X-Ray Phase Contrast Tomography Reveals Early Vascular Alterations and Neuronal Loss in a Multiple Sclerosis Model.

The degenerative effects of multiple sclerosis at the level of the vascular and neuronal networks in the central nervous system are currently the object of intensive investigation. Preclinical studies have demonstrated the efficacy of mesenchymal stem cell (MSC) therapy in experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis, but the neuropathology of specific lesions in EAE and the effects of MSC treatment are under debate. Because conventional imaging techniques entail protocols that alter the tissues, limiting the reliability of the results, we have used non-invasive X-ray phase-contrast tomography to obtain an unprecedented direct 3D characterization of EAE lesions at micro-to-nano scales, with simultaneous imaging of the vascular and neuronal networks. We reveal EAE-mediated alterations down to the capillary network. Our findings shed light on how the disease and MSC treatment affect the tissues, and promote X-ray phase-contrast tomography as a powerful tool for studying neurovascular diseases and monitoring advanced therapies.

Hard X-ray index of refraction tomography of a whole rabbit knee joint: A feasibility study.

We report results of the computed tomography reconstruction of the index of refraction in a whole rabbit knee joint examined at the photon energy of 51keV. Refraction based images make it possible to delineate the bone, cartilage, and soft tissues without adjusting the contrast window width and level. Density variations, which are related to tissue composition and are not visible in absorption X-ray images, are detected in the obtained refraction based images. We discuss why refraction-based images provide better detectability of low contrast features than absorption images.

Virtual unrolling and deciphering of Herculaneum papyri by X-ray phase-contrast tomography.

A collection of more than 1800 carbonized papyri, discovered in the Roman 'Villa dei Papiri' at Herculaneum is the unique classical library survived from antiquity. These papyri were charred during 79 A.D. Vesuvius eruption, a circumstance which providentially preserved them until now. This magnificent collection contains an impressive amount of treatises by Greek philosophers and, especially, Philodemus of Gadara, an Epicurean thinker of 1st century BC. We read many portions of text hidden inside carbonized Herculaneum papyri using enhanced X-ray phase-contrast tomography non-destructive technique and a new set of numerical algorithms for 'virtual-unrolling'. Our success lies in revealing the largest portion of Greek text ever detected so far inside unopened scrolls, with unprecedented spatial resolution and contrast, all without damaging these precious historical manuscripts. Parts of text have been decoded and the 'voice' of the Epicurean philosopher Philodemus is brought back again after 2000 years from Herculaneum papyri.

A single-image method for x-ray refractive index CT.

X-ray refraction-based computer tomography imaging is a well-established method for nondestructive investigations of various objects. In order to perform the 3D reconstruction of the index of refraction, two or more raw computed tomography phase-contrast images are usually acquired and combined to retrieve the refraction map (i.e. differential phase) signal within the sample. We suggest an approximate method to extract the refraction signal, which uses a single raw phase-contrast image. This method, here applied to analyzer-based phase-contrast imaging, is employed to retrieve the index of refraction map of a biological sample. The achieved accuracy in distinguishing the different tissues is comparable with the non-approximated approach. The suggested procedure can be used for precise refraction computer tomography with the advantage of a reduction of at least a factor of two of both the acquisition time and the dose delivered to the sample with respect to any of the other algorithms in the literature.

A track length estimator method for dose calculations in low-energy X-ray irradiations: implementation, properties and performance.

The track length estimator (TLE) method, an "on-the-fly" fluence tally in Monte Carlo (MC) simulations, recently implemented in GATE 6.2, is known as a powerful tool to accelerate dose calculations in the domain of low-energy X-ray irradiations using the kerma approximation. Overall efficiency gains of the TLE with respect to analogous MC were reported in the literature for regions of interest in various applications (photon beam radiation therapy, X-ray imaging). The behaviour of the TLE method in terms of statistical properties, dose deposition patterns, and computational efficiency compared to analogous MC simulations was investigated. The statistical properties of the dose deposition were first assessed. Derivations of the variance reduction factor of TLE versus analogous MC were carried out, starting from the expression of the dose estimate variance in the TLE and analogous MC schemes. Two test cases were chosen to benchmark the TLE performance in comparison with analogous MC: (i) a small animal irradiation under stereotactic synchrotron radiation therapy conditions and (ii) the irradiation of a human pelvis during a cone beam computed tomography acquisition. Dose distribution patterns and efficiency gain maps were analysed. The efficiency gain exhibits strong variations within a given irradiation case, depending on the geometrical (voxel size, ballistics) and physical (material and beam properties) parameters on the voxel scale. Typical values lie between 10 and 10(3), with lower levels in dense regions (bone) outside the irradiated channels (scattered dose only), and higher levels in soft tissues directly exposed to the beams.

