RESUMO
The histochemical distribution of mercury in the kidneys and gut of frogs (Rana ridibunda) exposed to inorganic mercury was analyzed with autometallography (AMG). It was found that most mercury in the kidneys accumulated in the proximal convoluted tubules as Hg-S nanocrystal, while control animals were totally void of AMG grains. In the gut the highest concentration of mercury was observed in the large intestine. The AMG grains were primarily located in the apical part of the absorptive cells, although rather high concentrations of silver enhanced mercury quantum dots were also detected in a special cell type of gut epithelium and the glycocalyx. A certain amount of AMG grains were detected in the lumen of the gut. We hypothesize that this pool of quantum dots results from sloughed off epithelial cells and macrophages. Such still intact cells and red blood cells containing AMG grains were also found in the lumen of the gut.
Assuntos
Compostos Inorgânicos/metabolismo , Mercúrio/metabolismo , Pontos Quânticos , Ranidae/metabolismo , Enxofre/metabolismo , Animais , Especificidade de ÓrgãosRESUMO
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.
Assuntos
Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Vesículas Sinápticas/metabolismo , Zinco/metabolismo , Animais , Proteínas de Transporte , Proteínas de Transporte de Cátions , Modelos Animais de Doenças , Imuno-Histoquímica/métodos , Proteínas de Membrana/deficiência , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Microscopia Imunoeletrônica/métodos , Neocórtex/patologia , Neurônios/metabolismo , Neurônios/ultraestrutura , Vesículas Sinápticas/ultraestrutura , Fatores de TempoRESUMO
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.
Assuntos
Doença de Alzheimer/metabolismo , Córtex Cerebral/metabolismo , Placa Amiloide/metabolismo , Zinco/deficiência , Zinco/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/genética , Animais , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Feminino , Alimentos Formulados , Masculino , Camundongos , Camundongos Transgênicos , Necessidades Nutricionais , Placa Amiloide/patologia , Presenilina-1/genética , Fatores de RiscoRESUMO
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.
Assuntos
Bismuto/metabolismo , Pontos Quânticos , Animais , Técnicas Histológicas , Rim/metabolismo , Rim/ultraestrutura , Fígado/metabolismo , Fígado/ultraestrutura , Masculino , Radioisótopos/metabolismo , Ratos , Ratos WistarRESUMO
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.
Assuntos
Autorradiografia/métodos , Pontos Quânticos , Animais , Células Cultivadas , Feminino , Ouro , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , NanopartículasRESUMO
Nerve cells are highly susceptible to ischemic and hypoxic injuries. The neuroglobin (Ngb), found in vertebrate nerve cells, has been suggested to protect nerve cells from ischemic episodes by a yet unknown mechanism. However, contradicting reports exist regarding localization and up-regulation of Ngb in response to hypoxia. The aim of the present study was to probe the distribution of Ngb proteins in mouse brain and retina by immunohistochemistry, and to quantify the levels of Ngb mRNA by reverse-transcription-polymerase chain reaction (RT-PCR) after short-term (2 h) exposure to 7.6% oxygen. We found Ngb to be present throughout the neocortex, most abundantly in the perirhinal, entorhinal and temporal cortical areas, the thalamus and hypothalamus, the choroid plexus, the olfactory bulb and the cranial nerve nuclei in the brainstem. Intense staining was observed in the mesencephalic central grey area and the Purkinje cells. Two-hour hypoxic exposure caused no detectable changes in staining intensity or spatial distribution of Ngb neither in the Purkinje cells nor in any other brain areas observed. The RT-PCR data supported the lack of differences in brain Ngb levels between normal and oxygen-deprived animals. In the retina, Ngb localization by immunohistochemistry was confined to the inner segments of the photoreceptors, the plexiform layers and the ganglion cells. Short-termed hypoxia did not change retinal Ngb levels as assessed by both techniques. The lack of Ngb up-regulation in the brain is consistent with results from previous long-term hypoxic experiments, suggesting that Ngb is not regulated by pure hypoxia in vivo.
Assuntos
Globinas/genética , Globinas/metabolismo , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células de Purkinje/fisiologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Neuroglobina , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
An easy to perform autometallographic technique (AMG) for capturing zinc ions in Alzheimer plaques is presented. The possibility of visualizing loosely bound or free zinc ions in tissue by immersion autometallography (iZnS(AMG)) is a relatively recent development. The iZnS(AMG) staining is caused by zinc-sulphur nanocrystals created in 1-2 mm thick brain slices that are immersed in a 0.1% sodium sulphide, 3% glutaraldehyde phosphate buffered solution, the NeoTimm Solution (NTS), for 3 days. When the zinc-sulphur nanocrystals are subsequently silver-enhanced by autometallography, the plaques are readily identified as spheres of dark interlacing strands of different sizes, embedded in the pattern of zinc-enriched terminals. The zinc specificity of the iZnS(AMG) technique was tested by immersion of brain slides in the chelator DEDTC prior to the NTS immersion. The iZnS(AMG) detection of zinc ions is easily standardized and can be used in the quantification of plaques with stereological methods. This technique is the first to detect zinc in plaques in the cerebellum of transgenic PS1/APP mice and the first to detect zinc ions in plaques and dystrophic neurites at electron microscopical levels.
