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.     

Autometallographic tracing of zinc ions in growing bone

It has previously been established that zinc (Zn) supplementation increases bone dimensions and strength in growing rats. The present study aims at describing differences in the localization of loosely bound or free zinc ions, as revealed by autometallography (AMG), that might take place in the skeleton of growing rats following alimentary zinc depletion and supplementation. Male Wistar rats, 4 weeks old, were randomly divided into three groups. The rats had free access to a semi-synthetic diet with different amounts of zinc added. Group 1 was given a zinc-free (2 mg zinc/kg) diet, group 2 a 47 mg zinc/kg diet, and group 3 a 60 mg zinc/kg diet. All animals were killed after 4 weeks. Animals from each group were transcardially perfused with a 0.1 % sodium sulphide solution according to the zinc specific Neo-Timm method causing zinc ions to be bound in AMG catalytic zinc-sulphur clusters. We found clusters of zinc ions localized in the mineralizing osteoid in all groups. No immediate differences in AMG staining intensity could be observed between the groups neither in the uncalcified bone nor in the osteoblasts. However, alimentary zinc supply resulted in an increase in the height of the total growth plate in a dose-dependent manner. Zinc ions were also observed in chondrocytes throughout the whole thickness of the articular and the epiphyseal cartilage as well as in the inner layer of the synovial membrane.     

In vivo liberation of silver ions from metallic silver surfaces

In vivo liberation of electrically charged silver atoms/silver ions from metallic silver pellets, silver grids and silver threads placed in the brain, skin and abdominal cavity was proved by way of the histochemical technique autometallography (AMG). A bio-film or “dissolution membrane” inserted between the metallic surface and macrophages was recognized on the surface of the implanted silver after a short period of time. Bio-released silver ions bound in silver–sulphur nanocrystals were traced within the first 24 h in the “dissolution membrane” and the “dissolucytotic” macrophages. In animals that had survived 10 days or more, silver nanocrystals were detected both extra- and intracellularly in places far away from the implant including regional lymph nodes, liver, kidneys and the central nervous system (CNS). The accumulated silver was always confined to lysosome-like organelles. Dissolucytotic silver was extracellularly related to collagen fibrils and fibres in connective tissue and basement membranes. Our study demonstrates that (1) the number of bio-released silver ions depends on the size of the surface of the implanted silver, (2) the spread of silver ions throughout the body takes place primarily not only through the vascular system, but also by retrograde axonal transport. It is concluded that implantation of silver or silver-plated devices is not recommendable.     

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.     

Chemical Blocking of Zinc Ions in CNS Increases Neuronal Damage Following Traumatic Brain Injury (TBI) in Mice

Background

Traumatic brain injury (TBI) is one of the leading causes of disability and death among young people. Although much is already known about secondary brain damage the full range of brain tissue responses to TBI remains to be elucidated. A population of neurons located in cerebral areas associated with higher cognitive functions harbours a vesicular zinc pool co-localized with glutamate. This zinc enriched pool of synaptic vesicles has been hypothesized to take part in the injurious signalling cascade that follows pathological conditions such as seizures, ischemia and traumatic brain injury. Pathological release of excess zinc ions from pre-synaptic vesicles has been suggested to mediate cell damage/death to postsynaptic neurons.

Methodology/Principal Findings

In order to substantiate the influence of vesicular zinc ions on TBI, we designed a study in which damage and zinc movements were analysed in several different ways. Twenty-four hours after TBI ZnT3-KO mice (mice without vesicular zinc) were compared to littermate Wild Type (WT) mice (mice with vesicular zinc) with regard to histopathology. Furthermore, in order to evaluate a possible neuro-protective dimension of chemical blocking of vesicular zinc, we treated lesioned mice with either DEDTC or selenite. Our study revealed that chemical blocking of vesicular zinc ions, either by chelation with DEDTC or accumulation in zinc-selenium nanocrystals, worsened the effects on the aftermath of TBI in the WT mice by increasing the number of necrotic and apoptotic cells within the first 24 hours after TBI, when compared to those of chemically untreated WT mice.

Conclusion/Significance

ZnT3-KO mice revealed more damage after TBI compared to WT controls. Following treatment with DEDTC or selenium an increase in the number of both dead and apoptotic cells were seen in the controls within the first 24 hours after TBI while the degree of damage in the ZnT3-KO mice remained largely unchanged. Further analyses revealed that the damage development in the two mouse strains was almost identical after either zinc chelation or zinc complexion therapy.     

