nanoGold and µGold inhibit autoimmune inflammation: a review.

The newest data on metallic gold have placed the noble metal central in the fight for the safe treatment of autoimmune inflammation. There are two different ways to use gold for the treatment of inflammation: gold microparticles > 20 µm and gold nanoparticles. The injection of gold microparticles (µGold) is a purely local therapy. µGold particles stay put where injected, and gold ions released from them are relatively few and taken up by cells within a sphere of only a few millimeters in diameter from their origin particles. The macrophage-induced release of gold ions may continue for years. Injection of gold nanoparticles (nanoGold), on the other hand, is spread throughout the whole body, and the bio-released gold ions, therefore, affect multitudes of cells all over the body, as when using gold-containing drugs such as Myocrisin. Since macrophages and other phagocytotic cells take up and transport nanoGold and remove it after a short period, repeated treatment is necessary. This review describes the details of the cellular mechanisms that lead to the bio-release of gold ions in µGold and nanoGold.

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

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

In vivo 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.

Effects of dissolucytotic gold ions on recovering brain lesions.

Recent experimental research has shown that metallic gold releases charged gold atoms when placed intracerebrally and that the liberated gold ions affect inflammation in the brain. The observations suggest that metallic gold can be used as a safe suppressor of inflammation in the central nervous system.

Protracted elimination of gold nanoparticles from mouse liver.

The present study aims at revealing the fate of 40-nm gold nanoparticles after intravenous injections. The gold nanoparticles were traced histochemically with light and transmission electron microscopy using autometallographic (AMG) staining, and the gold content in the liver was determined with inductively coupled plasma mass spectrometry (ICP-MS). Gold nanoparticles were identified in almost all Kupffer cells one day after the injection, but the fraction of gold-loaded cells gradually decreased to about one fifth after 6 months. Transmission electron microscopic analysis showed that the gold nanoparticles had accumulated inside the vesicular lysosome/endosome-like structures of the macrophages. At day 1, about 4.5 per thousand of the area of the liver sections was AMG-stained, after 1 month it had decreased to 0.7 per thousand, and thereafter no further significant reduction was recorded. Because ICP-MS only showed a 9% fall in the gold content over the observed 6 months, the AMG finding of a significant reduction in the stained area of the liver sections and number of macrophages loaded with gold nanoparticles reveals that over time an increasing part of the total amount of gold nanoparticles in the liver is contained in fewer macrophages accumulated in growing clusters.

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.

Kupffer cells are central in the removal of nanoparticles from the organism.

BACKGROUND: The study aims at revealing the fate of nanoparticles administered intravenously and intraperitoneally to adult female mice, some of which were pregnant. Gold nanoparticles were chosen as a model because these particles have been found to be chemically inert and at the same time are easily traced by autometallography (AMG) at both ultrastructural and light microscopic levels. RESULTS: Gold nanoparticles were injected intravenously (IV) or intraperitoneally (IP) and traced after 1, 4 or 24 hours. For IV injections 2 and 40 nm particles were used; for IP injections 40 nm particles only. The injected nanoparticles were found in macrophages only, and at moderate exposure primarily in the Kupffer cells in the liver. IV injections resulted in a rapid accumulation/clustering of nanoparticles in these liver macrophages, while the uptake in spleen macrophages was moderate. IP injections were followed by a delayed uptake in the liver and included a moderate uptake in macrophages located in mesenteric lymph nodes, spleen and small intestine. Ultrastructurally, the AMG silver enhanced nanocrystals were found in lysosome-like organelles of the Kupffer cells and other macrophages wherever located.Accumulations of gold nanoparticles were not found in any other organs analysed, i.e. kidneys, brain, lungs, adrenals, ovaries, placenta, and fetal liver, and the control animals were all void of AMG staining. CONCLUSION: Our results suggest that: (1) inert gold nanoparticles do not penetrate cell membranes by non-endocytotic mechanisms, but are rather taken up by endocytosis; (2) gold nanoparticles, independent of size, are taken up primarily by Kupffer cells in the liver and secondarily by macrophages in other places; (3) gold nanoparticles do not seem to penetrate the placenta barrier; (4) the blood-brain barrier seems to protect the central nervous system from gold nanoparticles; (5) 2 nanometer gold particles seem to be removed not only by endocytosis by macrophages, and we hypothesize that part of these tiny nanoparticles are released into the urine as a result of simple filtration in the renal glomeruli.

