and intracranial exposure, high amounts of bismuth were found in the reticular and hypothalamic nuclei, in the oculomotor and hypoglossal nuclei and in Purkinje cells. Within the central nervous system (CNS) retrograde axonal transport was seen after intracranial bismuth exposure. Axonal transport seems to influence the distribution of bismuth as the highest uptake of bismuth after intraperitoneal exposure was seen in the facial and the trigeminal motor nuclei, i.e. neurones with processes outside the blood-brain barrier, whereas these nuclei contained no bismuth following ic exposure. Ultrastructurally, accumulation of bismuth was seen in lysosomes.

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PubMed2005

Zinc-regulating proteins, ZnT-1, and metallothionein I/II are present in different cell populations in the mouse testis.

Zinc ions play an important role in testis development and spermatogenesis. Thus, nutritional zinc deficiency leads to aberrant testicular development, reduced spermatogenesis, and male sterility. The precise actions of zinc in mediating these functions and the mechanisms by which zinc is itself regulated in the testis, however, have not been adequately elucidated. We have assessed the distribution of the zinc-regulating proteins ZnT-1 and metallothionein I/II (MT I/II) in the mouse seminiferous tubule. Co-labeling for ZnT-1 and MT I/II demonstrated unique patterns of distribution for these proteins, with ZnT-1 present in Sertoli cells in addition to luminal spermatozoa and MT I/II restricted to spermatocytes. These findings were confirmed by dual-label immunofluorescence for ZnT-1 and the Sertoli cell marker, vimentin, and by immunoelectron microscopy. The differential expression patterns of ZnT-1 and MTs support the hypothesis that ZnT-1 and MTs play different roles in the regulation of intracellular zinc in this organ. The specific expression of ZnT-1 in the Sertoli cells, moreover, is consistent with their role in maintaining a nurturing, closely regulated environment for spermatogenesis.

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PubMed2005

Zinc-specific autometallographic in vivo selenium methods: tracing of zinc-enriched (ZEN) terminals, ZEN pathways, and pools of zinc ions in a multitude of other ZEN cells.

In vivo-applied sodium selenide or sodium selenite causes the appearance of zinc-selenium nanocrystals in places where free or loosely bound zinc ions are present. These nanocrystals can in turn be silver enhanced by autometallographic (AMG) development. The selenium method was introduced in 1982 as a tool for zinc-ion tracing, e.g., in vesicular compartments such as synaptic vesicles of zinc-enriched (ZEN) terminals in the central nervous system, and for visualization of zinc ions in ZEN secretory vesicles of, e.g., somatotrophic cells in the pituitary, zymogene granules in pancreatic acinar cells, beta-cells of the islets of Langerhans, Paneth cells of the crypts of Lieberkühn, secretory cells of the tubuloacinar glands of prostate, epithelium of parts of ductus epididymidis, and osteoblasts. If sodium selenide/selenite is injected into brain, spinal cord, spinal nerves containing sympathetic axons, or intraperitoneally, retrograde axonal transport of zinc-selenium nanocrystals takes place in ZEN neurons, resulting in accumulation of zinc-selenium nanocrystals in lysosomes of the neuronal somata. The technique is, therefore, also a highly specific tool for tracing ZEN pathways. The present review includes an update of the 1982 paper and presents evidence that only zinc ions are traced with the AMG selenium techniques if the protocols are followed to the letter.

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PubMed2004

Dynamic zinc pools in mouse choroid plexus.

We examined the presence of Zn-transporters (ZnT1, ZnT3, ZnT4, and ZnT6) proteins and zinc ions in rat choroid epithelium with immunohistochemistry and zinc selenide autometallography (ZnSe(AMG)). The four ZnT proteins were all expressed in the choroid epithelial cells. ZnT3 immunostaining was found in vesicle membranes in the apical part of the cells, associated to the microvillus membrane. Correspondingly, the ZnSe(AMG) technique revealed zinc ions in small vesicles, in microvilli, and multivesicular bodies in the epithelial cells. Traceable zinc ions were also found in lysosome-like organelles of fenestrated endothelial cells, but here no corresponding ZnT3 immunostaining was seen. The observations suggests that the choroid plexus is instrumental to regulation of the level of zinc ions in the cerebrospinal fluid.