锌及锌转运蛋白ZnT3在小鼠海马苔藓纤维的一致性分布

目的 研究游离锌离子和锌转运蛋白ZnT3在小鼠海马的定位以及二的分布是否具有一致性。方法 应用锌TSQ荧光技术、锌金属自显影技术检测含锌神经元内的游离锌离子;应用免疫电镜技术检测ZnT3在含锌神经元轴突终末的分布。结果 游离锌离子和ZnT3免疫反应产物的分布在海马苔藓纤维内的分布具有一致性。在齿状回和CA3区的苔藓纤维内,锌和ZnT3蛋白定位于轴突终末的突触小泡。富含锌离子的含锌神经元轴突终末与CA3区锥体细胞的胞体和树突形成突触。尚可见锌离子存在于突触间隙内。结论 ZnT3向突触小泡内转运锌离子使锌离子聚积在含锌神经元轴突终末的突触小泡内,发挥锌离子的神经生物学功能。     

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.     

Immobilization of amyloglucosidase from SSF of Aspergillus niger by crosslinked enzyme aggregate onto magnetic nanoparticles using minimum amount of carrier and characterizations

Immobilizations of enzymes are done for operational stability, recovery and re-use of the enzymes and easy separation of products. Amyloglucosidase (AMG) obtained from solid state fermentation (SSF) of Aspergillus niger was directly immobilized by novel technique of crosslinked enzyme aggregate onto magnetic nanoparticles. AMG was covalently linked to the magnetic nanoparticle (MNP) to form a monolayer of AMG (MNP–AMG), followed by crosslinked aggregates with free AMG (which was not immobilized) to yield MNP with high enzyme loading (MNP–AMG_n). Under optimized conditions, very high recovery (92.8%) of enzyme activity was obtained in MNP–AMG_n using 14 times less carrier compared to the quantity of carrier required by conventional method. MNP–AMG_n showed enhanced affinity for substrate, thermal stability, storage stability and reusability.     

Autometallography allows ultrastructural monitoring of zinc in the endocrine pancreas

Zinc is intimately involved in insulin metabolism, its major known role being the binding of insulin in osmotically stable hexamers in beta-cell granules. To investigate the anatomical distribution of zinc ions necessary for insulin binding we examined the rat pancreas by autometallography (AMG). AMG demonstrates chelatable zinc and is a sensitive marker for zinc in vesicles and also a surrogate marker for recently described zinc pumps regulating intravesicular zinc metabolism. Zinc ions were found in alpha- and beta-cell granules, primarily in the periphery of the granules. Only occasionally was zinc seen in other islet cell types. AMG allows the study of the microscopic and ultrastructural localisation of free zinc ions in the pancreas. The applicability of the method at the ultrastructural level in particular makes AMG a very sensitive tool in future studies on the role of zinc ions in the pancreas.     

Zinc transporter 3 immunohistochemical tracing of sprouting mossy fibres

Zinc transporter 3 (ZNT3) has been shown to transport zinc ions from the cytosol into presynaptic vesicles in the mammalian brain. Several studies have stated that the zinc ion containing synaptic vesicles of zinc-enriched neurons (ZEN) are loaded with ZNT3 proteins in their membranes. This fact makes it possible to trace sprouting mossy fibres in the temporal lobe epileptic hippocampus. In the present study, we examined the expression and distribution patterns of ZNT3 protein and chelatable zinc ions in the mouse hippocampus after pilocarpine treatment. Our results demonstrate that both ZNT3 immunostaining and autometallography reveal identical patterns of sprouting mossy fibres in the inner molecular layer in the mouse hippocampus. Using ZNT3 immuno-electron microscopic analysis we confirmed the presence of ectopic mossy fibre terminals in the inner molecular layer and found additionally by immuno-blotting a significant increase of ZNT3 in the pilocarpine-treated mouse hippocampi compared to age-matched controls. The increase of ZNT3 after pilocarpine treatment was time-dependent. The results support the notion that ZNT3 immunohistochemistry provides an excellent tool for tracing sprouting of ZEN terminals. The progressive increase of ZNT3 immunostaining in the temporal lobe epileptic hippocampus may relate to the increased levels of vesicular zinc ions during seizure.     