The lack of effects of zinc and nitric oxide in initial state of pilocarpine-induced seizures.

In this study we investigated whether intracerebroventricular (i.c.v.) injection of L-NAME (a nitric oxide synthase inhibitor) or CaEDTA (an extracellular zinc chelator) or the combination of the two could affect the initial phase of pilocarpine induced (2 h) seizures. Two groups of rats were used. Animals from both groups were given with i.c.v. injections of either saline (10 microl), L-NAME (150 microg/10 microl), CaEDTA (100 mM/10 microl) or L-NAME and CaEDTA. One group received pilocarpine HCl (380 mg/kg i.p.) the other served as control. Pilocarpine HCl was injected intraperitoneally 10 min later. The behavior of the animals was observed for 2h and the intensity of their seizures was scored. The rats were then sacrificed and their brains were removed and analyzed for zinc ions by using the immersion autometallography and the TSQ fluorescence staining. All the animals which received pilocarpine HCl developed seizures. Despite treatment with L-NAME and/or CaEDTA we found that the latency and the intensity of seizures were similar in both groups investigated. The distribution of stainable zinc ions and the intensity of staining in hippocampus were not affected by pilocarpine and found unchanged after L-NAME and/or CaEDTA injections in both the control animals and the pilocarpine treated animals. The data suggest that the nitric oxide system and zinc ions do not affect pilocarpine-induced seizures in their initial state.

Silver enhancement of quantum dots resulting from (1) metabolism of toxic metals in animals and humans, (2) in vivo, in vitro and immersion created zinc-sulphur/zinc-selenium nanocrystals, (3) metal ions liberated from metal implants and particles.

Autometallographic (AMG) silver enhancement is a potent histochemical tool for tracing a variety of metal containing nanocrystals, e.g. pure gold and silver nanoclusters and quantum dots of silver, mercury, bismuth or zinc, with sulphur and/or selenium. These nanocrystals can be created in many different ways, e.g. (1) by manufacturing colloidal gold or silver particles, (2) by treating an organism in vivo with sulphide or selenide ions, (3) as the result of a metabolic decomposition of bismuth-, mercury- or silver-containing macromolecules in cell organelles, or (4) as the end product of histochemical processing of tissue sections. Such nano-sized AMG nanocrystals can then be silver-amplified several times of magnitude by being exposed to an AMG developer, i.e. a normal photographic developer enriched with silver ions. The present monograph attempts to provide a review of the autometallographic silver amplification techniques known today and their use in biology. After achieving a stronghold in histochemistry by Timm's introduction of the "silver-sulphide staining" in 1958, the AMG technique has evolved and expanded into several different areas of research, including immunocytochemistry, tracing of enzymes at LM and EM levels, blot staining, retrograde axonal tracing of zinc-enriched (ZEN) neurons, counterstaining of semithin sections, enhancement of histochemical reaction products, marking of phagocytotic cells, staining of myelin, tracing of gold ions released from gold implants, and visualization of capillaries. General technical comments, protocols for the current AMG methods and a summary of the most significant scientific results obtained by this wide variety of AMG histochemical approaches are included in the present article.

Neurotoxic zinc translocation into hippocampal neurons is inhibited by hypothermia and is aggravated by hyperthermia after traumatic brain injury in rats.

Hypothermia reduces excitotoxic neuronal damage after seizures, cerebral ischemia and traumatic brain injury (TBI), while hyperthermia exacerbates damage from these insults. Presynaptic release of ionic zinc (Zn2+), translocation and accumulation of Zn2+ ions in postsynaptic neurons are important mechanisms of excitotoxic neuronal injury. We hypothesized that temperature-dependent modulation of excitotoxicity is mediated in part by temperature-dependent changes in the synaptic release and translocation of Zn2+. In the present studies, we used autometallographic (AMG) and fluorescent imaging of N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ) staining to quantify the influence of temperature on translocation of Zn2+ into hippocampal neurons in adult rats after weight drop-induced TBI. The central finding was that TBI-induced Zn2+ translocation is strongly influenced by brain temperature. Vesicular Zn2+ release was detected by AMG staining 1 h after TBI. At 30 degrees C, hippocampus showed almost no evidence of vesicular Zn2+ release from presynaptic terminals; at 36.5 degrees C, the hippocampus showed around 20% to 30% presynaptic vesicular Zn2+ release; and at 39 degrees C vesicular Zn2+ release was significantly greater (40% to 60%) than at 36.5 degrees C. At 6 h after TBI, intracellular Zn2+ accumulation was detected by the TSQ staining method, which showed that Zn2+ translocation also paralleled the vesicular Zn2+ release. Neuronal injury, assessed by counting eosinophilic neurons, also paralleled the translocation of Zn2+, being minimal at 30 degrees C and maximal at 39 degrees C. We conclude that pathological Zn2+ translocation in brain after TBI is temperature-dependent and that hypothermic neuronal protection might be mediated in part by reduced Zn2+ translocation.