Breast tumor segmentation in high resolution x-ray phase contrast analyzer based computed tomography.

PURPOSE: Phase contrast computed tomography has emerged as an imaging method, which is able to outperform present day clinical mammography in breast tumor visualization while maintaining an equivalent average dose. To this day, no segmentation technique takes into account the specificity of the phase contrast signal. In this study, the authors propose a new mathematical framework for human-guided breast tumor segmentation. This method has been applied to high-resolution images of excised human organs, each of several gigabytes. METHODS: The authors present a segmentation procedure based on the viscous watershed transform and demonstrate the efficacy of this method on analyzer based phase contrast images. The segmentation of tumors inside two full human breasts is then shown as an example of this procedure's possible applications. RESULTS: A correct and precise identification of the tumor boundaries was obtained and confirmed by manual contouring performed independently by four experienced radiologists. CONCLUSIONS: The authors demonstrate that applying the watershed viscous transform allows them to perform the segmentation of tumors in high-resolution x-ray analyzer based phase contrast breast computed tomography images. Combining the additional information provided by the segmentation procedure with the already high definition of morphological details and tissue boundaries offered by phase contrast imaging techniques, will represent a valuable multistep procedure to be used in future medical diagnostic applications.

Radiation dose in breast CT imaging with monochromatic x-rays: simulation study of the influence of energy, composition and thickness.

Recent developments have shown that high resolution phase contrast x-ray computed tomography (CT) of the breast can be performed at clinically compatible doses. Results have yet been obtained in vitro on full breasts, and the clinical translation of the technique seems more and more possible. This work presents a method to quickly estimate the average dose in the organ using the software GATE. The influence of different parameters on the dose distribution, like breast composition and thickness, and for preclinical test, the presence of a skin/PMMA external layer, has been investigated. Several correction factors, to be applied to the given dose database, are also introduced to allow the use of these results in geometries different from those studied here. An energy optimization study is presented that considers also the influence on the energy choice of x-ray detector. A simple analytical method to estimate the best energy that minimizes the dose-transmittance ratio in CT imaging is presented and compared with the results of simulations.

Analytical and experimental determination of signal-to-noise ratio and figure of merit in three phase-contrast imaging techniques.

We present a theoretical and experimental comparison of three X-ray phase-contrast techniques: propagation-based imaging, analyzer-based imaging and grating interferometry. The signal-to-noise ratio and the figure of merit are quantitatively compared for the three techniques on the same phantoms and using the same X-ray source and detector. Principal dependencies of the signal upon the numerous acquisition parameters, the spatial resolution and X-ray energy are discussed in detail. The sensitivity of each technique, in terms of the smallest detectable phase shift, is also evaluated.

High-resolution breast tomography at high energy: a feasibility study of phase contrast imaging on a whole breast.

Previous studies on phase contrast imaging (PCI) mammography have demonstrated an enhancement of breast morphology and cancerous tissue visualization compared to conventional imaging. We show here the first results of the PCI analyser-based imaging (ABI) in computed tomography (CT) mode on whole and large (>12 cm) tumour-bearing breast tissues. We demonstrate in this work the capability of the technique of working at high x-ray energies and producing high-contrast images of large and complex specimens. One entire breast of an 80-year-old woman with invasive ductal cancer was imaged using ABI-CT with monochromatic 70 keV x-rays and an area detector of 92×92 µm² pixel size. Sagittal slices were reconstructed from the acquired data, and compared to corresponding histological sections. Comparison with conventional absorption-based CT was also performed. Five blinded radiologists quantitatively evaluated the visual aspects of the ABI-CT images with respect to sharpness, soft tissue contrast, tissue boundaries and the discrimination of different structures/tissues. ABI-CT excellently depicted the entire 3D architecture of the breast volume by providing high-resolution and high-contrast images of the normal and cancerous breast tissues. These results are an important step in the evolution of PCI-CT towards its clinical implementation.