Assuntos
Doença de Alzheimer/metabolismo , Placa Amiloide/química , Zinco/análise , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Cristalografia/métodos , Feminino , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Nanotecnologia/métodos , Placa Amiloide/patologia , Placa Amiloide/ultraestrutura , Presenilina-1RESUMO
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.
Assuntos
Glucose/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Zinco/metabolismo , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Insulina/metabolismo , Secreção de Insulina , Insulinoma , Neoplasias Pancreáticas , Ratos , Coloração e RotulagemRESUMO
The distribution of mercury in the liver of the frog Rana ridibunda with the autometallographic method was investigated. The mercury specific autometallographic (HgS/SeAMG) technique is a sensitive histochemical approach for tracing mercury in tissues from mercury-exposed organisms. Mercury accumulates in vivo as mercury sulphur/mercury selenium nanocrystals that can be silver-enhanced. Thus, only a fraction of the Hg can be visualized. Six animals were exposed for one day and another group of six animals for 6 days in 1 ppm mercury (as HgCI2 ) dissolved in fresh water. A third group of six animals, served as controls, were sacrificed the day of arrival at the laboratory. First, mercury appears in the blood plasma and erythrocytes. Next, mercury moves to hepatocytes and in the apical part of the cells, that facing bile canaliculi. In a next step, mercury appears in the endothelial and Kupffer cells. It seems likely that, the mercury of hepatocytes moves through bile canaliculi to the gut, most probably bound to glutathione and/or other similar ligands. Most probably, the endothelial and Kupffer cells comprise the first line of defense against metal toxicity.
Assuntos
Monitoramento Ambiental/métodos , Poluentes Ambientais/análise , Fígado/química , Mercúrio/análise , Rana ridibunda , Animais , Feminino , Histocitoquímica/métodosRESUMO
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.
Assuntos
Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osteogênese/efeitos dos fármacos , Zinco/deficiência , Zinco/uso terapêutico , Animais , Bioensaio/métodos , Densidade Óssea/efeitos dos fármacos , Densidade Óssea/fisiologia , Matriz Óssea/química , Matriz Óssea/efeitos dos fármacos , Matriz Óssea/metabolismo , Osso e Ossos/citologia , Calcificação Fisiológica/efeitos dos fármacos , Calcificação Fisiológica/fisiologia , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Alimentos Formulados , Lâmina de Crescimento/citologia , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/metabolismo , Histocitoquímica/métodos , Íons/análise , Íons/metabolismo , Masculino , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/fisiologia , Ratos , Ratos Wistar , Resultado do Tratamento , Zinco/análiseRESUMO
Zinc ions in the secretory granules of beta-cells are known to glue insulin molecules, creating osmotically stable hexamers. When the secretory granules open to the surface, the zinc ion pressure decreases rapidly and pH levels change from acid to physiological, which results in free insulin monomers and zinc ions. The released zinc ions have been suggested to be involved in a paracrine regulation of alpha- and beta-cells. Since zinc is intimately involved in insulin metabolism and because zinc homeostasis is known to be disturbed in type 2 diabetes, we decided to study the ultrastructural localisation of zinc ions in insulin-resistant and type 2 diabetic rats as compared to controls. By means of autometallography, the only method available for demonstrating zinc ions at ultrastructural levels, we found zinc ions in the secretory granules and adjacent to the plasma membrane. The membrane-related staining outside the plasma membrane reflects release of zinc ions during exocytosis. No apparent difference was found in the ultrastructural localisation of zinc ions when we compared the obese Zucker (fa/fa) rats, representing the insulin resistance syndrome, and the GK rats, representing type 2 diabetes, with controls. This suggests that the ultrastructural localisation of zinc ions is unaffected by the development of type 2 diabetes in rats in a steady state of glycaemia.
