Myelin insulation as a risk factor for axonal degeneration in autoimmune demyelinating disease.

Axonal degeneration determines the clinical outcome of multiple sclerosis and is thought to result from exposure of denuded axons to immune-mediated damage. Therefore, myelin is widely considered to be a protective structure for axons in multiple sclerosis. Myelinated axons also depend on oligodendrocytes, which provide metabolic and structural support to the axonal compartment. Given that axonal pathology in multiple sclerosis is already visible at early disease stages, before overt demyelination, we reasoned that autoimmune inflammation may disrupt oligodendroglial support mechanisms and hence primarily affect axons insulated by myelin. Here, we studied axonal pathology as a function of myelination in human multiple sclerosis and mouse models of autoimmune encephalomyelitis with genetically altered myelination. We demonstrate that myelin ensheathment itself becomes detrimental for axonal survival and increases the risk of axons degenerating in an autoimmune environment. This challenges the view of myelin as a solely protective structure and suggests that axonal dependence on oligodendroglial support can become fatal when myelin is under inflammatory attack.

Nanometers-Thick Ferromagnetic Surface Produced by Laser Cutting of Diamond.

In this work, we demonstrate that cutting diamond crystals with a laser (532 nm wavelength, 0.5 mJ energy, 200 ns pulse duration at 15 kHz) produced a ≲20 nm thick surface layer with magnetic order at room temperature. We measured the magnetic moment of five natural and six CVD diamond crystals of different sizes, nitrogen contents and surface orientations with a SQUID magnetometer. A robust ferromagnetic response at 300 K was observed only for crystals that were cut with the laser along the (100) surface orientation. The magnetic signals were much weaker for the (110) and negligible for the (111) orientations. We attribute the magnetic order to the disordered graphite layer produced by the laser at the diamond surface. The ferromagnetic signal vanished after chemical etching or after moderate temperature annealing. The obtained results indicate that laser treatment of diamond may pave the way to create ferromagnetic spots at its surface.

High Iron and Iron Household Protein Contents in Perineuronal Net-Ensheathed Neurons Ensure Energy Metabolism with Safe Iron Handling.

A subpopulation of neurons is less vulnerable against iron-induced oxidative stress and neurodegeneration. A key feature of these neurons is a special extracellular matrix composition that forms a perineuronal net (PN). The PN has a high affinity to iron, which suggests an adapted iron sequestration and metabolism of the ensheathed neurons. Highly active, fast-firing neurons-which are often ensheathed by a PN-have a particular high metabolic demand, and therefore may have a higher need in iron. We hypothesize that PN-ensheathed neurons have a higher intracellular iron concentration and increased levels of iron proteins. Thus, analyses of cellular and regional iron and the iron proteins transferrin (Tf), Tf receptor 1 (TfR), ferritin H/L (FtH/FtL), metal transport protein 1 (MTP1 aka ferroportin), and divalent metal transporter 1 (DMT1) were performed on Wistar rats in the parietal cortex (PC), subiculum (SUB), red nucleus (RN), and substantia nigra (SNpr/SNpc). Neurons with a PN (PN+) have higher iron concentrations than neurons without a PN: PC 0.69 mM vs. 0.51 mM, SUB 0.84 mM vs. 0.69 mM, SN 0.71 mM vs. 0.63 mM (SNpr)/0.45 mM (SNpc). Intracellular Tf, TfR and MTP1 contents of PN+ neurons were consistently increased. The iron concentration of the PN itself is not increased. We also determined the percentage of PN+ neurons: PC 4%, SUB 5%, SNpr 45%, RN 86%. We conclude that PN+ neurons constitute a subpopulation of resilient pacemaker neurons characterized by a bustling iron metabolism and outstanding iron handling capabilities. These properties could contribute to the low vulnerability of PN+ neurons against iron-induced oxidative stress and degeneration.

Measuring the iron content of dopaminergic neurons in substantia nigra with MRI relaxometry.