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PubMed2004

Abundance of zinc ions in synaptic terminals of mocha mutant mice: zinc transporter 3 immunohistochemistry and zinc sulphide autometallography.

The mocha mouse is an autosomal recessive pigment mutant on mouse chromosome 10 caused by a deletion in the gene for the delta subunit of the adaptor-like complex AP-3. Based on zinc transporter 3 (ZnT3) immunohistochemistry, zinc TSQ fluorescence and a modified Timm method, previous studies found a lack of histochemically-detectable zinc and a substantial reduction in the ZnT3 immunoreactivity. It has, therefore, been suggested that the mocha mouse could serve as a model for studies of the significance of zinc ions in zinc-enriched (ZEN) neurons. We have chosen the mocha-zinc-model in a study of the significance of ZEN neurons in hypoxia-caused damage in mouse brain. In order to establish that the model was either void of zinc ions or had a significantly decreased level of zinc ions in their ZEN terminals, we repeated the studies that had lead to the above assumption, the only methodology difference being that we used the zinc specific Neo-Timm method instead of the Timm method applied in the original study. We found that, although the ZnS autometallography (AMG) technique revealed a reduction in staining intensity as compared to the littermate controls, there were still plenty of zinc ions in the ZEN terminals, in particular visible in telencephalic structures like neocortex and hippocampus. At ultrastructural levels the zinc ions were found in a pool of vesicles of the ZEN terminals as in the control animals, but additionally zinc ions could be traced in ZEN neuronal somata in the neocortex and hippocampus. The mossy fibres in the hippocampus of mocha mice also bind with TSQ, though less than in the controls. We found ZnS AMG grains in ZEN neuronal somata, which were also immunoreactive for ZnT3. Our study confirmed the decreased ZnT3 immunoreactivity in ZEN terminals of the mocha mouse found in the original study. Based on these findings, we suggest that the mocha mouse may not be an ideal model for studies of the histochemically-detectable zinc ion pool of the central nervous system.

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PubMed2004

Immersion autometallography: histochemical in situ capturing of zinc ions in catalytic zinc-sulfur nanocrystals.

In the mid-1980s, two versions of Timm's original immersion sulfide silver method were published. The authors used immersion of tissue in a sulfide solution as opposed to Timm, who used immersion of tissue blocks in hydrogen sulfide-bubbled alcohol. The autometallography staining resulting from the "sulfide only immersion" was not particularly impressive, but the significance of this return to an old approach became obvious when Wenzel and co-workers presented their approach in connection with introduction by the Palmiter group of zinc transporter 3 (ZnT3). The Wenzel/Palmiter pictures are the first high-resolution, high-quality pictures taken from tissues in which free and loosely bound zinc ions have been captured in zinc-sulfur nanocrystals by immersion. The trick was to place formalin-fixed blocks of mouse brains in a solution containing 3% glutaraldehyde and 0.1% sodium sulfide, ingredients used for transcardial perfusion in the zinc-specific NeoTimm method. That the NeoTimm technique results in silver enhancement of zinc-sulfur nanocrystals has been proved by proton-induced X-ray multielement analyses (PIXE) and in vivo chelation with diethyldithiocarbamate (DEDTC). The aims of the present study were (a) to make the immersion-based capturing of zinc ions in zinc-sulfur nanocrystals work directly on sections and slices of fixed brain tissue, (b) to work out protocols that ensure zinc specificity and optimal quality of the staining, (c) to apply "immersion autometallography" (iZnSAMG) to other tissues that contain zinc-enriched (ZEN) cells, and (d) to make the immersion approach work on unfixed fresh tissue.

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PubMed2004

Bismuth uptake in rat testicular macrophages: a follow-up observation suggesting that bismuth alters interactions between testicular macrophages and Leydig cells.