Assuntos
Diabetes Mellitus Tipo 2/patologia , Diabetes Mellitus/patologia , Ilhotas Pancreáticas/patologia , Obesidade , Zinco/análise , Animais , Diabetes Mellitus/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Teste de Tolerância a Glucose , Imuno-Histoquímica , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/ultraestrutura , Microscopia Imunoeletrônica , Ratos , Ratos ZuckerRESUMO
A new autometallographic (AMG) technique for staining myelin in formaldehyde- or paraformaldehyde- (PFA) fixed tissue is presented. The tissue sections were exposed to AMG development without prior treatment with silver salts. The method was examined on PFA-fixed tissue from mouse, rat, pig, and formaldehyde-fixed human autopsy material. Samples from brain, spinal cord, cranial, and spinal nerves were either cut on a vibratome, frozen and cryostat sectioned, or embedded and microtome sectioned, before AMG development and counterstaining. The AMG-myelin technique results in a specific black/dark-brown staining of myelin in all parts of the CNS and PNS. It works on all species examined, independent of the histological preparation techniques applied. The AMG staining is stable, stays unchanged through decades, allows counterstaining, and has previously been used with immunohistochemical techniques. On perfusion-fixed tissue the technique works without further fixation, but the intensity of the AMG-myelin staining is increased by increased postfixation time. Additionally, immersion fixation has to last for days depending on the size of the tissue block in order to obtain proper myelin staining. The most feasible explanation of the chemical events underlying the AMG-myelin technique is that nano-sized clusters of metallic silver are formed in the myelin as a result of chemical bounds with reducing capacity, exposed or created by the formaldehyde molecule. The AMG method is simple to perform and as specific as the conventional osmium and luxol fast blue stainings. The present technique is thus an effective, simple, inexpensive, and quick myelin staining method of formaldehyde- or PFA-fixed tissue.
Assuntos
Autorradiografia/métodos , Bainha de Mielina/química , Bainha de Mielina/metabolismo , Animais , Encéfalo/anatomia & histologia , Química Encefálica , Soluções Tampão , Citratos/química , Coloides/química , Corantes , Fixadores , Formaldeído , Humanos , Indicadores e Reagentes , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Wistar , Substâncias Redutoras , Prata/química , Coloração pela Prata , Especificidade da Espécie , Suínos , Porco Miniatura , Inclusão do Tecido , Fixação de TecidosRESUMO
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.
Assuntos
Proteínas de Transporte/análise , Proteínas de Membrana/análise , Proteínas de Membrana Transportadoras , Gânglio Cervical Superior/química , Proteínas de Transporte Vesicular , Zinco/análise , Animais , Axônios/química , Proteínas de Transporte de Cátions , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Microscopia de Fluorescência , Neuropeptídeo Y/análise , Ratos , Ratos Wistar , Tirosina 3-Mono-Oxigenase/análise , Proteínas Vesiculares de Transporte de AcetilcolinaRESUMO
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.
Assuntos
Bismuto/farmacocinética , Poluentes Ambientais/farmacocinética , Armas de Fogo , Íons , Animais , Animais Selvagens , Bismuto/metabolismo , Sistema Nervoso Central/metabolismo , Plexo Corióideo/metabolismo , Rim/metabolismo , Córtex Renal/metabolismo , Células Intersticiais do Testículo/metabolismo , Masculino , Metais Pesados , Neurônios/metabolismo , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Bismuth-containing drugs have several applications, one being their use against Helicobacter pylori-associated peptic ulcers, and bismuth has been discovered in macrophages at the base and margins of peptic ulcers. In the present study, the autometallographic technique for the histochemical demonstration of bismuth was applied, showing that bismuth citrate-exposed J774 cells accumulate the metal in their lysosomes. Such accumulations resulted in lysosomal rupture - assayed by the acridine orange uptake technique and flow cytofluorometry - and ensuing apoptotic cell death.
Assuntos
Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Compostos Organometálicos/farmacocinética , Compostos Organometálicos/toxicidade , Laranja de Acridina/química , Animais , Citometria de Fluxo , Corantes Fluorescentes/química , Lisossomos/patologia , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Compostos Organometálicos/efeitos adversos , Células Tumorais CultivadasRESUMO
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).
