Combined use of x-ray fluorescence microscopy, phase contrast imaging for high resolution quantitative iron mapping in inflamed cells

X-ray fluorescence microscopy (XRFM) is a powerful technique to detect and localize elements in cells. To derive information useful for biology and medicine, it is essential not only to localize, but also to map quantitatively the element concentration. Here we applied quantitative XRFM to iron in phagocytic cells. Iron, a primary component of living cells, can become toxic when present in excess. In human fluids, free iron is maintained at 10-18 M concentration thanks to iron binding proteins as lactoferrin (Lf). The iron homeostasis, involving the physiological ratio of iron between tissues/secretions and blood, is strictly regulated by ferroportin, the sole protein able to export iron from cells to blood. Inflammatory processes induced by lipopolysaccharide (LPS) or bacterial pathoge inhibit ferroportin synthesis in epithelial and phagocytic cells thus hindering iron export, increasing intracellular iron and bacterial multiplication. In this respect, Lf is emerging as an important regulator of both iron and inflammatory homeostasis. Here we studied phagocytic cells inflamed by bacterial LPS and untreated or treated with milk derived bovine Lf. Quantitative mapping of iron concentration and mass fraction at high spatial resolution is obtained combining X-ray fluorescence microscopy, atomic force microscopy and synchrotron phase contrast imaging

Mineral maturity and crystallinity index are distinct characteristics of bone mineral

The purpose of this study was to test the hypothesis that mineral maturity and crystallinity index are two different characteristics of bone mineral. To this end, Fourier transform infrared microspectroscopy (FTIRM) was used. To test our hypothesis, synthetic apatites and human bone samples were used for the validation of the two parameters using FTIRM. Iliac crest samples from seven human controls and two with skeletal fluorosis were analyzed at the bone structural unit (BSU) level by FTIRM on sections 2–4 lm thick. Mineral maturity and crystallinity index were highly correlated in synthetic apatites but poorly correlated in normal human bone. In skeletal fluorosis, crystallinity index was increased and maturity decreased, supporting the fact of separate measurement of these two parameters. Moreover, results obtained in fluorosis suggested that mineral characteristics can be modified independently of bone remodeling. In conclusion, mineral maturity and crystallinity index are two different parameters measured separately by FTIRM and offering new perspectives to assess bone mineral traits in osteoporosis

Iron Storage within Dopamine Neurovesicles Revealed by Chemical Nano-Imaging

Altered homeostasis of metal ions is suspected to play a critical role in neurodegeneration. However, the lack of analytical technique with sufficient spatial resolution prevents the investigation of metals distribution in neurons. An original experimental setup was developed to perform chemical element imaging with a 90 nm spatial resolution using synchrotron-based X-ray fluorescence. This unique spatial resolution, combined to a high brightness, enables chemical element imaging in subcellular compartments. We investigated the distribution of iron in dopamine producing neurons because iron-dopamine compounds are suspected to be formed but have yet never been observed in cells. The study shows that iron accumulates into dopamine neurovesicles. In addition, the inhibition of dopamine synthesis results in a decreased vesicular storage of iron. These results indicate a new physiological role for dopamine in iron buffering within normal dopamine producing cells. This system could be at fault in Parkinson's disease which is characterized by an increased level of iron in the substancia nigra pars compacta and an impaired storage of dopamine due to the disruption of vesicular trafficking. The re-distribution of highly reactive dopamine-iron complexes outside neurovesicles would result in an enhanced death of dopaminergic neurons

Nav1.7 is required for normal C-low threshold mechanoreceptor function in humans and mice

Patients with bi-allelic loss of function mutations in the voltage-gated sodium channel Nav1.7 present with congenital insensitivity to pain (CIP), whilst low threshold mechanosensation is reportedly normal. Using psychophysics (n = 6 CIP participants and n = 86 healthy controls) and facial EMG (n = 3 CIP participants and n = 8 healthy controls) we have found that these patients also have abnormalities in the encoding of affective touch which is mediated by the specialised afferents; C-low threshold mechanoreceptors (C-LTMRs). In the mouse we found that C-LTMRs express high levels of Nav1.7. Genetic loss or selective pharmacological inhibition of Nav1.7 in C-LTMRs resulted in a significant reduction in the total sodium current density, an increased mechanical threshold and reduced sensitivity to non-noxious cooling. The behavioural consequence of loss of Nav1.7 in C-LTMRs in mice was an elevation in the von Frey mechanical threshold and less sensitivity to cooling on a thermal gradient. Nav1.7 is therefore not only essential for normal pain perception but also for normal C-LTMR function, cool sensitivity and affective touch

The High Radiosensitizing Efficiency of a Trace of Gadolinium-Based Nanoparticles in Tumors

