Søren Juhl

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.

Ultrastructural localization of zinc ions in the rat prostate : An autometallographic study

The prostate contains high amounts of free zinc ions which are excreted into the seminal fluid. The extra‐ and intracellular distribution of zinc ions using the highly specific autometallographical (AMG) method is described.

How to detect gold, silver and mercury in human brain and other tissues by autometallographic silver amplification

Gold, silver, mercury and zinc bind chemically to sulphide or selenide ions and create crystal lattices that can be detected in histological sections by a silver amplification technique called autometallography (AMG). The technique specifically magnifies such nanometer–sized catalytic crystals. For each metal, a detailed protocol has been worked out. If several different AMG metals/metal molecules are present in the same tissue, it is possible to distinguish one from another. The AMG technique is based on the capability of small crystal lattices of the aforementioned metals and metal molecules to initiate AMG silver amplification. Electrons released from adhering hydroquinone molecules reduce silver ions that are integrally connected with the crystal lattices. In this manner, particles consisting of only a few atoms of, say, gold, or molecules of mercury selenide (Figure 1), can be silver amplified to a size at which they can be detected in the electron microscope, or even further to dimensions that can be observed in the light microscope. Thus the AMG technique opens up the possibility of visualizing gold, e.g. in the nervous system of rheumatic patients who have been treated with aurothiomalate. Mercury can similarly be visualized in tissues from individuals who have been exposed to mercury, either through leaching from amalgam dental fillings, through eating fish, or by occupational exposure, and silver in the central (CNS) and peripheral nervous systems (PNS) and other tissues from individuals exposed to silver in one form or another.

Autometallographic Silver Enhancement of Zinc Sulfide Crystals Created in Cryostat Sections from Human Brain Biopsies: A New Technique that Makes it Feasible to Demonstrate Zinc Ions in Tissue Sections from Biopsies and Early Autopsy Material

We present a new technique that allows zinc ions in synaptic and secretory vesicles of biopsy and early autopsy material (>2 hr post mortem) to be transformed to nanometer-sized zinc sulfide crystal lattices for subsequent autometallographic (AMG) development. Human brain biopsies, or other tissue samples containing zinc-enriched (ZEN) cells, are frozen in liquid nitrogen or by CO2 gas immediately after removal. The tissue blocks are cut in a cryostat and the sections placed on glass slides. The slides are transferred to an H2S exposure chamber placed in a −15C freezer. After 1–24 hr of gas exposure the sections are removed from the chamber, fixed while thawing, and dehydrated. The sections are then exposed to an AMG developer. AMG causes silver enhancement of zinc sulfide crystal lattices created in the tissues through the H2S exposure, making them visible. It is imperative that the tissues are frozen instantaneously after removal, because loosely bound or free zinc ions start leaving their vesicular compartment soon after death. The AMG technique can, despite inadequate fixation and damage to the tissue caused by freezing, also be used to trace zinc ions at ultrastructural levels, and it is demonstrated that zinc ions in the human neocortex are located in synaptic vesicles. In the few human biopsies analyzed thus far, the light microscopic pattern created by the silver-enhanced ZEN terminals resembles that seen in the neocortex of rat brain. The technique has been applied to cryostat sections from neocortex biopsies of five individuals undergoing brain surgery. Biopsies from three patients resulted in satisfactory AMG-stained sections. Rat brains removed and frozen immediately after decapitation constituted the material on which the present technique was developed. Such material results in an almost uniform high quality of staining, and we found that unexposed sections can be stored for at least 5 months at −80C without ensuing significant loss of AMG staining intensity. (J Histochem Cytochem 45:1503–1510, 1997)

Distinct differences in partial oxygen pressure at micrometer ranges in the rat hippocampal region