Bismuth ions are metabolized into autometallographic traceable bismuth-sulphur quantum dots

Bismuth - sulphur quantum dots can be silver enhanced by autometallography (AMG). In the present study, autometallographic silver enhanced bismuth-sulphur nanocrystals were isolated from unfixed cryo-sections of kidneys and livers of rats exposed to bismuth (Bi207) subnitrate. After being subjected to AMG all the organic material was removed by sonication and enzymatic digestion and the silver enhanced Bi-S quantum dots spun down by an ultracentrifuge and analyzed by scintillation. The analysis showed that the autometallographic technique traces approximately 94% of the total bismuth. This implies that the injected bismuth is ultimately captured in bismuth-sulphur quantum dots, i.e., that Bi-S nanocrystals are the end product of bismuth metabolism.     

A novel approach to the identification and quantitative elemental analysis of amyloid deposits—Insights into the pathology of Alzheimer’s disease

There is considerable interest in the role of metals such as iron, copper, and zinc in amyloid plaque formation in Alzheimer’s disease. However to convincingly establish their presence in plaques in vivo, a sensitive technique is required that is both quantitatively accurate and avoids isolation of plaques or staining/fixing brain tissue, since these processes introduce contaminants and redistribute elements within the tissue. Combining the three ion beam techniques of scanning transmission ion microscopy, Rutherford back scattering spectrometry and particle induced X-ray emission in conjunction with a high energy (MeV) proton microprobe we have imaged plaques in freeze-dried unstained brain sections from CRND-8 mice, and simultaneously quantified iron, copper, and zinc. Our results show increased metal concentrations within the amyloid plaques compared with the surrounding tissue: iron (85 ppm compared with 42 ppm), copper (16 ppm compared to 6 ppm), and zinc (87 ppm compared to 34 ppm).     

Selective Electrodiffusion of Zinc Ions in a Zrt-, Irt-like Protein,ZIPB

All living cells need zinc ions to support cell growth. Zrt-, Irt-like proteins (ZIPs) represent a major route for entry of zinc ions into cells, but how ZIPs promote zinc uptake has been unclear. Here we report the molecular characterization of ZIPB from Bordetella bronchiseptica, the first ZIP homolog to be purified and functionally reconstituted into proteoliposomes. Zinc flux through ZIPB was found to be nonsaturable and electrogenic, yielding membrane potentials as predicted by the Nernst equation. Conversely, membrane potentials drove zinc fluxes with a linear voltage-flux relationship. Direct measurements of metal uptake by inductively coupled plasma mass spectroscopy demonstrated that ZIPB is selective for two group 12 transition metal ions, Zn²⁺ and Cd²⁺, whereas rejecting transition metal ions in groups 7 through 11. Our results provide the molecular basis for cellular zinc acquisition by a zinc-selective channel that exploits in vivo zinc concentration gradients to move zinc ions into the cytoplasm.     

Local‐field effect on the fluorescence relaxation of Tm3+:LaF3 nanocrystals immersed in liquid medium

Tm³⁺:LaF₃ nanocrystals were synthesized with hydrothermal technique. Local‐field effect on the radiative relaxation rate was studied in the system of Tm³⁺:LaF₃ nanocrystals immersed in several liquid media. The fluorescence lifetime was measured. It was found that the fluorescence decay presented the characteristics of second‐order exponential decay, for which the contribution from the ions inside the nanocrystal and ions at the interface of the nanocrystal were distinguished. Investigating the experimental results with proposed models, we found that the surface effect had to be eliminated. For rare earth doped LaF₃ nanocrystals, real‐cavity model well explains the influence of surrounding medium on the fluorescence relaxation rate. Copyright © 2009 John Wiley & Sons, Ltd.     

Zinc ion binding to human brain calcium binding proteins. Calmodulin and S100b protein

Comparative studies have been performed on the binding properties of zinc ions to human brain calmodulin and S100b protein. Calmodulin is characterized by two sets of Zn²⁺ binding sites, with K_D ranging from 8.10^?5M to 3.10^?4M. The S100b protein also exhibited two sets of zinc binding sites, with a much higher affinity. K_D = 10^?7 ? 10^?6M. We suggest that S100b protein should no longer be considered only as a “calcium binding protein” but also as a “zinc binding protein”, and that Zn²⁺ ions are involved in the functions of the S100 proteins.     

