Human Pineal Gland Involutionary Process: New Findings.

In this work, we report preliminary results about the involution of the human pineal gland involution. The detailed analysis of pineal structure was done on autopsy material of 77 persons in age 27-96 using x-ray phase-contrast tomography, histology, and immunohistochemistry. Our study suggests that the pineal gland alteration in older adults may be more profound than has been reported to date. We identified and described a new form of pineal gland involution that eventually led to the total degradation of the pineal gland. To our knowledge, this study is the first to report on the complete replacement of pineal gland parenchyma with connective tissue in older adults.

Micromorphology of pineal gland calcification in age-related neurodegenerative diseases.

BACKGROUND: The formation of concrements in human pineal gland (PG) is a physiological process and, according to many researchers, is associated with the involution of PG structures. The majority of scientific publications concern progressive calcification of PG, leaving out studies on the destruction of already formed calcified concrements. Our study fills the gap in knowledge about calcified zones destruction in PG in normal aging and neuropathological conditions, which has not been addressed until now. PURPOSE: Our objective is to gain insight into human PG tissue impairment in both normal aging and neurodegenerative conditions. X-ray phase-contrast tomography (XPCT) allowed us to study PG tissue degeneration at high spatial resolution and, for the first time, to examine the damaged PG concrements in detail. Our research finding could potentially enhance the understanding of the PG involvement in the process of aging as well as in Alzheimer's disease (AD) and vascular dementia (VD). METHODS: The research was carried out on human PG autopsy material in normal aging, VD, and AD conditions. Laboratory-based micro-computed tomography (micro-CT) was used to collect and evaluate samples of native, uncut, and unstained PG with different degrees of pineal calcification. The detailed high-resolution 3D images of the selected PGs were produced using synchrotron-based XPCT. Histology and immunohistochemistry of soft PG tissue confirmed XPCT results. RESULTS: We performed via micro-CT the evaluation of the morphometric parameters of PG such as total sample volume, calcified concrements volume, and percentage of concrements in the total volume of the sample. XPCT imaging revealed high-resolution details of age-related PG alteration. In particular, we noted signs of moderate degradation of concrements in some PGs from elderly donors. In addition, our analysis revealed noticeable degenerative change in both concrements and soft tissue of PGs with neuropathology. In particular, we observed a hollow core and separated layers as well as deep ragged cracks in PG concrements of AD and VD samples. In parenchyma of some samples, we detected wide pinealocyte-free fluid-filled areas adjacent to the calcified zones. CONCLUSION: The present work provides the basis for future scientific research focused on the dynamic nature of PG calcium deposits and PG soft tissue in normal aging and neurodegenerative diseases.

3D Spatial Distribution of Nanoparticles in Mice Brain Metastases by X-ray Phase-Contrast Tomography.

Characterizing nanoparticles (NPs) distribution in multiple and complex metastases is of fundamental relevance for the development of radiological protocols based on NPs administration. In the literature, there have been advances in monitoring NPs in tissues. However, the lack of 3D information is still an issue. X-ray phase-contrast tomography (XPCT) is a 3D label-free, non-invasive and multi-scale approach allowing imaging anatomical details with high spatial and contrast resolutions. Here an XPCT qualitative study on NPs distribution in a mouse brain model of melanoma metastases injected with gadolinium-based NPs for theranostics is presented. For the first time, XPCT images show the NPs uptake at micrometer resolution over the full brain. Our results revealed a heterogeneous distribution of the NPs inside the melanoma metastases, bridging the gap in spatial resolution between magnetic resonance imaging and histology. Our findings demonstrated that XPCT is a reliable technique for NPs detection and can be considered as an emerging method for the study of NPs distribution in organs.

Assessment and Imaging of Intracellular Magnesium in SaOS-2 Osteosarcoma Cells and Its Role in Proliferation.

Magnesium is an essential nutrient involved in many important processes in living organisms, including protein synthesis, cellular energy production and storage, cell growth and nucleic acid synthesis. In this study, we analysed the effect of magnesium deficiency on the proliferation of SaOS-2 osteosarcoma cells. When quiescent magnesium-starved cells were induced to proliferate by serum addition, the magnesium content was 2-3 times lower in cells maintained in a medium without magnesium compared with cells growing in the presence of the ion. Magnesium depletion inhibited cell cycle progression and caused the inhibition of cell proliferation, which was associated with mTOR hypophosphorylation at Serine 2448. In order to map the intracellular magnesium distribution, an analytical approach using synchrotron-based X-ray techniques was applied. When cell growth was stimulated, magnesium was mainly localized near the plasma membrane in cells maintained in a medium without magnesium. In non-proliferating cells growing in the presence of the ion, high concentration areas inside the cell were observed. These results support the role of magnesium in the control of cell proliferation, suggesting that mTOR may represent an important target for the antiproliferative effect of magnesium. Selective control of magnesium availability could be a useful strategy for inhibiting osteosarcoma cell growth.

Investigation of the human pineal gland 3D organization by X-ray phase contrast tomography.

