Gorm Danscher

Intravesicular localization of zinc in rat telencephalic boutons. A histochemical study

A transition metal (presumably zinc) has been localized inside the synaptic vesicles of rat telencephalic boutons which make asymmetric synaptic contacts (Gray type I). A modification of Timms sulphide-silver method for electron microscopy was applied to different areas of the rat forebrain: olfactory bulb, septum, caudate-putamen, amygdaloid complex, neocortex, entorhinal cortex and different parts of the hippocampal formation, viz. subiculum, stratum radiatum and oriens of both regio superior (CA1) and regio inferior hippocampi (CA3), the mossy fibre zone and the hilus fascia dentata and stratum moleculare in the gyrus dentatus. It was observed that the Timm staining in all these areas was located in synaptic boutons. Labelled boutons displayed a population of round clear vesicles and few dense core vesicles and made asymmetric synaptic contacts on dendritic spines. Silver granules in these boutons were confined to only a minor fraction of the round clear vesicles (approximately 10%). Labelled boutons occasionally showed silver granules in the synaptic clefts. The possible involvement of vesicular zinc in synaptic transmission is discussed.

Localization of gold in biological tissue

SummaryA detailed description is given of a method by which gold can be visualized in frozen, paraffin and Epon sections. Histological sections from animals treated with gold compounds are exposed to UV-light from 30 min to several hours. The reduced, metallic gold is then visualized by means of a photographic developer containing silver lactate. Light- and electronmicroscope photographs showing gold in different organs from rats and mice treated with aurothioglucose, aurothiosulfate and aurothiomalate are presented.

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

BackgroundThe 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.ResultsGold 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.ConclusionOur 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.

An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues.

SummaryModifications of the Timm sulphide silver method for the demonstration of heavy metals are described.To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixative is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.

Histochemically-reactive zinc in amyloid plaques, angiopathy, and degenerating neurons of Alzheimer's diseased brains

Excess brain zinc has been implicated in Alzheimers neuropathology. Here we evaluated that hypothesis by searching the brains of Alzheimers patients for abnormal zinc deposits. Using histochemical methods, we found vivid Zn2+ staining in the amyloid deposits of dense-core (senile) plaques, in the amyloid angiopathy surrounding diseased blood vessels, and in the somata and dendrites of neurons showing the characteristic neurofibrillary tangles (NFT) of Alzheimers. In contrast, brains from age-matched, non-demented subjects showed only occasional staining for Zn2+ in scattered neurons and possible plaques. A role of abnormal zinc metabolism in Alzheimers neuropathology is suggested.

Evidence that synaptically-released zinc contributes to neuronal injury after traumatic brain injury

Prior evidence indicates that synaptically-released zinc enters postsynaptic neurons in toxic excess during ischemia and seizures. In addition, prevention of this zinc translocation has been shown to be neuroprotective in both ischemia and seizures. Here we show evidence that the same translocation of zinc from presynaptic boutons into postsynaptic neurons occurs after mechanical injury to the brain. Specifically, using a rat model of traumatic brain injury, we show that trauma is associated with (i) loss of zinc from presynaptic boutons (ii) appearance of zinc in injured neurons, and (iii) neuroprotection by intraventricular administration of a zinc chelator just prior to brain impact. The possible use of zinc chelators for neuroprotection after head trauma is considered.

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.

Light and electron microscopic localization of silver in biological tissue

SummaryA method is described that visualizes trace amounts of silver in frozen, paraffin and epon sections from biological tissue. After exposure to light, which ensures reduction of silver ions that are not bound to sulphide, histological sections from animals treated with silver compounds are exposed to a photographic developer containing silver ions. Tissue silver acts as a catalyst for the hydroquinone reduction of silver ions to metallic silver which then accumulates at the site of the trace deposit. Light and electron micrographs showing silver in different organs from albino rats treated with silver lactate are presented. Localization of silver in motor neurons of the spinal gray matter and pons indicates a transport of silver over the blood-brain barrier. Silver precipitates in fetal liver suggest that silver ions can penetrate the placental barrier.

Increased amount of zinc in the hippocampus and amygdala of Alzheimer's diseased brains: A proton-induced X-ray emission spectroscopic analysis of cryostat sections from autopsy material

Zinc has been implicated as a contributing cause of the neuropathology of Alzheimers disease (AD), but consensus on the zinc content of AD brains has not yet been established. In the present study, multi-element PIXE was used to measure zinc in cryostat sections of brain tissue from AD patients and from normal control subjects. Compared to their age-matched controls, the AD patients showed an increase in zinc in the hippocampal and amygdalar regions. The instrumental PIXE assays do not show whether the zinc changes are due to altered zinc in the boutons of Zinc-ENriched (ZEN) neurons, i.e., zinc ions in synaptic vesicles, or to changes in the amount of zinc tightly bound to macromolecules. We hypothesise that the increased zinc level is caused by an increase in the amount of ZEN terminals. Such an increase could be the result of a sprout of ZEN terminals in diseased areas of the brain.

The dithizone, Timm's sulphide silver and the selenium methods demonstrate a chelatable pool of zinc in CNS

SummaryFrom rats intravitally treated with dithizone (diphenyl-thiocarbazone) brains and spinal cords were removed and freeze-dried. The dithizonates present in the CNS tissue were extracted with carbon tetrachloride and subjected to a multielement analysis (proton activation, PIXE). It was found that the extract contained two metals. Most of the metal was zinc, but small traces of copper were also dectected. Because prior treatment with the chelating agent, dithizone, can block both the Timm and the selenium metal staining methods, it is suggested that the three techniques label predominantly zine in the neuropil (DTS-zine).