Localization of ZnT7 and zinc ions in mouse retina--immunohistochemistry and selenium autometallography.

Zinc transporter 7 (ZnT7, Slc30a7), a member of the Slc30 family, is involved in mobilizing zinc ions from the cytoplasm into the Golgi apparatus. In the present study, we examined the distribution and localization of ZnT7 and the labile zinc ions in the mouse retina using immunohistochemistry and in vivo zinc-selenium autometallography (ZnSe(AMG)). Our results showed that ZnT7 is abundantly expressed in the ganglion cells and pigment epithelial cells of the mouse retina. ZnT7 is also expressed in the amacrine cells and the layer of optic fibers of the mouse retina, but to a lesser extent. Weak staining of ZnT7 was detected in the inner plexiform layer, outer plexiform layer, and outer segment of the photoreceptors. However, ZnT7 was not detected in the outer nuclear layer and inner segment of the photoreceptors. A high level of labile zinc pool was detected in the pigment epithelial cells, the inner segment of the photoreceptors, and the marginal region of the inner nuclear layer. Less amount of labile zinc ions were detected in the ganglion cells of the retina. These observations strongly suggest that ZnT7 may play critical roles in retinal zinc homeostasis and that chelatable zinc pools may have multiple functions in the retina.

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.

Adrenalectomy-induced ZnT3 downregulation in mouse hippocampus is followed by vesicular zinc depletion.

The effects of adrenalectomy (ADX) on the vesicular zinc content of zinc-enriched (ZEN) terminals in mouse hippocampus were investigated at light microscopic levels using zinc transporter-3 immunohistochemistry (ZnT3IHC) and zinc selenium autometallography (ZnSeAMG). ZnT3 resides in the synaptic vesicle membranes of ZEN neurons and is believed to move zinc ions into the vesicles. ZnT3IHC staining closely corresponds to the ZnSeAMG staining, but in the present study we present evidence of a delayed decrease of ZEN zinc, as compared to downregulation of the ZnT3 protein following ADX. Twenty-four hours after adrenalectomy the level of ZnT3IHC was visibly reduced while the ZnSeAMG staining intensity seemed unchanged. After 10 and 30 days, however, downregulation of ZnT3 was paralleled by a distinct reduction in ZnSeAMG staining. The total protein concentration of ZnT3 was reduced by about 53%, and the total zinc concentration in the hippocampus of the same mice was reduced by 43-64%, 30 days after the adrenalectomy. The present results support previous results suggesting that ZnT3 is responsible for transport of zinc ions into a pool of synaptic vesicles in ZEN terminals.

Abundant expression of zinc transporters in Bergman glia of mouse cerebellum.

Zinc transporters (ZnTs) are membrane proteins involved in zinc ion transportation in mammalian cells. Seven members of ZnT family, ZnT1-7, have been cloned and characterized. These transporter proteins have different cellular and sub-cellular locations, suggesting that they may play different roles in zinc homeostasis in normal and pathological conditions in different tissues. Cerebellum is one of the most zinc-enriched regions in the central nervous system, but little is known about zinc metabolism in the cerebellum. In the present study, we investigated the detailed distributions of four members (ZnT1, ZnT3, ZnT4 and ZnT6) of the ZnT family, in the mouse cerebellum. Immunostaining and confocal microscopic observations revealed a similar staining pattern of ZnTs in the molecular layer and the Purkinje cell layer. Double labeling with anti-S-100beta or anti-MAP2 and anti-ZnTs clearly showed that the Bergman glial cell bodies in the Purkinje cell layer and their radial processes in the molecular layer exhibited strong immunofluorescence of all the tested ZnTs. However, the somata of the Purkinje cells contained a moderate immunostaining for ZnT1, but virtually lack of other three ZnTs. In the granular layer, ZnTs appeared with different immunostaining patterns. ZnT1 was expressed in a small number of neuronal cell bodies and their primary dendrites, whereas ZnT3 and ZnT4 were present in nerve terminals but not in the neuronal somata. ZnT6 was undetectable in either the cell bodies or processes in the granular layer. The present results indicate that the Bergman glial cells may play an important role in zinc metabolism in the mouse cerebellar cortex.