In Parkinson's disease, the depletion of iron-rich dopaminergic neurons in nigrosome 1 of the substantia nigra precedes motor symptoms by two decades. Methods capable of monitoring this neuronal depletion, at an early disease stage, are needed for early diagnosis and treatment monitoring. Magnetic resonance imaging (MRI) is particularly suitable for this task due to its sensitivity to tissue microstructure and in particular, to iron. However, the exact mechanisms of MRI contrast in the substantia nigra are not well understood, hindering the development of powerful biomarkers. In the present report, we illuminate the contrast mechanisms in gradient and spin echo MR images in human nigrosome 1 by combining quantitative 3D iron histology and biophysical modeling with quantitative MRI on post mortem human brain tissue. We show that the dominant contribution to the effective transverse relaxation rate (R2*) in nigrosome 1 originates from iron accumulated in the neuromelanin of dopaminergic neurons. This contribution is appropriately described by a static dephasing approximation of the MRI signal. We demonstrate that the R2* contribution from dopaminergic neurons reflects the product of cell density and cellular iron concentration. These results demonstrate that the in vivo monitoring of neuronal density and iron in nigrosome 1 may be feasible with MRI and provide directions for the development of biomarkers for an early detection of dopaminergic neuron depletion in Parkinson's disease.

Iron concentrations in neurons and glial cells with estimates on ferritin concentrations.

BACKGROUND: Brain iron is an essential as well as a toxic redox active element. Physiological levels are not uniform among the different cell types. Besides the availability of quantitative methods, the knowledge about the brain iron lags behind. Thereby, disclosing the mechanisms of brain iron homeostasis helps to understand pathological iron-accumulations in diseased and aged brains. With our study we want to contribute closing the gap by providing quantitative data on the concentration and distribution of iron in neurons and glial cells in situ. Using a nuclear microprobe and scanning proton induced X-ray emission spectrometry we performed quantitative elemental imaging on rat brain sections to analyze the iron concentrations of neurons and glial cells. RESULTS: Neurons were analyzed in the neocortex, subiculum, substantia nigra and deep cerebellar nuclei revealing an iron level between [Formula: see text] and [Formula: see text]. The iron concentration of neocortical oligodendrocytes is fivefold higher, of microglia threefold higher and of astrocytes twofold higher compared to neurons. We also analyzed the distribution of subcellular iron concentrations in the cytoplasm, nucleus and nucleolus of neurons. The cytoplasm contains on average 73% of the total iron, the nucleolus-although a hot spot for iron-due to its small volume only 6% of total iron. Additionally, the iron level in subcellular fractions were measured revealing that the microsome fraction, which usually contains holo-ferritin, has the highest iron content. We also present an estimate of the cellular ferritin concentration calculating [Formula: see text] ferritin molecules per [Formula: see text] in rat neurons. CONCLUSION: Glial cells are the most iron-rich cells in the brain. Imbalances in iron homeostasis that lead to neurodegeneration may not only be originate from neurons but also from glial cells. It is feasible to estimate the ferritin concentration based on measured iron concentrations and a reasonable assumptions on iron load in the brain.

Note: Performance of a novel electrostatic quadrupole doublet for nuclear microprobe application.

The newly designed and constructed electrostatic quadrupole doublet (EQD) at the University of North Texas (UNT) has achieved mass independent focusing of MeV particles to a spot size of 3.3 × 3.5 µm. The EQD is compared to the Louisiana magnetic doublet which is also in use at UNT. Characteristics such as demagnification and sensitivity to aberrations are simulated by the matrix raytracing software, propagation of rays and aberrations by matrices and compared for each focusing system. Particle induced x-ray emission (PIXE) maps of a 2000 mesh Cu grid are compared and show that both doublets produce suitable spot sizes for microprobe analysis. A coarser, 200 mesh grid and incident beams of 2 MeV H+ and O+ show the EQD to be stigmatic given common aperture sizes and lens potentials.

Ion exchanger in the brain: Quantitative analysis of perineuronally fixed anionic binding sites suggests diffusion barriers with ion sorting properties.

