Changes in the vesicular zinc pattern following traumatic brain injury.

The present study aims at evaluating the significance of zinc ions on the development of brain damage in a model of traumatic brain injury (TBI). The zinc ion specific autometallographic technique, the ZnSe(AMG) method, using silver enhancement of in vivo-captured zinc ions bound in zinc-selenium nanocrystals was applied to follow changes in the vesicular zinc pattern. Balb/c mice, ZnT3 knockout (ZnT3-Ko) mice, a mouse genetically knocked out for the protein ZnT3 responsible for sequestering zinc into synaptic vesicles, and littermates from the genetically un-manipulated mother type mice, wild type (Wt), were used. The Wt and the Balb/c mice exhibited instantaneously a boost in the zinc staining adjacent to the lesion involving all six neocortical layers. Ultra-structural analyses revealed that the in vivo created ZnSe nanocrystals were still confined to the vesicles of the zinc-enriched (ZEN) neurons in the neuropil. No differences between the Balb/c and Wt mice were seen at any time points. In the ZnT3-Ko mice the ZEN terminals stayed void of AMG grains, but a number of neuronal somata around the lesion became loaded with ZnSe nanocrystals. These silver-enhanced ZnSe nanocrystals were confined to the cytoplasm of the somata and their proximal dendrites. No such soma staining was seen in the Wt or Balb/c mice. We speculate that vesicular zinc may not contribute to neuronal damage following TBI.

Amyloid plaques arise from zinc-enriched cortical layers in APP/PS1 transgenic mice and are paradoxically enlarged with dietary zinc deficiency.

The ZnT3 zinc transporter is uniquely expressed in cortical glutamatergic synapses where it organizes zinc release into the synaptic cleft and mediates beta-amyloid deposition in transgenic mice. We studied the association of zinc in plaques in relation to cytoarchitectural zinc localization in the APP/PS1 transgenic mouse model of Alzheimer's disease. The effects of low dietary zinc for 3 months upon brain pathology were also studied. We determined that synaptic zinc distribution within cortical layers is paralleled by amyloid burden, which is heaviest for both in layers 2-3 and 5. ZnT3 immunoreactivity is prominent in dystrophic neurites within amyloid plaques. Low dietary zinc caused a significant 25% increase in total plaque volume in Alzheimer's mice using stereological measures. The level of oxidized proteins in brain tissue did not changed in animals on a zinc-deficient diet compared with controls. No obvious changes were observed in the autometallographic pattern of zinc-enriched terminals in the neocortex or in the expression levels of zinc transporters, zinc importers or metallothioneins. A small decrease in plasma zinc induced by the low-zinc diet was consistent with the subclinical zinc deficiency that is common in older human populations. While the mechanism remains uncertain, our findings indicate that subclinical zinc deficiency may be a risk factor for Alzheimer's pathology.

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.

Electron Microscopical Autometallography: Immunogold-Silver Staining (IGSS) and Heavy-Metal Histochemistry

Immunogold-silver staining (IGSS) utilizes a histochemical method called autometallography (AMG) to amplify tiny gold particles to sizes easily visible both in light and electron microscopy. In both applications it is advisable to use the smallest possible gold diameters (1-6 nm) to obtain the highest sensitivity, thus, allowing minute amounts of the target substance to be demonstrated. Gold labels smaller than 10 nm in diameter have been clearly shown to give the highest labeling densities of antigen-antibody binding sites. AMG can be used for the detection of catalytic crystal lattices of metallic gold and silver, and sulfides or selenides of mercury, silver, copper, bismuth, and zinc. The method has its roots in "physical development" technique, transplanted from photography to histology by Liesegang at the beginning of this century. In 1981, a series of papers were published by one of us with the purpose of introducing a reliable and easy-to-handle technique for light microscopical and ultrastructural studies. AMG has a multitude of applications apart from its use in detecting tissue metals. These include the highly sensitive and efficient in situ colloidal gold tracing of peptides, proteins, and amines by immunocytochemistry using the IGSS method, of carbohydrates by lectin IGSS, and of nucleic acids by IGSS in situ hybridization, IGSS in situ polymerase chain reaction, and IGSS in situ self-sustained sequence replication-based amplification (in situ 3SR) techniques, the last two even performing with single-copy sensitivity. Applications of pre- and postembedding AMG for semithin and ultrathin tissue sections are described.