Does selenium deficiency unmask mercury toxicity in motor neurons?

OBJECTIVE: Inorganic mercury enters in particular motor neurons and has been implicated in motor neuron diseases. One way that cells protect themselves from mercury toxicity is via selenium, so we sought to determine whether the motor neurons of mice on a low selenium diet would be more susceptible to mercury toxicity. METHODS: Recently weaned mouse pups were placed on diets containing either low, normal or high levels of selenium. Twenty days later, half were exposed to mercury vapor. Ninety days after exposure, their spinal motor neurons and phrenic motor axons were examined histologically. Mercury in the spinal cord was sought using autometallography. RESULTS: Neither low nor high selenium diets combined with mercury vapor had any clinical effect on the mice. Mercury was seen within the spinal motor neurons of all exposed mice. Spinal motor neurons and phrenic motor axons however appeared normal in morphology and size across the groups. CONCLUSION: Diets low or high in selenium did not damage motor neurons with or without mercury. This suggests that changes in the selenium environment are unlikely to precipitate mercury toxicity in motor neurons.

In vivo distribution of bismuth in the mouse brain: influence of long-term survival and intracranial placement on the uptake and transport of bismuth in neuronal tissue.

Bismuth is used for a multitude of industrial purposes and has partly replaced toxic heavy metals such as lead and mercury in e.g. lubricants and shotgun pellets. In medicine, bismuth-compounds have long been used to remedy gastrointestinal disorders; lately in combination with antibiotics to treat Helicobacter pylori associated peptic ulcers. An epidemic episode of bismuth-induced encephalopathy in France in the 1970s revealed the neurotoxic potential of bismuth. This incidence, involving almost 1000 patients, remains unexplained and the contribution of other factors besides bismuth has been postulated. Recently an autometallographic technique made it possible to detect bismuth in morphologically intact tissue. In the present study, autometallographicly detectable bismuth was seen throughout the brain following intraperitoneal and intracranial exposure. The neuronal staining pattern seems highly organized with some areas heavily stained and others with low or no staining. Long-term (8 months) intraperitoneal exposure led to higher bismuth uptake than short-term (2 weeks) exposure. Following both intraperitoneal and intracranial exposure, high amounts of bismuth were found in the reticular and hypothalamic nuclei, in the oculomotor and hypoglossal nuclei and in Purkinje cells. Within the central nervous system (CNS) retrograde axonal transport was seen after intracranial bismuth exposure. Axonal transport seems to influence the distribution of bismuth as the highest uptake of bismuth after intraperitoneal exposure was seen in the facial and the trigeminal motor nuclei, i.e. neurones with processes outside the blood-brain barrier, whereas these nuclei contained no bismuth following ic exposure. Ultrastructurally, accumulation of bismuth was seen in lysosomes.

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.

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.

Dynamic zinc pools in mouse choroid plexus.

We examined the presence of Zn-transporters (ZnT1, ZnT3, ZnT4, and ZnT6) proteins and zinc ions in rat choroid epithelium with immunohistochemistry and zinc selenide autometallography (ZnSe(AMG)). The four ZnT proteins were all expressed in the choroid epithelial cells. ZnT3 immunostaining was found in vesicle membranes in the apical part of the cells, associated to the microvillus membrane. Correspondingly, the ZnSe(AMG) technique revealed zinc ions in small vesicles, in microvilli, and multivesicular bodies in the epithelial cells. Traceable zinc ions were also found in lysosome-like organelles of fenestrated endothelial cells, but here no corresponding ZnT3 immunostaining was seen. The observations suggests that the choroid plexus is instrumental to regulation of the level of zinc ions in the cerebrospinal fluid.