International audienceWe recently developed the synthesis of ultrasmall gadolinium-based nanoparticles (GBN), (hydrodynamic diameter <5 nm) characterized by a safe behavior after intravenous injection (renal clearance, preferential accumulation in tumors). Owing to the presence of gadolinium ions, GBN can be used as contrast agents for magnetic resonance imaging (MRI) and as radiosensitizers. The attempt to determine the most opportune delay between the intravenous injection of GBN and the irradiation showed that a very low content of radiosensitizing nanoparticles in the tumor area is sufficient (0.1 μg/g of particles, i.e. 15 ppb of gadolinium) for an important increase of the therapeutic effect of irradiation. Such a promising and unexpected result is assigned to a suited distribution of GBN within the tumor, as revealed by the X-ray fluorescence (XRF) maps

II Congrés Internacional sobre Traducció : abril 1994 : actes

Machine learning-based approach unravels distinct pathological signatures induced by patient-derived α-synuclein seeds in monkeys. Dopaminergic neuronal cell death, associated with intracellular α-synuclein (α-syn)-rich protein aggregates [termed "Lewy bodies" (LBs)], is a well-established characteristic of Parkinson's disease (PD). Much evidence, accumulated from multiple experimental models, has suggested that α-syn plays a role in PD pathogenesis, not only as a trigger of pathology but also as a mediator of disease progression through pathological spreading. Here, we have used a machine learning-based approach to identify unique signatures of neurodegeneration in monkeys induced by distinct α-syn pathogenic structures derived from patients with PD. Unexpectedly, our results show that, in nonhuman primates, a small amount of singular α-syn aggregates is as toxic as larger amyloid fibrils present in the LBs, thus reinforcing the need for preclinical research in this species. Furthermore, our results provide evidence supporting the true multifactorial nature of PD, as multiple causes can induce a similar outcome regarding dopaminergic neurodegeneratio

In situ biospectroscopic investigation of rapid ischemic and postmortem induced biochemical alterations in the rat brain

© 2014 American Chemical Society. Rapid advances in imaging technologies have pushed novel spectroscopic modalities such as Fourier transform infrared spectroscopy (FTIR) and X-ray absorption spectroscopy (XAS) at the sulfur K-edge to the forefront of direct in situ investigation of brain biochemistry. However, few studies have examined the extent to which sample preparation artifacts confound results. Previous investigations using traditional analyses, such as tissue dissection, homogenization, and biochemical assay, conducted extensive research to identify biochemical alterations that occur ex vivo during sample preparation. In particular, altered metabolism and oxidative stress may be caused by animal death. These processes were a concern for studies using biochemical assays, and protocols were developed to minimize their occurrence. In this investigation, a similar approach was taken to identify the biochemical alterations that are detectable by two in situ spectroscopic methods (FTIR, XAS) that occur as a consequence of ischemic conditions created during humane animal killing. FTIR and XAS are well suited to study markers of altered metabolism such as lactate and creatine (FTIR) and markers of oxidative stress such as aggregated proteins (FTIR) and altered thiol redox (XAS). The results are in accordance with previous investigations using biochemical assays and demonstrate that the time between animal death and tissue dissection results in ischemic conditions that alter brain metabolism and initiate oxidative stress. Therefore, future in situ biospectroscopic investigations utilizing FTIR and XAS must take into consideration that brain tissue dissected from a healthy animal does not truly reflect the in vivo condition, but rather reflects a state of mild ischemia. If studies require the levels of metabolites (lactate, creatine) and markers of oxidative stress (thiol redox) to be preserved as close as possible to the in vivo condition, then rapid freezing of brain tissue via decapitation into liquid nitrogen, followed by chiseling the brain out at dry ice temperatures is required

Manganese enhanced MRI in rat hippocampus: a correlative study with synchrotron X-ray microprobe

Manganese enhanced MRI (MEMRI) offers many possibilities such as tract tracing and functional imaging in vivo. Mn is however neurotoxic and may induce symptoms similar to those associated with Parkinson's disease (manganism). The mechanisms of Mn-induced neurotoxicity are not clear. In this study, we combine synchrotron X-ray fluorescence microprobe (SR-XRF) and MEMRI techniques to investigate spatial distribution of Mn within the rat hippocampus and how Mn interacts with Ca, Fe and Zn at a cellular level. Images were acquired in the rat hippocampus (n=23) and using two injection routes: intra-cerebral (MnCl(2): 50 mM, 10 μL) and intra-peritoneal (MnCl(2): 100 mM, 30 mg/kg). For both injection routes, Mn is found in dentate gyrus and in CA3: control: 2.5 ± 1.6, intra-peritoneal: 5.0 ± 2.4, and intra-cerebral: 25.1 ± 9.2 μg/g. Mn follows Zn distribution and has a negative impact on the total amount of Zn and Fe. The Mn-enhanced MRI contrast is well correlated with the total Mn amount measured with SR-XRF (R(2)=0.93; p<0.002). After intra-cerebral injection, the hippocampal fissure is found to accumulate a large amount of Mn and yields a hypointense MRI signal, which may be ascribed to a reduction in T2. This study shows that SR-XRF is well suited to investigate Mn distribution at a mesoscale and that MRI is sensitive to low Mn concentrations. As perturbations in metal homeostasis may alter brain function, the injected dose of Mn in MEMRI studies needs to be carefully adjusted to obtain reliable functional information

Methodological Study Using XAS of an Arsenic Based Antileukemia Treatment

International audienc