Autometallographic tracing of bismuth in human brain autopsies.

For decades, drugs containing bismuth have been used to treat gastrointestinal disorders. Although a variety of adverse effects, including neurological syndromes, have been recorded, the biological/toxicological effects of bismuth ions are far from disclosed. Until recently, only quantitative assessments were possible, but resent research has made histochemical tracing of bismuth possible. The technique involves silver enhancement of bismuth crystallites by autometallography (AMG). In the present study, the localization of bismuth was traced by AMG in sections of paraffin-embedded brain tissue obtained by autopsy from 6 patients suffering from bismuth intoxication in a period ranging from 1975 through 1977. Tissue was analyzed at light and electron microscopical levels, and the presence of bismuth further confirmed by proton-induced x-ray emission (PIXE). Clinical data and bismuth concentrations in blood, cerebellum, and thalamus were measured by atomic absorption spectrophotometry (AAS) and are reported here. Histochemical analyses demonstrate that bismuth accumulated in neurons and glia cells in the brain regions examined (neocortex, cerebellum, thalamus, hippocampus). Cerebellar blood vessels stained most intensely. The PIXE and AAS data correlated with the histochemical staining patterns and intensities. At the ultrastructural level, bismuth was found to accumulate intracellularly in lysosomes and extracellularly in the basement membranes of some vessels.

The Timm-stained hippocampus of the European hedgehog: a basal mammalian form.

A quantitative and qualitative description has been made of the components of the Timm-stained hippocampus of the European hedgehog. While the laminar organization and the relative size of the major subdivisions of the hippocampus (i.e., area dentata, Ammon's horn, and subiculum) are similar to those of the albino laboratory rat, the relative proportions and the staining characteristics of some of the components of the subdivisions are different. The differences are particularly evident in Ammon's horn where regions are poorly differentiated along the dentatosubicular axis and the mossy fiber zone is relatively extensive. The description characterizes a hippocampal form that can be used as a basal reference in comparative studies of the mammalian hippocampus.

Amygdaloid efferents through the stria terminalis in the rat give origin to zinc-containing boutons.

Many regions of the basal forebrain are innervated by zinc-containing axonal boutons. In the present work, the lesion/degeneration method, coupled with histochemical staining for zinc-containing boutons, was used to determine the origins and efferent pathways of these zinc-containing projections to the basal forebrain. Knife cuts of the stria terminalis or extensive electrolytic lesions of the amygdala resulted in the bleaching of the staining for zinc (Timm stain) and terminal degeneration (Fink-Heimer method) ipsilaterally in the following areas: granule cell layer of the accessory olfactory bulb, shell of nucleus accumbens, bed nucleus of the stria terminalis, striohypothalamic nucleus, retrochiasmatic area, ventromedial hypothalamic nucleus (in the cell-sparse shell), medial tuberal nucleus, terete hypothalamic nucleus, and ventral premammillary nucleus. Small lesions made with ibotenic acid in the posteromedial part of the amygdalohippocampal area caused bleaching of the stain for zinc in the accessory olfactory bulb, in the medial zone of the bed nucleus of the stria terminalis, and in the ventral premammillary nucleus. Lesions in either the ventral subiculum or the anterolateral part of the amygdalohippocampal area caused bleaching in the ventromedial hypothalamic nucleus. Lesions in the hippocampus or in the neocortex did not produce bleaching of the stain for zinc in the above-mentioned terminal fields. The present results agree with previous studies on amygdaloid efferents and suggest that neurons in the amygdalohippocampal area and, possibly, in the ventral subiculum give origin to zinc-containing boutons.

