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.

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 and . 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 ferritin molecules per 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.

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Iron is essential for neurons and glial cells, playing key roles in neurotransmitter synthesis, energy production and myelination. In contrast, high concentrations of free iron can be detrimental and contribute to neurodegeneration, through promotion of oxidative stress. Particularly in Parkinson’s disease (PD) changes in iron concentrations in the substantia nigra (SN) was suggested to play a key role in degeneration of dopaminergic neurons in nigrosome 1. However, the cellular iron pathways and the mechanisms of the pathogenic role of iron in PD are not well understood, mainly due to the lack of quantitative analytical techniques for iron quantification with subcellular resolution. Here, we quantified cellular iron concentrations and subcellular iron distributions in dopaminergic neurons and different types of glial cells in the SN both in brains of PD patients and in non-neurodegenerative control brains (Co). To this end, we combined spatially resolved quantitative element mapping using micro particle induced X-ray emission (µPIXE) with nickel-enhanced immunocytochemical detection of cell type-specific antigens allowing to allocate element-related signals to specific cell types. Distinct patterns of iron accumulation were observed across different cell populations. In the control (Co) SNc, oligodendroglial and astroglial cells hold the highest cellular iron concentration whereas in PD, the iron concentration was increased in most cell types in the substantia nigra except for astroglial cells and ferritin-positive oligodendroglial cells. While iron levels in astroglial cells remain unchanged, ferritin in oligodendroglial cells seems to be depleted by almost half in PD. The highest cellular iron levels in neurons were located in the cytoplasm, which might increase the source of non-chelated Fe3+, implicating a critical increase in the labile iron pool. Indeed, neuromelanin is characterised by a significantly higher loading of iron including most probable the occupancy of low-affinity iron binding sites. Quantitative trace element analysis is essential to characterise iron in oxidative processes in PD. The quantification of iron provides deeper insights into changes of cellular iron levels in PD and may contribute to the research in iron-chelating disease-modifying drugs.

We report on a comparative study by Transmission Electron Microscopy (HRTEM) and Scanning Transmission Ion Microscopy (STIM) combined with Rutherford Backscattering Spectrometry (RBS) and Particle Induced X-Ray Emission (PIXE) on ultra-thin and thin cross-sections, respectively, of various skin samples (porcine skin, healthy human skin, human skin grafted on a severe combined immuno-deficient mouse model) to which we applied topically various formulations containing titanium dioxide (TiO2) nanoparticles with primary particle sizes in the range from 20-100 nm. Whereas the HRTEM and STIM/PIXE images reveal clear differences - mainly related to the different thickness of the cross-sections - they unambiguously show that penetration of TiO2 nanoparticles is restricted to the topmost 3-5 corneocyte layers of the stratum corneum (SC)