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.     

Autometallographic tracing of quantum dots

A short clarifying view of how semiconductor quantum dots (QDs) can be made visible in tissue sections by autometallographic (AMG) silver enhancement and how the introduction of AMG enhanceable gold nanoparticles into isolated cells can be used to follow the fate of these marked cells in organisms and cell cultures. As the AMG approach for visualizing quantum dots is extremely sensitive, QDs less than one nanometer can be made visible at both LM and EM levels.     

Lack of plasma protein hemopexin dampens mercury-induced autoimmune response in mice

Several factors affect the autoimmune response, including iron-dependent modulation of T cells. Hemopexin is the plasma protein with the highest binding affinity to heme. It mediates heme-iron recovery in the liver, thus controlling heme-iron availability in peripheral cells. The aim of the present study was to investigate the role of hemopexin in the progress of an autoimmune response. To this end, we chose a mouse model of mercury-induced autoimmunity and evaluated the susceptibility of hemopexin-null mice to mercury treatment compared with wild-type controls. In this study we show that lack of hemopexin dampens mercury-induced autoimmune responses in mice. Hemopexin-null mice produced fewer antinuclear autoantibodies and had reduced deposits of immune complexes in the kidney after mercuric chloride treatment compared with wild-type mice. These features were associated with a reduction in activated T cells and lower absolute B cell number in spleen and impaired IgG1 and IgG2a production. In contrast, in hemopexin-null mice the response to OVA/CFA immunization was maintained. In addition, hemopexin-null mice had reduced transferrin receptor 1 expression in T cells, possibly due to the increase in heme-derived iron. Interestingly, CD4(+)T cells isolated from mercury-treated hemopexin-null mice show reduced IFN-gamma-dependent STAT1 phosphorylation compared with that of wild-type mice. Our data suggest that hemopexin, by controlling heme-iron availability in lymphocytes, modulates responsiveness to IFN-gamma and, hence, autoimmune responses.     

Gold ions bio-released from metallic gold particles reduce inflammation and apoptosis and increase the regenerative responses in focal brain injury

Traumatic brain injury results in loss of neurons caused as much by the resulting neuroinflammation as by the injury. Gold salts are known to be immunosuppressive, but their use are limited by nephrotoxicity. However, as we have proven that implants of pure metallic gold release gold ions which do not spread in the body, but are taken up by cells near the implant, we hypothesize that metallic gold could reduce local neuroinflammation in a safe way. Bio-liberation, or dissolucytosis, of gold ions from metallic gold surfaces requires the presence of disolycytes i.e. macrophages and the process is limited by their number and activity. We injected 20-45 mum gold particles into the neocortex of mice before generating a cryo-injury. Comparing gold-treated and untreated cryolesions, the release of gold reduced microgliosis and neuronal apoptosis accompanied by a transient astrogliosis and an increased neural stem cell response. We conclude that bio-liberated gold ions possess pronounced anti-inflammatory and neuron-protective capacities in the brain and suggest that metallic gold has clinical potentials. Intra-cerebral application of metallic gold as a pharmaceutical source of gold ions represents a completely new medical concept that bypasses the blood-brain-barrier and allows direct drug delivery to inflamed brain tissue.     

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.     

Histochemical localization of zinc ions in the epididymis of the rat

Summary In the present study, the autometallograpic zinc sulphide technique, an improved version of the original Timm sulphide-silver method, was used. This technique reveals a particular pool of ionic zinc that is chelatable by diethyldithiocarbamate. At the light microscopical level, no reaction for zinc was found in tissues of young prepubertal rats. In adult mating and non-mating rats low zinc staining was found in the head and intermediate epididymis whereas the tail of the epididymis demonstrated high levels of zinc ions. Sections from the epididymal tail revealed a compartmentalization, based on pronounced differences in staining intensity along the epididymal ducts. At higher magnification zinc ions were found in the apical part of the principal cell and in the lumen. At the ultrastructural level autometallographic grains were located in vesicles and in lysosome-like structures of the apical parts of the principal cells. The luminal grains were found either associated with sperm cells, with the surface of the large microvilli (stereocilia), or free in the seminal fluid. The variation in content of zinc ions in the epididymal epithelium and lumen suggests that zinc ions are secreted into the lumen from the epididymal tail and may somehow be involved in maturation of the sperm cells.     

Zinc fluxes during acute and chronic exposure of INS-1E cells to increasing glucose levels.