The positive effects of zinc on skeletal strength in growing rats.

The aim of the present study was to assess the skeletal effects of alimentary zinc depletion and supplementation in an animal model of intact, growing rats. The study was planned as a dose-response study. Thirty-six male Wistar rats, 4 weeks old, were divided into three groups of 12 rats each. The rats had free access to a semisynthetic diet with different amounts of zinc added. Group 1 was given a zinc-free diet containing 2 mg zinc/kg, group 2 was given a normal-zinc diet containing 47 mg zinc/kg; and group 3 was given a zinc-supplemented diet containing 60 mg zinc/kg. All animals were killed 4 weeks after initiation of the experiment and the right femora were removed. The biomechanical effects were measured at the following skeletal sites: femoral diaphysis; femoral neck; and distal femoral metaphysis. In addition, static histomorphometry was performed at the middiaphyseal region. Biomechanical testing revealed a significant zinc-induced increase in bone strength at all sites investigated. It also showed that zinc influenced bone strength in a dose-dependent manner except at the distal metaphysis, where there was no significant difference between the group fed normal-zinc diet and the group fed a hyper-zinc diet. Zinc also improved the rates of growth in the rats. The body weights and length of femora increased dose-dependently. Static histomorphometry showed that zinc exerted its main effect on the periosteal envelope, thereby increasing bone area, tissue area, and axial moment of inertia. We conclude that alimentary zinc supplementation in growing rats induces an increase of bone strength in both the femoral neck and the femoral diaphysis. These results further support the view that zinc has a positive effect on bone metabolism which mimics that of growth hormone (GH) or insulin-like growth factor 1 (IGF-1).

Labeling of the neurons of origin of zinc-containing pathways by intraperitoneal injections of sodium selenite.

Intraperitoneal injections of sodium selenite result in the formation of zinc-selenium complexes in zinc-containing axonal boutons ("Timm stainable boutons"), and the zinc-selenium precipitate can be rendered visible in histological sections by silver enhancement. In this work we present evidence, in the rat, that zinc-selenium precipitates formed in vivo after intraperitoneal injections of sodium selenite are translocated by colchicine-sensitive retrograde transport to neural perikarya when animals are allowed to survive 12-24 h after the selenite administration. Silver enhancement renders the perikaryal precipitates visible and thus demonstrates the perikarya of all zinc-containing neurons in the CNS simultaneously. Large populations of zinc-containing neurons identified by the method are found in layers II, III, and VI of all neocortical areas, in the superficial and deep layers of the prepyriform areas and, with a high degree of regional differentiation, in the retrosplenial, entorhinal, para- and presubicular cortices, the hippocampal formation and the amygdaloid complex. Zinc-containing cells were absent from the caudate-putamen, nucleus accumbens and septal complex. Labeled zinc-containing cells are absent in non-telencephalic parts of the brain. The findings indicate that the zinc-containing circuitry of the brain mainly serves in telencephalic information processing.

Zinc transporter 3 and zinc ions in the rodent superior cervical ganglion neurons.