Recent studies suggest that bismuth accumulates in Leydig cells. In addition, a reduced level of serum testosterone and a statistically significant reduction of Leydig cells have been observed. It was therefore hypothesized that Bi has a direct toxic effect on rat Leydig cells. We have now developed a method for double labeling of bismuth and ED-2 (a marker for testicular macrophages). The present data demonstrate that the heavily bismuth-loaded cells in rat testis, originally interpreted as being Leydig cells, are bismuth-loaded macrophages. Consequently, our data suggest a modified hypothesis regarding bismuth-induced interactions between testicular macrophages and Leydig cells.

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PubMed2003

Autometallographic tracing of mercury in pilot whale tissues in the Faroe Islands.

OBJECTIVES: Autometallography (AMG) was applied for tracing mercury in long-finned pilot whales (Globicephala melas) harvested in the Faroe Islands. RESULTS: Ample mercury accumulation was found in kidney tubules, in contrast to the largely unstained glomeruli. Hepatocytes, as well as liver macrophages, exhibited high mercury uptake. The muscle tissue accumulated only scant amounts of mercury, primarily around the nuclei of the striated muscle cells. At the ultrastructural level, mercury was found to accumulate intracellularly in lysosomes, and extracellularly in the basement membranes of vessels. The results were verified by proton induced X-ray emission (PIXE) analysis, and it was established that the tissue contained no other AMG traceable metals. CONCLUSION: The use of AMG analysis on autopsies and biopsies is suggested as a tool for evaluating mercury pollution.

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PubMed2003

Gastrointestinal and systemic uptake of bismuth in mice after oral exposure.

Bismuth compounds have been used in medicine for more than 200 years. In recent years, bismuth has gained renewed interest as a remedy for eradication of gastrointestinal pathogens, especially Helicobacter pylori. In this study we describe the anatomical distribution of bismuth in the gastrointestinal tract and other organs after oral exposure in a mouse model. After exposure of the experimental animals to ranitidine bismuth citrate or bismuth citrate, we used the autometallographic silver enhancement technique to demonstrate the presence of bismuth in tissue samples from the gastrointestinal tract, liver, spleen, thymus, kidney and lymph nodes. We exposed cultured murine peritoneal macrophages to bismuth citrate and examined the bismuth accumulation over time. We found that in the mouse bismuth is absorbed systemically after a single dose of either compound, ranitidine bismuth more easily than bismuth citrate. Uptake could be shown in the stomach, duodenum, ileum and kidney for hours after exposure. Weeks after the exposure, deposits of bismuth were found in lymph nodes, liver, spleen and kidney as well as in macrophages in the gastrointestinal lamina propria. At the subcellular level, bismuth was found exclusively in lysosomes, primarily in macrophages and dendritic cells. Subsequent analyses of macrophage cultures showed lysosomal accumulations to be time and dose dependent.

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PubMed2002

Bismuth-induced neuronal cell death in rat dorsal root ganglion: a stereological study.

The goal of the present study has been to quantify the morphological changes in myelinated nerve roots and dorsal root ganglion (DRG) cells in rats exposed to bismuth subnitrate. Male Wistar rats (n =30) were divided into three groups of ten animals. The ten animals in each group were given intraperitoneal injections of one of the following: (1) 500 mg/kg bismuth subnitrate, (2) 1,000 mg/kg bismuth subnitrate, or (3) saline. The mean total cell number of B-cells in the DRG was significantly smaller in the two treated groups, 18% (2P <0.001) and 23% (2P <0.001), respectively, than it was in the control group. In addition, there was an 11% reduction in the number of A-cells (2P =0.039) in rats exposed to the highest concentration of bismuth. Bismuth did not affect the total number or mean cross sectional area of axons and myelin sheaths of the myelinated nerve fibers in the ventral or dorsal nerve root of the DRG. This is the first study to investigate pathological changes of the peripheral nervous system after bismuth intoxication.

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PubMed2002

Bismuth tracing in organotypic cultures of rat hippocampus.