A mapping at micrometer ranges of the partial oxygen pressure in the rat hippocampus was performed. The oxygen tension in the rat hippocampal region was measured using a glass oxygen microsensor in 30-microm steps along straight lines at a set of stereotactic coordinates. In the hippocampus the pattern of the oxygen tensions reflected the autometallographic zinc sulphide (AMG(ZnS)) pattern, i.e. the pattern of zinc enriched (ZEN) terminals. The highest levels of oxygen tension were recorded in the areas that are most heavily stained with the autometallographic zinc sulphide (AMG(ZnS)) method, like hilus fasciae dentatae. The zinc ions located in synaptic vesicles of the ZEN terminals can also be demonstrated by AMG silver amplification in brains from animals in vivo treated with sodium selenite. This method depends on the presence of a substantial reduction capacity of the tissues as selenite ions (SeO(2)(3)-) must to be reduced to selenide ions (Se2-) before the catalytic zinc selenide crystals can be formed. At some point, either during the transport from the infusion site to the actual target tissue or in the target tissue itself, selenium is reduced from Se(+ IV) to Se(- II). The importance of the reduction capacity of the target tissue in this process is demonstrated by the fact that areas found to have the highest concentration of zinc ions, e.g. hilus fasciae dentatae and the mossy fibres of CA3, are almost unstained after 1 h of i.p. Na2SeO3 exposure. An explanation of this phenomenon could be that the reduction process Se(+ IV) <==> Se(- II) leading to the formation of Se2- is moved to the left by the presence of oxygen, thus inhibiting the precipitation of ZnSe crystals. It is suggested that the subtle oxygen pressure pattern found in the rat hippocampus might also reflect essential biological zinc-related mechanisms vital to brain function.

Histochemical demonstration of zinc ions in human epididymis using autometallography

SummaryA recently described autometallographic technique, allowing demonstration of chelatable zinc in human biopsy material, was applied to cryostat sections from biopsies of human epididymis. Sections from the rat epididymis were used as control materials to examine the quality of the method compared with a previously used autometallographic method. The human epididymis exhibits heavy staining in the head of the epididymis and only small amounts of zinc in the body and tail of the organ. The zinc staining was found in the apical part of the ciliated cells and in the lumen. The present technique can be used to localize zinc ions at ultrastructural levels. Zinc grains were localized in lysosome-like bodies and secretory granules of the ciliated cells. The luminal staining was present as free, evenly dispersed zinc grains or attached to sperm cells and stereocilia in the lumen. The large differences in staining patterns along the epididymal tract in humans and rats suggest that zinc ions are important for the maturation of sperm cells

Computer-assisted visualization of the rat epididymis: a methodological study based on paraffin sections autometallographically stained for zinc ions

A concept for the computer-assisted visualization of tubular organs is presented. Unmarked histological zinc-stained serial sections from the epididymis of the Wistar rat were aligned to demonstrate the concept. Virtual images were made through the aligned sections and served as controls for the alignment process. Animation of the serial sections and the virtual images revealed new information about the structure of the organ under investigation. The analysis was used to upgrade the anatomical knowledge of rat epididymis by describing how the epididymal duct runs through the structure. The proximal parts of the epididymis contain large communicating septa of connective tissue dividing the caput and the upper part of the corpus epididymidis into segments. The tortuousness was high in the caput with many turns within a small area of the epididymis, whereas longer loops were found in the lower part of the corpus and cauda epididymidis. The tube of the vas deferens was found to become an integrated part of the ductal system in the cauda epididymidis, although it was histologically easy to distinguish from the epididymal duct. The total number of cross-sections of the ductus epididymidis in the 2254, 15-mu m-thick, tissue sections analysed was 104 700, giving a minimum length of the ductal system of 1.5 m.

Efficiency of autometallographic detection of mercury in the rat kidney

SummaryThe autometallographic silver enhancement method is a method for subcellular localization of some heavy metals, such as mercury. However, no quantitative estimate has been made of the amount of mercury demonstrated by the method. In this study, pellets of autometallographic silver grains were prepared from unfixed kidney slices of rats exposed i.p. to mercury chloride containing trace amounts of 203Hg. The slices were silver-enhanced, and subsequently all organic material was removed by enzymatic digestion. During all stages of the experiment the solutions and tissue were gamma-counted. The analysis showed that the final pellets contained approximately 30% of the mercury compared to that found in the slices prior to development and that the mercury was probably located in lysosomes.

PC-assisted three-dimensional description of organs containing tubular structures, applied on the epididymis of the rat

A concept for three-dimensional computer-assisted reconstruction of tubular organs, e.g. the epididymis, is described. Histologic serial sections without artificial landmarks from the epididymis of the Wistar rat were aligned. Virtual images through the aligned sections served as a control of the alignment process and can reveal new information about the structure of the organ under investigation. The method can be used for improving the anatomical description of the epididymis, i.e. how the ductus epididymidis is coiled along this organ. Other tubular tissues and organs can be investigated and analysed with this PC-assisted method, e.g. testis and kidney.