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.

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).

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.

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.

Uptake of bismuth in motor neurons of mice after single oral doses of bismuth compounds.

Bismuth, a component of many gastrointestinal medications, is a heavy metal little studied as regards nervous system uptake. We were interested to see if low doses of intragastric bismuth entered the nervous system, and if dietary selenium influenced the amount of bismuth detected. Mice were given 40 to 1200 mg/kg of bismuth subnitrate (BSN), bismuth subsalicylate (BSS), colloidal bismuth subcitrate (CBS), or ranitidine bismuth citrate (RBC) intragastrically. Mice on low- or high-selenium diets were given 4 to 32 mg/kg of bismuth from RBC. One week later, sections of nervous tissue were stained with autometallography to detect bismuth grains (Bi(AMG)). Bismuth was found in neurons with axons outside of the nervous system, in particular motor neurons, and in cells outside the blood-brain barrier. The lowest bismuth dose which resulted in Bi(AMG) in motor neurons was 696 mg/kg from BSN, 57 mg/kg from BSS, 29 mg/kg from CBS, and 26 mg/kg from RBC. No bismuth was seen in motor neurons of mice on the low-selenium diet. Intragastric doses of bismuth therefore enter mouse motor neurons, and the amount detectable varies with dietary selenium.

Histochemical tracing of bismuth in testis from rats exposed intraperitoneally to bismuth subnitrate.

The histochemical silver amplification technique autometallography (AMG), was used to trace bismuth in the testis of Wistar rats injected intraperitoneally with bismuth subnitrate. In the seminiferous tubules, bismuth was located in lysosomes of Sertoli cells closely associated with heads of spermatids in the late stages of the spermatogenesis, i.e. shortly before the release of Step 19 spermatids in Stage XIII. No bismuth-specific AMG silver grains were detected in the spermatogenic cell line. However, tails of free sperm cells located in the tubular lumen showed autometallographic grains in close contact to the nine outer microtubule doublets in the axonema. Leydig cells concentrated huge amounts of AMG-bismuth in their lysosomes. Furthermore, parallel exposure to selenium significantly increased the amount of histochemically traceable bismuth in the rat testis.

The effect of selenium on the localization of autometallographic mercury in dorsal root ganglia of rats.

The autometallographic technique was used to demonstrate the localization of mercury in dorsal root ganglia of adult Wistar rats. The animals were either exposed to mercury vapour, 100 micrograms Hg m-3, 6 h day-1, 5 days per week, or treated with organic mercury in the drinking water, 20 mg CH3HgCl per litre, for 4 weeks. The effect of orally administered sodium selenite on the pattern of intracellular distribution of mercury in these two situations was investigated. In rats exposed to mercury vapour alone, faint staining was present in ganglion cells. The selenite induced a conspicuous increase in the number of stained cells and in the intracellular staining intensity. In rats treated with organic mercury, mercury deposits were detected within ganglion cells and macrophages. The number of mercury-containing cells was increased by co-administration of selenite. In addition, satellite cells, the capsule and vessel walls were faintly stained. Twenty weeks after cessation of the organic mercury treatment, mercury staining was reduced. Again, selenite treatment enhanced staining intensity. When studied using the electron microscope, mercury was restricted to lysosomes, irrespective of treatments. The present study shows that the deposition of autometallographic mercury in the dorsal root ganglia depends on the chemical type of mercury, the co-administration of selenite and the length of the survival period.

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.

Localization of mercury in CNS of the rat. IV. The effect of selenium on orally administered organic and inorganic mercury.

The distribution and exact cellular localization of mercury in the brain and upper cervical spinal cord of the adult male Wistar rat has been determined using the autometallographic silver-enhancement technique. A detailed atlas of mercury-containing nuclei following oral administration of HgCl2 (20 mg x liter-1 or CH3HgCl (20 mg x liter-1) was prepared. The effect of orally administered Na2SeO3 (2 mg x liter-1) on these patterns was investigated. In animals treated with CH3HgCl, sodium selenite induced a conspicuous increase in mercury staining of nerve cell bodies in specific areas of the central nervous system (CNS) including laminae III-VI in the cerebral cortex, thalamus, hypothalamus, and brain stem nuclei. In the cerebellum, the cortical Purkinje cells and nerve cells in the deep nuclei were targets for appreciable mercury accumulations after CH3HgCl. Again, these deposits were increased by coadministration of selenite. In the spinal cord following administration of CH3HgCl alone, staining was limited to the gray matter. The intensity of this staining was increased by selenite and deposits also appeared in the white matter. Mercury accumulations were present in scattered glia cells in the cuneate and gracile fasciculi. Treatment with HgCl2 alone or in combination with selenite yielded no staining of the Purkinje cells, nor did selenite result in an increase in the density of other stained cell bodies throughout the CNS, as was the case with organic mercury. The most intense neuronal staining was seen in sections taken from rats treated with a combination of CH3HgCl and selenite. Lesser staining was seen in neuroglia, ependymal, and choroidal cells. In the latter two cell types, staining intensity was unaffected by selenite treatment. In HgCl2-treated rats the same cell types were targets for mercury deposits although staining was to a significantly lesser degree. Concurrent treatment with selenite had no visible effect on the staining pattern. Ultrastructurally, the bulk of the mercury was located in lysosomes. Administration of CH3HgCl combined with selenite caused mercury to appear in the nuclei of neurons. Selenium treatment delayed the functional toxic effects of CH3HgCl. Sections prepared from animals treated separately with selenium or demineralized water (used as the solvent for all compounds) were devoid of mercury deposits.