Bismuth is known to have neurotoxic side effects in humans and animals. In the 1970s France experienced about a thousand cases of patients suffering from bismuth-induced encephalopathy. Studies suggest that bismuth may provoke a selective degeneration of CA1 pyramidal cells in the organotypic cultures of rat hippocampus. A currently established technique for the histochemical visualization of bismuth was applied on hippocampal tissue cultures allowing the tracing of bismuth in concentrations hitherto not possible. The accumulation and subcellular localization of bismuth is demonstrated in the tissue cultures of rat hippocampus. CA1 pyramidal cells in the rat hippocampus exhibit the highest uptake of bismuth. High bismuth citrate concentrations (10 microM) are able to totally destroy the cytoarchitecture of the hippocampus. At ultrastructural levels bismuth was found to be located exclusively in lysosome-like organelles.

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PubMedDate not supplied

Influence of bismuth on the number of neurons in cerebellum and hippocampus of normal and hypoxia-exposed mouse brain: a stereological study.

The industrial use of bismuth is increasing. In medicine, bismuth compounds have long been used in the treatment of gastrointestinal disorders, recently in combination with antibiotics for the treatment of Helicobacter pylori-associated peptic ulcers. Bismuth-induced encephalopathy is a known side-effect. One of the symptoms of bismuth encephalopathy is ataxia, suggesting possible cerebellar involvement. The introduction of autometallography (AMG) for tracing BiS/BiSe nanocrystals has provided histochemical evidence supporting the cerebellum being involved in bismuth encephalopathy, but the effect of bismuth on the neuron number in the cerebellum has never been evaluated. In vitro studies have indicated that CA1 neurons may be targets for bismuth intoxication, but results have been conflicting. Recently, the loss of dorsal root ganglion cells was reported after moderate bismuth exposure. This raises the question whether the use of another neurotoxic stimulus, such as hypoxia, amplifies the toxic effects of bismuth. Despite AMG-detectable bismuth accumulations, stereological examinations revealed no statistically significant decrease in the number of Purkinje, CA1 or CA3 neurons or in the volume of the cerebellar granule layer. Surprisingly, intermittent hypoxia led to a statistically significant loss of Purkinje cells without affecting the hippocampus. Bismuth neither ameliorated nor exacerbated the hypoxic effects on the cerebellum.

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PubMedDate not supplied

Decreased serum testosterone levels in rats exposed intraperitoneally to bismuth subnitrate.

The consumption of bismuth is increasing and knowledge of the potential teratogenic and reproductive damage of bismuth exposure is fragmentary. In the present study we used autometallography (AMG)--the histochemical silver amplification technique--to trace bismuth in the testis and pituitary glands of Wistar rats injected intraperitoneally with bismuth subnitrate. Large amounts of bismuth AMG grains were concentrated in the lysosomes of Leydig cells, and serum testosterone levels were reduced when compared with controls. No histochemical traces of bismuth were found in the anterior lobe of the pituitary gland. Compared with their corresponding controls, neither follicle-stimulating hormone nor luteinizing hormone were affected. The selective uptake of bismuth in Leydig cells, followed by decreased testosterone levels, emphasizes a potential hazard of bismuth-provoked male reproductive impairment.

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PubMedDate not supplied

Leydig cell death in rats exposed to bismuth subnitrate.

In a previous study, rats were treated with one injection of 500 mg kg(-1) bismuth subnitrate intraperitoneally and maintained for 2 weeks. Bismuth was observed in Leydig cells, with a subsequent reduction in serum testosterone levels. In the present study, stereological procedures were used to estimate the number of Leydig cells in the right testis from the retained rats used in the previously published study. The mean number of Leydig cells in the control group was estimated to be 18.7 x 10(6) (coefficient of variation = 0.11), which is comparable to previous estimations. In the group exposed to bismuth the mean was 15.5 x 10(6) (coefficient of variation = 0.18). The observed 17% difference between the two groups was statistically significant (2P = 0.019). The inter-individual variation was largest in the bismuth-exposed group. Testis weight and body weight were not significantly reduced after bismuth exposure. These findings support the hypothesis that bismuth has a direct toxic effect on rat Leydig cells and underscores the potential risks of bismuth on male reproduction.

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