Theoretical comparison of three X-ray phase-contrast imaging techniques: propagation-based imaging, analyzer-based imaging and grating interferometry.

Various X-ray phase-contrast imaging techniques have been developed and applied over the last twenty years in different domains, such as material sciences, biology and medicine. However, no comprehensive inter-comparison exists in the literature. We present here a theoretical study that compares three among the most used techniques: propagation-based imaging (PBI), analyzer-based imaging (ABI) and grating interferometry (GI). These techniques are evaluated in terms of signal-to-noise ratio, figure of merit and spatial resolution. Both area and edge signals are considered. Dependences upon the object properties (absorption, phase shift) and the experimental acquisition parameters (energy, system point-spread function etc.) are derived and discussed. The results obtained from this analysis can be used as the reference for determining the most suitable technique for a given application.

Dietary composition programmes placental phenotype in mice.

Dietary composition during pregnancy influences fetal and adult phenotype but its effects on placental phenotype remain largely unknown. Using molecular, morphological and functional analyses, placental nutrient transfer capacity was examined in mice fed isocaloric diets containing 23%, 18% or 9% casein (C) during pregnancy. At day 16, placental transfer of glucose, but not methyl-aminoisobutyric acid (MeAIB), was greater in C18 and C9 than C23 mice, in association with increased placental expression of the glucose transporter Slc2a1/GLUT1, and the growth factor Igf2. At day 19, placental glucose transport remained high in C9 mice while MeAIB transfer was less in C18 than C23 mice, despite greater placental weights in C18 and C9 than C23 mice. Placental System A amino acid transporter expression correlated with protein intake at day 19. Relative growth of transport verses endocrine zones of the placenta was influenced by diet at both ages without changing the absolute volume of the transport surface. Fetal weight was unaffected by diet at day 16 but was reduced in C9 animals by day 19. Morphological and functional adaptations in placental phenotype, therefore, occur to optimise nutrient transfer when dietary composition is varied, even subtly. This has important implications for the intrauterine programming of life expectancy.

Placental-specific Igf2 deficiency alters developmental adaptations to undernutrition in mice.

The pattern of fetal growth is a major determinant of the subsequent health of the infant. We recently showed in undernourished (UN) mice that fetal growth is maintained until late pregnancy, despite reduced placental weight, through adaptive up-regulation of placental nutrient transfer. Here, we determine the role of the placental-specific transcript of IGF-II (Igf2P0), a major regulator of placental transport capacity in mice, in adapting placental phenotype to UN. We compared the morphological and functional responses of the wild-type (WT) and Igf2P0-deficient placenta in WT mice fed ad libitium or 80% of the ad libitium intake. We observed that deletion of Igf2P0 prevented up-regulation of amino acid transfer normally seen in UN WT placenta. This was associated with a reduction in the proportion of the placenta dedicated to nutrient transport, the labyrinthine zone, and its constituent volume of trophoblast in Igf2P0-deficient placentas exposed to UN on d 16 of pregnancy. Additionally, Igf2P0-deficient placentas failed to up-regulate their expression of the amino acid transporter gene, Slc38a2, and down-regulate phosphoinositide 3-kinase-protein kinase B signaling in response to nutrient restriction on d 19. Furthermore, deleting Igf2P0 altered maternal concentrations of hormones (insulin and corticosterone) and metabolites (glucose) in both nutritional states. Therefore, Igf2P0 plays important roles in adapting placental nutrient transfer capacity during UN, via actions directly on the placenta and/or indirectly through the mother.

A simplified approach for computed tomography with an X-ray grating interferometer.