Assuntos
Fêmur/efeitos dos fármacos , Zinco/farmacologia , Animais , Peso Corporal , Relação Dose-Resposta a Droga , Fêmur/fisiologia , Masculino , Ratos , Ratos WistarRESUMO
Electrophysiological experiments have shown that zinc ions modulate glutamate and GABA receptors in brain slices. All the zinc-enriched neuronal pathways in the brain analyzed up until now have been found to be glutaminergic. Many years ago, zinc-enriched terminals with flat vesicles and symmetric synapses were found to be present in rat spinal cord by Henrik Daa Schrøder, and recently these findings have been supported by immunohistochemical and electron microscopical data in lamprey, mouse and rat. In the present study we expanded these observations by revealing a colocalization of zinc ions, zinc transporter-3 (ZnT3) and glutamic acid decarboxylase (GAD) in synaptic vesicles of zinc-enriched terminals throughout the mouse spinal cord. Confocal analysis of ZnT3 and GAD immunofluorescence was used at light microscopical levels, and a combination of zinc selenium autometallography and GAD immunocytochemistry at electron microscopic levels. Zinc-enriched/GABAergic terminals were observed in all laminae of the spinal gray matter, but most densely populated were laminae I and III in the dorsal horn. In the lateral and ventral funiculi of the white matter, rows of inhibitory zinc-enriched boutons were seen radiating from the gray matter. Ultrastructurally, colocalization of zinc ions and GAD immunoreactivity was seen in a pool of presynaptic terminals in the above locations. Some zinc-enriched terminals were not GAD-positive and some GAD-positive terminals were void of zinc ions. The majority of the zinc-enriched, not GABAergic terminals could be classified as excitatory based on their morphology, i.e. round clear vesicles and symmetric synapses. We conclude that a majority of the spinal cord zinc-enriched terminals are GABAergic. The zinc-enriched terminals with excitatory morphology are most likely glutaminergic, a few have an inhibitory morphology but are not GABAergic. These are most likely glycinergic.
Assuntos
Inibição Neural/fisiologia , Terminações Pré-Sinápticas/metabolismo , Medula Espinal/metabolismo , Transmissão Sináptica/fisiologia , Zinco/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Proteínas de Transporte/metabolismo , Imunofluorescência , Glutamato Descarboxilase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Neurônios Motores/metabolismo , Neurônios Motores/ultraestrutura , Fibras Nervosas Mielinizadas/metabolismo , Fibras Nervosas Mielinizadas/ultraestrutura , Células do Corno Posterior/metabolismo , Células do Corno Posterior/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Compostos de Selênio/metabolismo , Medula Espinal/ultraestrutura , Compostos de Zinco/metabolismoRESUMO
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.
Assuntos
Terminações Nervosas/fisiologia , Inibição Neural/fisiologia , Medula Espinal/fisiologia , Zinco/metabolismo , Animais , Proteínas de Transporte/metabolismo , Glutamato Descarboxilase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Isoformas de Proteínas/metabolismo , Compostos de Selênio , Medula Espinal/metabolismo , Medula Espinal/ultraestrutura , Distribuição TecidualRESUMO
Zinc enriched neurons have a pool of synaptic vesicles which contain free or loosely-bound zinc ions. The movement of the vesicular zinc ions into the synaptic clefts has been previously studied by microdialysis, fluorescence postmortem staining for zinc and radioactive zinc isotope. In this study the zinc fluorescence probe N-6-metoxy-p-toluensulfonamide quinoline (TSQ) has been applied as a tracer of synaptic release of zinc ions. This fluorochrome permeates cell membranes and when exposed to living brain slices gives rise to a staining pattern similar to that seen with autometallography. In the living brain slices, fluorescence emission persists after exposure to calcium saturated ethylen diamino-tetra-acetic acid (Ca-EDTA) because this chelator does not penetrate cell membranes, while sodium dethyldithiocarbamate (DEDTC), that does penetrate membranes, partially suppressed the fluorescence emission. Stimulation of slices bathed in the non-permeant chelator Ca-EDTA with 50 mM potassium chloride leads to a rapid and complete disappearance of fluorescence. In the absence of Ca-EDTA, however, potassium stimulation induces a sudden transitory increase in fluorescence. This increase is caused by a translocation of the fluorochrome (TSQ) zinc molecules from the weakly acid interior of the synaptic vesicles to the neutral extracellular space, whereby the fluorescence emission of the molecules is enhanced sufficiently to be recorded by a high sensitivity TV camera.
Assuntos
Aminoquinolinas/metabolismo , Membrana Celular/metabolismo , Corantes Fluorescentes/metabolismo , Neurofisiologia/métodos , Vesículas Sinápticas/metabolismo , Compostos de Tosil/metabolismo , Zinco/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Ácido Edético/farmacologia , Processamento Eletrônico de Dados/instrumentação , Processamento Eletrônico de Dados/métodos , Processamento de Imagem Assistida por Computador/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Lagartos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Microscopia de Vídeo/instrumentação , Microscopia de Vídeo/métodos , Neurofisiologia/instrumentação , Cloreto de Potássio/farmacologia , Coelhos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/efeitos dos fármacos , Telencéfalo/citologia , Telencéfalo/efeitos dos fármacos , Telencéfalo/metabolismoRESUMO
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.