Previous studies have revealed that zinc-enriched (ZEN) terminals are present in all parts of the CNS though with great differences in intensity. The densest populations of both ZEN terminals and ZEN somata are found in telencephalic structures, but also structures like the spinal cord demonstrate impressive ZEN systems spreading terminals several segments around the respective ZEN somata. The present study evaluates whether sympathetic neurons in the superior cervical ganglia (SCG) are ZEN neurons, i.e. contain vesicles that have zinc transporter 3 (ZnT3) proteins in their membranes and contain zinc ions. ZnT3 immunoreactivity (IR) was found in the somata and processes in the postganglionic neurons of mouse SCG. Only a small fraction of neurons (less than 5%), expressed varying degrees of ZnT3. Colchicine treatment, however, increased the number of ZnT3-positive neurons three-fold, suggesting an accumulation of ZnT3 protein in the somata. A small proportion of the postganglionic axons revealed dotted accumulations of ZnT3 IR along their courses. Double labeling showed that all ZnT3-positive neurons and axons were also tyrosine hydroxylase-positive with strong immunofluorescence, while no colocalization was found between ZnT3 and the vesicular acetylcholine transporter (VAChT) or neuropeptide Y IR. VAChT-positive preganglionic neurons were found to terminate on ZnT3 neuronal somata. 6-Methoxy 8-para toluene sulfonamide quinoline fluorescence and zinc selenium autometallography (ZnSe(AMG)) revealed that a subgroup of SCG cells contained free or loosely bound zinc ions. It is therefore concluded that ZnT3 and zinc ions are present in a subpopulation of TH-positive, NPY-negative neurons in the rodent SCG, supporting the notion that vesicular zinc ions may play a special role in the peripheral sympathetic adrenergic system.

Inhibitory zinc-enriched terminals in mouse spinal cord.

The ultrastructural localization of zinc transporter-3, glutamate decarboxylase and zinc ions in zinc-enriched terminals in the mouse spinal cord was studied by zinc transporter-3 and glutamate decarboxylase immunohistochemistry and zinc selenium autometallography, respectively. The distribution of zinc selenium autometallographic silver grains, and zinc transporter-3 and glutamate decarboxylase immunohistochemical puncta in both ventral and dorsal horns as seen in the light microscope corresponded to their presence in the synaptic vesicles of zinc-enriched terminals at ultrastructural levels. The densest populations of zinc-enriched terminals were seen in dorsal horn laminae I, III and IV, whereas the deeper laminae V and VI contained fewer terminals. At ultrastructural levels, zinc-enriched terminals primarily formed symmetrical synapses on perikarya and dendrites. Only relatively few asymmetrical synapses were observed on zinc-enriched terminals. In general, the biggest zinc-enriched terminals contacted neuronal somata and large dendritic elements, while medium-sized and small terminals made contacts on small dendrites. The ventral horn was primarily populated by big and medium-sized zinc-enriched terminals, whereas the dorsal horn was dominated by medium-sized and small zinc-enriched terminals. The presence of boutons with flat synaptic vesicles with zinc ions and symmetric synaptic contacts suggests the presence of inhibitory zinc-enriched terminals in the mammalian spinal cord, and this was confirmed by the finding that zinc ions and glutamate decarboxylase are co-localized in these terminals. The pattern of zinc-enriched boutons in both dorsal and ventral horns is compatible with evidence suggesting that zinc may be involved in both sensory transmission and motor control.

Zinc-rich synaptic boutons in human temporal cortex biopsies.

The distribution of zinc-rich synaptic boutons in biopsies of the temporal cortex from epileptic patients who had undergone surgery is described. Unfixed cryostat sections were exposed to H(2)S vapour to precipitate endogenous zinc, which was subsequently shown by silver enhancement. In the temporal cortex, the stain for zinc was arranged in bands: stain was heavy in layers II and VI, moderate-to-heavy in layers I, III and V, and low in layer IV. The white matter was virtually devoid of staining. At the electron microscope level, labelling was found in synaptic boutons that made asymmetric synaptic contacts. Immunohistochemical staining for glutamate receptor subunits GluR2/3 was observed in cell bodies in layers II, III, V and VI, coincident with the layers that showed heavy staining for zinc. Immunostaining for glutamate receptor subunit GluR1 was prominent in non-pyramidal neurons in deep cortical layers. These results support findings in other mammals and indicate that the human neocortex may contain an extensive system of zinc-rich cortico-cortical connections. This system may be altered in pathological conditions.

Silver amplification of mercury sulfide and selenide: a histochemical method for light and electron microscopic localization of mercury in tissue.