We present a simplified acquisition and processing method for X-ray grating interferometry computed tomography (CT). The proposed approach eliminates the need to scan the gratings, thus allowing for a faster CT acquisition compared to methods presently in use. The contrast in the reconstructed images can be expressed as a linear combination of the absorption and refraction within the sample. Experimental images of a test phantom made of known materials and a human bone-cartilage sample prove the correctness of the method and show very good agreement with the theory. The here proposed approach might be highly interesting in many fields where a reduced imaging acquisition time is requested and/or where the radiation dose delivered to the sample has to be kept low, such as, for example, in in-vivo imaging.

Environmental regulation of placental phenotype: implications for fetal growth.

Environmental conditions during pregnancy determine birthweight, neonatal viability and adult phenotype in human and other animals. In part, these effects may be mediated by the placenta, the principal source of nutrients for fetal development. However, little is known about the environmental regulation of placental phenotype. Generally, placental weight is reduced during suboptimal conditions like maternal malnutrition or hypoxaemia but compensatory adaptations can occur in placental nutrient transport capacity to help maintain fetal growth. In vivo studies show that transplacental glucose and amino acid transfer adapt to the prevailing conditions induced by manipulating maternal calorie intake, dietary composition and hormone exposure. These adaptations are due to changes in placental morphology, metabolism and/or abundance of specific nutrient transporters. This review examines environmental programming of placental phenotype with particular emphasis on placental nutrient transport capacity and its implications for fetal growth, mainly in rodents. It also considers the systemic, cellular and molecular mechanisms involved in signalling environmental cues to the placenta. Ultimately, the ability of the placenta to balance the competing interests of mother and fetus in resource allocation may determine not only the success of pregnancy in producing viable neonates but also the long-term health of the offspring.

Imprinted genes and the epigenetic regulation of placental phenotype.

Imprinted genes are expressed in a parent-of-origin manner by epigenetic modifications that silence either the paternal or maternal allele. They are widely expressed in fetal and placental tissues and are essential for normal placental development. In general, paternally expressed genes enhance feto-placental growth while maternally expressed genes limit conceptus growth, consistent with the hypothesis that imprinting evolved in response to the conflict between parental genomes in the allocation of maternal resources to fetal growth. Using targeted deletion, uniparental duplication, loss of imprinting and transgenic approaches, imprinted genes have been shown to determine the transport capacity of the definitive mouse placenta by regulating its growth, morphology and transporter abundance. Imprinted genes in the placenta are also responsive to environmental challenges and adapt placental phenotype to the prevailing nutritional conditions, in part, by varying their epigenetic status. In addition, interplay between placental and fetal imprinted genes is important in regulating resource partitioning via the placenta both developmentally and in response to environmental factors. By balancing the opposing parental drives on resource allocation with the environmental signals of nutrient availability, imprinted genes, like the Igf2-H19 locus, may act as nutrient sensors and optimise the fetal acquisition of nutrients for growth. These genes, therefore, have a major role in the epigenetic regulation of placental phenotype with long term consequences for the developmental programming of adult health and disease.

Adaptations in placental phenotype support fetal growth during undernutrition of pregnant mice.

Undernutrition during pregnancy reduces birth weight and programmes adult phenotype with consequences for life expectancy, but its effects on the phenotype of the placenta, responsible for supplying nutrients for fetal growth, remain largely unknown. Using molecular, morphological and functional analyses, placental phenotype was examined in mice during restriction of dietary intake to 80% of control from day 3 of pregnancy. At day 16, undernutrition reduced placental, but not fetal, weight in association with decreased junctional zone volume and placental expression of glucose transporter Slc2a1. At day 19, both placental and fetal weights were reduced in undernourished mice (91% and 87% of control, respectively, P < 0.01), as were the volume and surface area of the labyrinthine zone responsible for placental nutrient transfer (85% and 86%, respectively, P < 0.03). However, unidirectional materno-fetal clearance of tracer glucose was maintained and methyl-aminoisobutyric acid increased 166% (P < 0.005) per gram of undernourished placenta, relative to controls. This was associated with an 18% and 27% increased placental expression of glucose and system A amino acid transporters Slc2a1 and Slc38a2, respectively, at day 19 (P < 0.04). At both ages, undernutrition decreased expression of the placental specific transcript of the Igf2 gene by 35% (P < 0.01), although methylation of its promoter was unaffected. The placenta, therefore, adapts to help maintain fetal growth when its own growth is compromised by maternal undernutrition. Consequently, placental phenotype is responsive to environmental conditions and may help predict the risk of adult disease programmed in utero.

