Arne Andreasen

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.

Three-Dimensional Reconstruction of the Mouse Nephron

Renal function is crucially dependent on renal microstructure which provides the basis for the regulatory mechanisms that control the transport of water and solutes between filtrate and plasma and the urinary concentration. This study provides new, detailed information on mouse renal architecture, including the spatial course of the tubules, lengths of different segments of nephrons, histotopography of tubules and vascular bundles, and epithelial ultrastructure at well-defined positions along Henles loop and the distal convolution of nephrons. Three-dimensional reconstruction of 200 nephrons and collecting ducts was performed on aligned digital images, obtained from 2.5-mum-thick serial sections of mouse kidneys. Important new findings were highlighted: (1) A tortuous course of the descending thin limbs of long-looped nephrons and a winding course of the thick ascending limbs of short-looped nephrons contributed to a 27% average increase in the lengths of the corresponding segments, (2) the thick-walled tubules incorporated in the central part of the vascular bundles in the inner stripe of the outer medulla were identified as thick ascending limbs of long-looped nephrons, and (3) three types of short-looped nephron bends were identified to relate to the length and the position of the nephron and its corresponding glomerulus. The ultrastructure of the tubule segments was identified and suggests important implications for renal transport mechanisms that should be considered when evaluating the segmental distribution of water and solute transporters within the normal and diseased kidney.

Zopiclone in the treatment of sleep abnormalities in fibromyalgia.

The clinical and polysomnographical effects of zopiclone were evaluated in 41 patients with fibromyalgia in a double blind controlled study. A significant improvement was observed regarding tiredness during the day and subjective sleep complaints, but no effects on pain or stiffness were observed. The sleep structure remained unchanged during treatment. Zopiclone seems to be of value in treating the sleep complaints in patients with fibromyalgia.

Aquaporin-1 Is not Expressed in Descending Thin Limbs of Short-Loop Nephrons

In mammalian kidneys, aquaporin-1 is responsible for water reabsorption along the proximal tubule and is also thought to be involved in the concentration of urine that occurs in the medulla. It has been suggested, however, that aquaporin-1 is not expressed in the last part of the descending thin limbs of short loop nephrons in rats and mice, and its expression in this region in humans has not been studied. We examined the expression of aquaporin-1 and the urea transporter UT-A2 in serial sections of mouse nephrons in the inner stripe of the outer medulla using immunohistochemistry. In contrast to previous observations, we demonstrate a complete absence of aquaporin-1 along the entire length of descending thin limbs of 90% of short loop nephrons. Conversely, as expected, we identified aquaporin-1 in proximal tubules, descending thin limbs of long loop nephrons, and medullary descending vasa recta. We also observed this abrupt transition from aquaporin-1-positive proximal tubules to aquaporin-1-negative descending thin limbs of short loop nephrons in sections of human and rat kidneys. UT-A2 was restricted to the last 28% to 44% of the descending thin limbs of all short loop nephrons. Because the majority of nephrons are of the short loop variety, our findings suggest that the mechanisms of water transport in the descending thin limbs of short loop nephrons should be reevaluated. Likewise, the roles of aquaporin-1 and UT-A2 in the countercurrent multiplier and water conversation may need to be readdressed.

Digital Three-Dimensional Reconstruction and Ultrastructure of the Mouse Proximal Tubule

Mice are prime targets of experimental gene modification and have become object of an increasing number of biologic studies in renal physiology, development, and molecular biology. Phenotypic changes in response to gene modification require detailed information on normal structure. However, detailed analyses of normal mouse kidney structure and organization are lacking. This study describes the 3D organization and ultrastructural, segmental variation of the mouse kidney proximal tubule. A total of 160 proximal tubules in three C57/BL/6J mouse kidneys were analyzed on 800 serial sections from each kidney from the surface to the inner stripe of the outer zone of medulla. All tubules were reconstructed in 3D and visualized by interactive computer graphics. A quantitative ultrastructural analysis of the mouse proximal tubule at every 300 to 400 micro m was performed. The 3D representation revealed a distinct organization of the mouse proximal tubule, each occupying a separate domain within the cortex. Superficial proximal tubules have long straight parts converging into clusters within the medullary rays. Tubules originating deeper within the cortex become longer and increasingly tortuous. In the medullary rays, these are arranged in layers outside the clusters of more superficial tubules. In contrast to rat and human kidney, no major segmental variation in the ultrastructure of the proximal tubule was identified, and no parameters enabled definition of distinct segments in this strain of mice. In conclusion, significant new information on the 3D organization of the murine proximal tubule has been obtained. Quantitative, ultrastructural analyses of mouse proximal tubules reveal substantial differences compared with other species.

Rapid disappearance of zinc positive terminals in focal brain ischemia

The study was undertaken to determine if the levels of vesicular zinc in neuronal terminals would decrease in response to focal brain ischemia. The middle cerebral artery was occluded distal to the striatal branches in male spontaneously hypertensive rats. At 7, 15, 30, 45, 60, 90, 120 min; 3, 6, 12, 24, 48 h and 7 days later the animals were sacrificed and the brains were stained for zinc-sulfides, cell bodies and AChE-positive cholinergic fibers. The density of zinc positive terminals significantly decreased in the neocortical ischemic zone 7 min after middle cerebral artery occlusion (MCAO). In the neocortical layers II and III most zinc positive neuronal terminals disappeared at 7 min after MCAO whereas the zinc positive terminals in layers V and VI remained positive at least 2 h. Beginning at 1 h after MCAO and progressing to 24 h a significant decrease in the density of zinc positive terminals was observed in the dorsolateral striatum, and ventrobasal thalamic nucleus, both major projection areas of the sensorimotor cortex. The disappearance of zinc positive neuronal terminals in the ischemic neocortex and related areas, is most likely due to a neuronal release of vesicular zinc in response to hypoxia. The high extracellular concentration of zinc is thought to be both neuroprotective by blocking the NMDA receptor and neurotoxic by activating neuronal influx of Ca2+ through voltage gated calcium channels. It seems evident that the latter effect of zinc is contributing to the neuronal death in focal brain ischemia.

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)

Interactive 3D computer model of the human corneolimbal region: crypts, projections and stem cells

Purpose:  This study aims to clarify the existence of and to map the localization of different proposed stem cell niches in the corneal limbal region.

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 localization of zinc ions in the epididymis of the rat

SummaryIn the present study, the autometallograpic zinc sulphide technique, an improved version of the original Timm sulphide-silver method, was used. This technique reveals a particular pool of ionic zinc that is chelatable by diethyldithiocarbamate. At the light microscopical level, no reaction for zinc was found in tissues of young prepubertal rats. In adult mating and non-mating rats low zinc staining was found in the head and intermediate epididymis whereas the tail of the epididymis demonstrated high levels of zinc ions. Sections from the epididymal tail revealed a ‘compartmentalization’, based on pronounced differences in staining intensity along the epididymal ducts. At higher magnification zinc ions were found in the apical part of the principal cell and in the lumen. At the ultrastructural level autometallographic grains were located in vesicles and in lysosome-like structures of the apical parts of the principal cells. The luminal grains were found either associated with sperm cells, with the surface of the large microvilli (stereocilia), or free in the seminal fluid. The variation in content of zinc ions in the epididymal epithelium and lumen suggests that zinc ions are secreted into the lumen from the epididymal tail and may somehow be involved in maturation of the sperm cells.