Synchrotron-based infrared and X-ray imaging shows focalized accumulation of Cu and Zn co-localized with beta-amyloid deposits in Alzheimer's disease

Alzheimer's disease (AD) is characterized by the misfolding and plaque-like accumulation of a naturally occurring peptide in the brain called amyloid beta (Abeta). Recently, this process has been associated with the binding of metal ions such as iron (Fe), copper (Cu), and zinc (Zn). It is thought that metal dyshomeostasis is involved in protein misfolding and may lead to oxidative stress and neuronal damage. However, the exact role of the misfolded proteins and metal ions in the degenerative process of AD is not yet clear. In this study, we used synchrotron Fourier transform infrared micro-spectroscopy (FTIRM) to image the in situ secondary structure of the amyloid plaques in brain tissue of AD patients. These results were spatially correlated with metal ion accumulation in the same tissue sample using synchrotron X-ray fluorescence (SXRF) microprobe. For both techniques, a spatial resolution of 5-10 microm was achieved. FTIRM results showed that the amyloid plaques have elevated beta-sheet content, as demonstrated by a strong amide I absorbance at 1625cm(-1). Using SXRF microprobe, we find that AD tissue also contains "hot spots" of accumulated metal ions, specifically Cu and Zn, with a strong spatial correlation between these two ions. The "hot spots" of accumulated Zn and Cu were co-localized with beta-amyloid plaques. Thus for the first time, a strong spatial correlation has been observed between elevated beta-sheet content in Abeta plaques and accumulated Cu and Zn ions, emphasizing an association of metal ions with amyloid formation in AD.     

Development and Validation of an Anodic Stripping Voltammetric Method for Determination of Zn<Superscript>2+</Superscript> Ions in Brain Microdialysate Samples

An easy, rapid, and sensitive anodic stripping voltammetric method with a controlled growth mercury drop electrode has been developed and validated for the determination of Zn²⁺ ions in brain microdialysate samples obtained from rats. The considered level of the zinc concentration in the dialysate was 0.5–6 ppb. In the investigated method, the stripping step was carried out by using a differential pulse potential-time voltammetric excitation signal. The optimal experimental conditions as well as the instrumental and accumulation parameters and supporting electrolyte composition were investigated. The optimized method was validated for precision, linearity, and accuracy. Mean recovery 82–110% was achieved, the precision expressed by CV not greater than 7.6% and the linearity given by correlation coefficient not lower than 0.9988. The limit of detection was 0.1 ppb. No interferences were observed. Due to high linearity, precision, and sensitivity, the developed method may be successfully applied in the determination of zinc ions in microdialysate brain samples. The results obtained for the first time demonstrate detailed characteristics of the determination of zinc in the brain microdialysate fluid by the ASV method. It may be applied in a wide range of physiological and pharmacological studies which focus on very low zinc concentration/alteration in various compartments of the organisms.     

Quantification of vesicular zinc in the rat brain

By means of the Neo-Timm method it has recently been shown that zinc is present in a fraction of the round clear synaptic vesicles of certain boutons located primarily in telencephalic structures (Pérez-Clausell and Danscher 1985). It is believed that this zinc belongs to a fraction of the total brain zinc which is histochemically active (Frederickson and Danscher 1988) in that it can be visualized by means of e.g. the Neo-Timm and selenium methods (autometallography). The present study is based on the suggestion that the autometallographically developed zinc patterns represent a histochemical quantitative expression of this fraction of the total brain zinc. The different colours of the zinc pattern reflect local variations in the concentration of zinc containing vesicles. Large boutons with a high content of stained vesicles will show up darkly because of fusion of adjoining silver grains while smaller boutons with fewer zinc containing vesicles give rise to yellow staining of various shades. We have exploited this difference in staining of pattern by applying computerized optic densitometry to light microscopic sections treated according to the Neo-Timm and the selenium methods, respectively.     

Quantification of vesicular zinc in the rat brain

Summary By means of the Neo-Timm method it has recently been shown that zinc is present in a fraction of the round clear synaptic vesicles of certain boutons located primarily in telencephalic structures (Pérez-Clausell and Danscher 1985). It is believed that this zinc belongs to a fraction of the total brain zinc which is histochemically active (Frederickson and Danscher 1988) in that it can be visualized by means of e.g. the Neo-Timm and selenium methods (autometallography). The present study is based on the suggestion that the autometallographically developed zinc patterns represent a histochemical quantitative expression of this fraction of the total brain zinc. The different colours of the zinc pattern reflect local variations in the concentration of zinc containing vesicles. Large boutons with a high content of stained vesicles will show up darkly because of fusion of adjoining silver grains while smaller boutons with fewer zinc containing vesicles give rise to yellow staining of various shades. We have exploited this difference in staining pattern by applying computerized optic densitometry to light microscopic sections treated according to the Neo-Timm and the selenium methods, respectively.     

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.