A method for light and electron microscopic demonstration of mercury sulfides and mercury selenides in mammalian tissue is presented. Silver ions adhering to the surface of submicroscopic traces of mercury sulfides or selenides in the tissue are reduced to metallic silver by hydroquinone. Physical development thereupon renders deposits of mercury sulfides or mercury selenide visible as spheres of solid silver. Examples of localization of mercury in the central nervous system and various organs from animals exposed to mercury chloride or methyl mercury chloride with or without additional sodium selenide treatment are presented. Selenium treatment results in a considerable increase in the amount of mercury that can be made visible by silver amplification. After mercury chloride treatment, most of the mercury is localized in lysosomes and is only rarely seen in secretory granules. After simultaneous selenium treatment, mercury is also found in nuclei of proximal tubule cells in the kidney and in macrophages. The "sulfide-osmium" method for ultrastructural localization of mercury suggested by Silberberg, Lawrence, and Leider (Arch Environ Health 19:7, 1969) and the light microscopic method using a photographic emulsion suggested by Umeda, Saito, and Saito (Jpn J Exp Med 39:17, 1969) have been experimentally analyzed and commented on.

Ultrastructural autometallography: a method for silver amplification of catalytic metals.

The autometallographic technique involves application of a silver bromide-containing emulsion on the surface of ultrathin sections placed on grids that are subsequently exposed to a photographic developer. In tissue sections from animals treated intravitally with gold, silver, or mercury compounds, accumulations of the metals are visualized by autometallography and can be used for quantitative studies. After amplification, sections can be stained with lead citrate and uranyl acetate. Using autometallography, particles of colloidal gold dispersed in a film of gelatin showed a time-dependent growth and were gradually amplified up to 3.5-fold after 15 min of development. Hence the method may prove useful tracing colloidal gold particles in sections with low particle density, and be a powerful tool for revealing metals in biological tissues.

Light microscopic visualization of colloidal gold on resin-embedded tissue.

RNase labeled with colloidal gold was used as a model for the present technique evolved for the light microscopic localization of gold-labeled substances in semithin resin-embedded sections. Tissue sections placed on glass slides were treated with the gold-enzyme complex and subsequently exposed to a photographic developed containing silver lactate. During the development gold particles are encapsulated in growing shells of metallic silver and gradually made visible in the light microscope. The amplification method can be applied to paraffin-embedded and frozen sections as well. This technique may prove useful as a supplement to studies utilizing colloidal gold or silver as markers normally used at the electron microscopic level.

Zinc-containing 7S-NGF complex. Evidence from zinc histochemistry for localization in salivary secretory granules.

7S-NGF is a pro-protein containing a neurotrophic subunit, beta-NGF, which has been localized by immunocytochemistry to the granules of granular convoluted tubule (GCT) cells in certain murine salivary glands [Watson et al., Anat Rec (1985) 213:365]. The 7S-NGF pro-protein contains zinc and is stabilized by zinc ions [Pattison and Dunn, Biochemistry (1976) 15:3696]. In the present work, dithizone, toluene sulfonamide quinoline (TSQ), and neo-Timm's methods for zinc were used to determine whether zinc histochemistry could be used to visualize the zinc associated with the 7S-NGF complex and, if so, whether zinc histochemistry might corroborate the reported localization of the 7S-NGF complex in GCT secretory granules. The results indicate that intensity of zinc staining varies with the reported variations in NGF levels in different salivary glands, and that the zinc is selectively concentrated in the GCT secretory granules. We suggest that zinc histochemistry may be a useful marker for the presence of the zinc-stabilized 7S-NGF pro-protein.

Retrograde tracing of zinc-containing neurons by selenide ions: a survey of seven selenium compounds.

The autometallographic retrograde tracing of zinc-containing neurons by intracerebral injection of sodium selenite (Na2SeO3), introduced by Danscher in 1982, has recently been described in more detail. Intracerebral injections of both sodium selenide (Na2Se) and sodium selenite (Na2SeO3) have been successfully used; however, sodium selenite had a rather toxic effect on the injected tissue. In the present study, we tested seven different selenium compounds to find the most suitable compound for retrograde tracing of zinc-positive pathways. Among the tested compounds, sodium selenide (Na2Se) caused insignificant necrosis within the injection site and was easily transported retrogradely when handled anaerobically. Sodium selenide is therefore recommended as the compound of choice.