Pineal gland (PG) is a part of the human brain epithalamus that plays an important role in sleep, circadian rhythm, immunity, and reproduction. The calcium deposits and lesions in PG interfere with normal function of the organ and can be associated with different health disorders including serious neurological diseases. At the moment, the detailed mechanisms of PG calcifications and PG lesions formation as well as their involvement in pathological processes are not fully understood. The deep and comprehensive study of the structure of the uncut human PG with histological details, poses a stiff challenge to most imaging techniques, due to low spatial resolution, low visibility or to exceedingly aggressive sample preparation. Here, we investigate the whole uncut and unstained human post-mortem PGs by X-ray phase contrast tomography (XPCT). XPCT is an advanced 3D imaging technique, that permits to study of both soft and calcified tissue of a sample at different scales: from the whole organ to cell structure. In our research we simultaneously resolved 3D structure of parenchyma, vascular network and calcifications. Moreover, we distinguished structural details of intact and degenerated PG tissue. We discriminated calcifications with different structure, pinealocytes nuclei and the glial cells processes. All results were validated by histology. Our research clear demonstrated that XPCT is a potential tool for the high resolution 3D imaging of PG morphological features. This technique opens a new perspective to investigate PG dysfunction and understand the mechanisms of onset and progression of diseases involving the pineal gland.

X-ray phase contrast tomography for the investigation of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motor neurons. Pre-clinical studies drive the development of animal models that well mimic ALS disorder and enable both the dissection of disease processes and an early assessment of therapy efficacy. A comprehensive knowledge of neuronal and vascular lesions in the brain and spinal cord is an essential factor to understand the development of the disease. Spatial resolution and bidimensional imaging are important drawbacks limiting current neuroimaging tools, while neuropathology relies on protocols that may alter tissue chemistry and structure. In contrast, recent ex vivo studies in mice demonstrated that X-ray phase-contrast tomography enables study of the 3D distribution of both vasculature and neuronal networks, without sample sectioning or use of staining. Here we present our findings on ex vivo SOD1G93A ALS mice spinal cord at a micrometric scale. An unprecedented direct quantification of neuro-vascular alterations at different stages of the disease is shown.

Single cell versus large population analysis: cell variability in elemental intracellular concentration and distribution.

The quantification of elemental concentration in cells is usually performed by analytical assays on large populations missing peculiar but important rare cells. The present article aims at comparing the elemental quantification in single cells and cell population in three different cell types using a new approach for single cells elemental analysis performed at sub-micrometer scale combining X-ray fluorescence microscopy and atomic force microscopy. The attention is focused on the light element Mg, exploiting the opportunity to compare the single cell quantification to the cell population analysis carried out by a highly Mg-selective fluorescent chemosensor. The results show that the single cell analysis reveals the same Mg differences found in large population of the different cell strains studied. However, in one of the cell strains, single cell analysis reveals two cells with an exceptionally high intracellular Mg content compared with the other cells of the same strain. The single cell analysis allows mapping Mg and other light elements in whole cells at sub-micrometer scale. A detailed intensity correlation analysis on the two cells with the highest Mg content reveals that Mg subcellular localization correlates with oxygen in a different fashion with respect the other sister cells of the same strain. Graphical abstract Single cells or large population analysis this is the question!

Characterization of mouse spinal cord vascular network by means of synchrotron radiation X-ray phase contrast tomography.

High resolution Synchrotron-based X-ray Phase Contrast Tomography (XPCT) allows the simultaneous detection of three dimensional neuronal and vascular networks without using contrast agents or invasive casting preparation. We show and discuss the different features observed in reconstructed XPCT volumes of the ex vivo mouse spinal cord in the lumbo-sacral region, including motor neurons and blood vessels. We report the application of an intensity-based segmentation method to detect and quantitatively characterize the modification in the vascular networks in terms of reduction in experimental visibility. In particular, we apply our approach to the case of the experimental autoimmune encephalomyelitis (EAE), i.e. human multiple sclerosis animal model.

Quantitative chemical imaging of the intracellular spatial distribution of fundamental elements and light metals in single cells.

We report a method that allows a complete quantitative characterization of whole single cells, assessing the total amount of carbon, nitrogen, oxygen, sodium, and magnesium and providing submicrometer maps of element molar concentration, cell density, mass, and volume. This approach allows quantifying elements down to 10(6) atoms/µm(3). This result was obtained by applying a multimodal fusion approach that combines synchrotron radiation microscopy techniques with off-line atomic force microscopy. The method proposed permits us to find the element concentration in addition to the mass fraction and provides a deeper and more complete knowledge of cell composition. We performed measurements on LoVo human colon cancer cells sensitive (LoVo-S) and resistant (LoVo-R) to doxorubicin. The comparison of LoVo-S and LoVo-R revealed different patterns in the maps of Mg concentration with higher values within the nucleus in LoVo-R and in the perinuclear region in LoVo-S cells. This feature was not so evident for the other elements, suggesting that Mg compartmentalization could be a significant trait of the drug-resistant cells.