We report a qualitative study on central nervous system (CNS) damage that demonstrates the ability of X-ray phase contrast tomography (XPCT) to confirm data obtained with standard 2D methodology and permits the description of additional features that are not detected with 2D or other 3D techniques. In contrast to magnetic resonance or computed tomography, XPCT makes possible the high-resolution 3D imaging of soft tissues classically considered “invisible” to X-rays without the use of additional contrast agents, or without the need for intense processing of the tissue required by 2D techniques. Most importantly for studies of CNS diseases, XPCT enables a concomitant multi-scale 3D biomedical imaging of neuronal and vascular networks ranging from cells through to the CNS as a whole. In the last years, we have used XPCT to investigate neurodegenerative diseases, such as Alzheimer’s disease (AD) and multiple sclerosis (MS), to shed light on brain damage and extend the observations obtained with standard techniques. Here, we show the cutting-edge ability of XPCT to highlight in 3D, concomitantly, vascular occlusions and damages, close associations between plaques and damaged vessels, as well as dramatic changes induced at neuropathological level by treatment in AD mice. We corroborate data on the well-known blood-brain barrier dysfunction in the animal model of MS, experimental autoimmune encephalomyelitis, and further show its extent throughout the CNS axis and at the level of the single vessel/capillary.

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 SOD1^G93A ALS mice spinal cord at a micrometric scale. An unprecedented direct quantification of neuro-vascular alterations at different stages of the disease is shown.

The Hartmann wavefront sensor is able to measure, separately and in absolute, the real δ and imaginary part β of the X-ray refractive index. While combined with tomographic setup, the Hartman sensor opens many interesting opportunities behind the direct measurement of the material density. In order to handle the different ways of using an X-ray wavefront sensor in imaging, we developed a 3D wave propagation model based on Fresnel propagator. The model can manage any degree of spatial coherence of the source, thus enabling us to model experiments accurately using tabletop, synchrotron or X-ray free-electron lasers. Beam divergence is described in a physical manner consistent with the spatial coherence. Since the Hartmann sensor can detect phase and absorption variation with high sensitivity, a precise simulation tool is thus needed to optimize the experimental parameters. Examples are displayed.

Synchrotron X-ray Phase Contrast micro-Tomography (SXrPC mu T) is a powerful tool in the investigation of biological tissues, including the central nervous system (CNS), and it allows to simultaneously detect the vascular and neuronal network avoiding contrast agents or destructive sample preparations. However, specific sample preparation procedures aimed to optimize the achievable contrast-and signal-to-noise ratio (CNR and SNR, respectively) are required. Here we report and discuss the effects of perfusion with two different fixative agents (ethanol and paraformaldehyde) and with a widely used contrast medium (MICROFIL (R)) on mouse spinal cord. As a main result, we found that ethanol enhances contrast at the grey/white matter interface and increases the contrast in correspondence of vascular features and fibres, thus providing an adequate spatial resolution to visualise the vascular network at the microscale. On the other hand, ethanol is known to induce tissue dehydration, likely reducing cell dimensions below the spatial resolution limit imposed by the experimental technique. Nonetheless, neurons remain well visible using either perfused paraformaldehyde or MICROFIL (R) compound, as these latter media do not affect tissues with dehydration effects. Paraformaldehyde appears as the best compromise: it is not a contrast agent, like MICROFIL (R), but it is less invasive than ethanol and permits to visualise well both cells and blood vessels. However, a quantitative estimation of the relative grey matter volume of each sample has led us to conclude that no significant alterations in the grey matter extension compared to the white matter occur as a consequence of the perfusion procedures tested in this study.

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.