Perineuronal nets (PNs) are a specialized form of brain extracellular matrix, consisting of negatively charged glycosaminoglycans, glycoproteins and proteoglycans in the direct microenvironment of neurons. Still, locally immobilized charges in the tissue have not been accessible so far to direct observations and quantifications. Here, we present a new approach to visualize and quantify fixed charge-densities on brain slices using a focused proton-beam microprobe in combination with ionic metallic probes. For the first time, we can provide quantitative data on the distribution and net amount of pericellularly fixed charge-densities, which, determined at 0.4-0.5 M, is much higher than previously assumed. PNs, thus, represent an immobilized ion exchanger with ion sorting properties high enough to partition mobile ions in accord with Donnan-equilibrium. We propose that fixed charge-densities in the brain are involved in regulating ion mobility, the volume fraction of extracellular space and the viscosity of matrix components.

A digitizer based compact digital spectrometer for ion beam analysis using Field Programmable Gate Arrays and various energy algorithms.

We report on the implementation of a compact multi-detector fully digital spectrometer and data acquisition system at a nuclear microprobe for ion beam analysis and imaging. The spectrometer design allows for system scalability with no restriction on the number of detectors. It consists of four-channel high-speed digitizer modules for detector signal acquisition and one low-speed digital-to-analog converter (DAC) module with two DAC channels and additional general purpose inputs∕outputs to control ion beam scanning and data acquisition. Each digitizer module of the spectrometer provides its own Field Programmable Gate Array (FPGA) as digital signal processing unit to analyze detector signals as well as to synchronize the ion beam position in hard real-time. With the customized FPGA designs for all modules, all calculation intensive tasks are executed inside the modules, which reduces significantly the data stream to and CPU load on the control computer. To achieve an optimal energy resolution for all detector∕preamplifier pulse shape characteristics, a user-definable infinite impulse response filter with high throughput for energy determination was implemented. The new spectrometer has an online data analysis feature, a compact size, and is able to process any type of detector signals such as particle induced x-ray emission, Rutherford backscattering spectrometry, or scanning transmission ion microscopy.

On the quantification of intracellular proteins in multifluorescence-labeled rat brain slices using slide-based cytometry.

In several brain regions, a subpopulation of neurons exists being characterized by the expression of a peculiar form of extracellular matrix, a so-called perineuronal net (PNN). We have previously shown that the PNN can bind large amounts of iron due to its polyanionic charge. Because free iron can generate reactive oxygen species thus being potentially toxic, the PNN may have a protective function by "scavenging" this free iron. Because of this ability, we have hypothesized that PNN-related neurons have an altered iron-specific metabolism. Thus, to compare the intracellular concentrations of iron containing proteins, specifically, the iron storage protein ferritin H between neurons with and without a PNN, we have used slide-based cytometry with image-based threshold-boundary cell detection on brain sections. In tissue sections, the integrity of the extracellular matrix, especially the characteristic PNNs, is preserved, which is necessary for the identification of the two neuronal subpopulations. A multilabeling approach was chosen to select neurons (neuronal marker NeuN), to classify the neurons according to their subtype (matrix marker Wisteria floribunda agglutinin), and to quantify the protein concentration (protein marker). Using this novel method, we were able to detect a relative difference in protein concentration as low as 12% between the two subpopulations of neurons in the neuronal population of the rat parietal cortex.

Scanning transmission ion micro-tomography (STIM-T) of biological specimens.

Computed tomography (CT) was applied to sets of Scanning Transmission Ion Microscopy (STIM) projections recorded at the LIPSION ion beam laboratory (Leipzig) in order to visualize the 3D-mass distribution in several specimens. Examples for a test structure (copper grid) and for biological specimens (cartilage cells, cygospore) are shown. Scanning Transmission Micro-Tomography (STIM-T) at a resolution of 260 nm was demonstrated for the first time. Sub-micron features of the Cu-grid specimen were verified by scanning electron microscopy. The ion energy loss measured during a STIM-T experiment is related to the mass density of the specimen. Typically, biological specimens can be analysed without staining. Only shock freezing and freeze-drying is required to preserve the ultra-structure of the specimen. The radiation damage to the specimen during the experiment can be neglected. This is an advantage compared to other techniques like X-ray micro-tomography. At present, the spatial resolution is limited by beam position fluctuations and specimen vibrations.