Bismuth autometallography: protocol, specificity, and differentiation.

We provide a detailed protocol of the autometallographic bismuth technique and evaluate the specificity of the technique. We show by the multi-element technique "proton-induced X-ray microanalysis" (PIXE) that the autometallographic grains contain silver, bismuth, and sulfur, proving that autometallography can be used for specific tracing of bismuth bound as bismuth sulfide clusters in tissue sections from Bi-exposed animals or humans. In sections from animals exposed concurrently to selenium and bismuth, the autometallographic grains also contain selenium. This demonstrates that, if present in excess in the organisms, selenium will bind to exogenous bismuth, creating bismuth selenide clusters. As a further possible control for specificity and as a tool for differentiating among autometallographically detectable metals in sections containing more than one, we describe how bismuth sulfide clusters can be removed from Epon-embedded tissue sections by potassium cyanide.

Silver enhancement of tissue mercury: demonstration of mercury in autometallographic silver grains from rat kidneys.

The autometallographic silver enhancement method has been applied increasingly to detect trace amounts of mercury in preparations of biological tissue. It has, however, been difficult to establish the presence of a core of mercury within the silver grain by direct methods such as energy dispersive X-ray analysis. In the present work, a sample of autometallographic silver grains was prepared from kidneys of rats exposed to mercury in the drinking water. Frozen sections from the kidneys were silver-enhanced and subsequently all organic material was removed by enzymatic digestion. The remaining pellet of silver grains was analyzed by proton-induced X-ray emission (PIXE) and mercury was demonstrated in an amount of 0.1-0.5% compared to silver. In addition, it was demonstrated that two pools of catalytic mercury compounds exist, probably corresponding to sulfide- and selenium-bound mercury.

Autometallographic silver enhancement of zinc sulfide crystals created in cryostat sections from human brain biopsies: a new technique that makes it feasible to demonstrate zinc ions in tissue sections from biopsies and early autopsy material.

We present a new technique that allows zinc ions in synaptic and secretory vesicles of biopsy and early autopsy material (< 2 hr post mortem) to be transformed to nanometer-sized zinc sulfide crystal lattices for subsequent autometallographic (AMG) development. Human brain biopsies, or other tissue samples containing zinc-enriched (ZEN) cells, are frozen in liquid nitrogen or by CO2 gas immediately after removal. The tissue blocks are cut in a cryostat and the sections placed on glass slides. The slides are transferred to an H2S exposure chamber placed in a -15 C freezer. After 1-24 hr of gas exposure the sections are removed from the chamber, fixed while thawing, and dehydrated. The sections are then exposed to an AMG developer. AMG causes silver enhancement of zinc sulfide crystal lattices created in the tissues through the H2S exposure, making them visible. It is imperative that the tissues are frozen instantaneously after removal, because loosely bound or free zinc ions start leaving their vesicular compartment soon after death. The AMG technique can, despite inadequate fixation and damage to the tissue caused by freezing, also be used to trace zinc ions at ultrastructural levels, and it is demonstrated that zinc ions in the human neocortex are located in synaptic vesicles. In the few human biopsies analyzed thus far, the light microscopic pattern created by the silver-enhanced ZEN terminals resembles that seen in the neocortex of rat brain. The technique has been applied to cryostat sections from neocortex biopsies of five individuals undergoing brain surgery. Biopsies from three patients resulted in satisfactory AMG-stained sections. Rat brains removed and frozen immediately after decapitation constituted the material on which the present technique was developed. Such material results in an almost uniform high quality of staining, and we found that unexposed sections can be stored for at least 5 months at -80 C without ensuing significant loss of AMG staining intensity.