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.

Evidence from dithizone and selenium zinc histochemistry that perivascular mossy fiber boutons stain preferentially "in vivo".

This paper describes a perivascular staining pattern that is obtained when dithizone or sodium selenite are used to label zinc intravitally. Our observations indicate that the perivascular staining is a result of zinc labeling in mossy fiber boutons adjacent to capillaries and suggest that there might be a special blood brain barrier in the mossy fiber regions.

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.

Cytochemical demonstration of mercury deposits in trout liver and kidney following methyl mercury intoxication: differentiation of two mercury pools by selenium.

The amount and the ultrastructural distribution of mercury was studied in seven different organs of rainbow trout (Salmo gairdneri) fingerlings following exposure to methyl mercury (MeHg)-contaminated fodder for periods of 2 and 7 weeks. The amounts of mercury retained by the whole fish and the selected organs were determined by measuring the uptake of 203Hg-labeled MeHg. Spleen, liver, and kidney had the highest concentrations after both experimental periods, while the largest relative increases were found in brain, muscle, and kidney. The subcellular distribution of mercury accumulations was demonstrated cytochemically in liver and kidney using the silver enhancement method by which accumulations of mercury-sulfides and/or mercury-selenides are made visible for light and electron microscopy. When sections prepared from the liver and kidney from fish, injected with selenium 2 hr prior to being killed, were compared with those of fish not treated with selenium, two distinct pools of mercury could be demonstrated, the HgS pool, and the HgSe pool. The HgS pool, supposed to represent inorganic mercury, was found exclusively within lysosomes. The increase of this pool from 2 to 7 weeks was most pronounced in the kidney. The HgSe pool, supposed to represent methyl mercury, was shown by the presence of silver deposits at new locations as well as by an increase in the amount of deposits within lysosomes. The new locations included (1) secretory-like vesicles and the bile canaliculi of the liver, suggesting a biliary excretion of this mercury pool; (2) microvilli and endosomes of kidney tubular cells, suggesting a glomerular filtration and subsequent reabsorption; and (3) mitochondria of proximal tubule cells.

Effects of selenium on toxicity and ultrastructural localization of silver in cultured macrophages.

The effects of selenium on cellular toxicity and histochemical distribution of silver were examined in a cell culture system of mouse peritoneal macrophages. Selenium caused a significant delay in the appearance of coagulation necrosis induced by high silver concentrations and reduced the cytostatic effect of lower doses of silver when long-term toxicity was examined. Furthermore, selenium increased the amount of silver that could be visualized by autometallography. The additional silver made available for this histochemical demonstration was located in the cytosol as well as in lysosomes, the sole localization of silver when selenium was not administered.

Entorhinal and prepiriform cortices of the European hedgehog. A histochemical and densitometric study based on a comparison between Timm's sulphide silver method and the selenium method.

The Timm and selenium staining techniques, based on silver amplification of endogenous zinc, produce an electron-dense precipitate in boutons. The staining characteristics of the two methods were compared by examining two allocortical regions, prepiriform cortex and entorhinal cortex, in the brain of the European hedgehog. In the 3 layers of prepiriform cortex and the 6 layers of entorhinal cortex the methods revealed sublayers, which allows a precise delimitation of areas 28M, 28L, a short transition zone, and the prepiriform cortex. The lamination of the entorhinal cortex of the hedgehog is similar to that found in the rat, but appears less distinct. This may point to a lower degree of afferent organization. For light microscopical investigations, the selenium technique appears superior to Timm's method because it produces a more distinct zonation pattern.

Selenium in the anterior pituitary of rats exposed to sodium selenite: light and electron microscopic localization.

Metal selenide accumulations were demonstrated in the anterior pituitary of the rat by a histochemical technique at light and electron microscopic levels. After administration of sodium selenite either by drinking water (2.5 to 15 ppm) or by ip injection (5 to 20 mg/kg body wt), intracellular accumulations were found in secretory granules and lysosomes of the somatotrophs, thyrotrophs, corticotrophs, and the gonadotrophs. The amount of countable deposits increased with increasing doses, whether selenite was given in drinking water or by ip injection. Localization of deposits was independent of route of administration. Following a single ip injection of 5 mg sodium selenite/kg, a steadily increasing amount of visible deposits was seen throughout the first week. After this peak the deposits started to decrease but could still be found after 2 weeks. Selenium may possibly create bonds to endogenous zinc in the anterior pituitary as has been suggested for the brain.

Selenium in the Paneth cells.

Two histochemical methods, Timm's sulphide silver method and the selenium method reveal the presence of two pools of metal ions in the Paneth cells. Ultrastructurally one pool is located in the secretion granules, the other in the cytoplasm.