Zinc in beta-cell secretory vesicles is essential for insulin hexamerization, and tight vesicular zinc regulation is mandatory. Little is known about zinc ion fluxes across the secretory vesicle membrane and the influence of changes in the extracellular environment on vesicular zinc. Our study aim was to investigate the effect of acute and chronic exposure to various glucose concentrations on zinc in secretory vesicles, the relation between zinc and insulin, and the presence of two zinc transporters, ZnT1 and ZnT4, in INS-1E cells. Zinc ions were demonstrated and semi-quantified using zinc-sulfide autometallography. Insulin content and secreted insulin were measured. Measurements were made on INS-1E cells after exposure to 2.0, 6.6, 16.7, and 24.6 mmol/l glucose for 1, 24, and 96 hours. 1h: Increasing glucose resulted in no changes in intravesicular zinc ions at 2, and 24.6 mmol/l glucose, but a slight increase at 16.7 mmol/l glucose. 24 and 96 h: Increasing glucose led to decreased vesicular zinc ion content accompanied by a decrease in insulin content. ZnT1 and ZnT4 were present in the cytoplasm. Our results demonstrate that intra-vesicular zinc ions respond to changes in the extra-cellolar glucose concentration, especially during chronic high glucose concentrations, where the content of vesicular zinc ions decreases.     

Metallic gold slows disease progression, reduces cell death and induces astrogliosis while simultaneously increasing stem cell responses in an EAE rat model of multiple sclerosis

Multiple sclerosis (MS) is the most common neurodegenerative disease in the Western world affecting younger, otherwise healthy individuals. Today no curative treatment exists. Patients suffer from recurring attacks caused by demyelination and underlying neuroinflammation, ultimately leading to loss of neurons. Recent research shows that bio-liberation of gold ions from metallic gold implants can ameliorate inflammation, reduce apoptosis and promote proliferation of neuronal stem cells (NSCs) in a mouse model of focal brain injury. Based on these findings, the present study investigates whether metallic gold implants affect the clinical signs of disease progression and the pathological findings in experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. Gold particles 20–45?μm suspended in hyaluronic acid were bilaterally injected into the lateral ventricles (LV) of young Lewis rats prior to EAE induction. Comparing gold-treated animals to untreated and vehicle-treated ones, a statistically significant slowing of disease progression in terms of reduced weight loss was seen. Despite massive inflammatory infiltration, terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed reduced apoptotic cell death in disease foci in the brain stem of gold-treated animals, alongside an up-regulation of glial fibrillary acidic protein-positive reactive astrocytes near the LV and in the brain stem. Cell counting of frizzled-9 and nestin-stained cells showed statistically significant up-regulation of NSCs migrating from the subventricular zone. Additionally, the neuroprotective proteins Metallothionein-1 and -2 were up-regulated in the corpus callosum. In conclusion, this study is the first to show that the presence of small gold implants affect disease progression in a rat model of MS, increasing the neurogenic response and reducing the loss of cells in disease foci. Gold implants might thus improve clinical outcome for MS patients and further research into the long-term effects of such localized gold treatment is warranted.     

In vivo cellular uptake of bismuth ions from shotgun pellets

Shotgun pellets containing bismuth (Bi) are widely used and may cause a rather intense exposure of some wild animals to Bi. A Bi shotgun pellet was implanted intramuscularly in the triceps surae muscle of 18 adult male Wistar rats. Another group of 9 animals had a Bi shotgun pellet implanted intracranially in the neocortex. Eight weeks to 12 months later the release of Bi ions was analysed by autometallography (AMG) of tissue sections from different organs (brain, spinal cord, kidney, liver, testes). In the group with intramuscular Bi shotgun pellets no AMG staining could be found for the first 2-4 months; 6 months after exposure Bi was traced in the kidney. Twelve months after the implantation the kidneys were heavily loaded and Bi was also traced in testosterone-producing Leydig cells, in glial cells and in neurons of brain and spinal cord. In the central nervous system (CNS) motor neurons were the most loaded. In rats with intracranially implanted shotgun pellets a massive uptake of Bi was observed involving both glia and neurons throughout the brain. The cells close to the shotgun pellet had the highest uptake. The animals showed a pronounced Bi uptake in the ependyma cells lining the ventricular system and in the cubic epithelia covering the choroid plexus. Dissemination of Bi ions to the rest of the body was demonstrated by AMG tracing of Bi accumulations in the tubular cells of the kidney. These findings emphasize that metallic Bi, including shotgun pellets, represents sites of intense Bi pollution if implanted or shot into a living organism, and further that such metallic Bi bodies, if they enter the CNS, cause a spread of Bi ions throughout it.