Placental efficiency and adaptation: endocrine regulation.

Size at birth is critical in determining life expectancy and is dependent primarily on the placental supply of nutrients. However, the fetus is not just a passive recipient of nutrients from the placenta. It exerts a significant acquisitive drive for nutrients, which acts through morphological and functional adaptations in the placenta, particularly when the genetically determined drive for fetal growth is compromised by adverse intrauterine conditions. These adaptations alter the efficiency with which the placenta supports fetal growth, which results in optimal growth for prevailing conditions in utero. This review examines placental efficiency as a means of altering fetal growth, the morphological and functional adaptations that influence placental efficiency and the endocrine regulation of these processes.

Disproportional effects of Igf2 knockout on placental morphology and diffusional exchange characteristics in the mouse.

Both complete knockout of the Igf2 gene (Igf2null(+/-)) and knockout of its placental specific transcript alone (Igf2P0(+/-)) lead to fetal growth restriction in mice. However, in the Igf2null(+/-) this growth restriction occurs concurrently in gestation with placental growth restriction, whereas, placental growth restriction precedes fetal growth restriction in the Igf2P0(+/-) mouse. Previous studies have shown that the Igf2P0(+/-) placenta has proportionate reductions in its cellular compartments and its diffusional exchange characteristics. Yet, nothing is known about the structural development or diffusional exchange characteristics of the Igf2null(+/-) mouse. Hence, this study compares the structural properties (using stereology) and diffusional exchange characteristics (using measurement of permeability-surface area product, P.S, of three inert hydrophilic tracers) of the Igf2null(+/-) and the Igf2P0(+/-) placenta to identify the role of Igf2 in the development of the labyrinthine exchange membrane and its functional consequences. Our data show disproportionate effects of complete Igf2 ablation on the compartments of the placenta, not seen when the placental-specific transcript alone is deleted. Furthermore, although the theoretical diffusing capacity (calculated from the stereological data) of the Igf2null(+/-) placenta was reduced relative to control, there was no effect of the complete knockout on permeability surface area available for small hydrophilic tracers. This is in contrast to the Igf2P0(+/-) placenta, where theoretical diffusion capacity and P.S values were reduced similarly. Total ablation of the Igf2 gene from the fetoplacental unit in the mouse therefore results in a disproportionate growth of placental compartments whereas, deleting the placental specific transcript of Igf2 alone results in proportional placental growth restriction. Thus, placental phenotype depends on the degree of Igf2 gene ablation and the interplay between placental and fetal Igf2 in the mouse.

Adaptations in placental nutrient transfer capacity to meet fetal growth demands depend on placental size in mice.

Experimental reduction in placental growth often leads to increased placental efficiency measured as grams of fetus produced per gram of placenta, although little is known about the mechanisms involved. This study tested the hypothesis that the smallest placenta within a litter is the most efficient at supporting fetal growth by examining the natural intra-litter variation in placental nutrient transfer capacity in normal pregnant mice. The morphology, nutrient transfer and expression of key growth and nutrient supply genes (Igf2P0, Grb10, Slc2a1, Slc2a3, Slc38a1, Slc38a2 and Slc38a4) were compared in the lightest and heaviest placentas of a litter at days 16 and 19 of pregnancy, when mouse fetuses are growing most rapidly in absolute terms. The data show that there are morphological and functional adaptations in the lightest placenta within a litter, which increase active transport of amino acids per gram of placenta and maintain normal fetal growth close to term, despite the reduced placental mass. The specific placental adaptations differ with age. At E16, they are primarily morphological with an increase in the volume fraction of the labyrinthine zone responsible for nutrient exchange, whereas at E19 they are more functional with up-regulated placental expression of the glucose transporter gene, Slc2a1/GLUT1 and one isoform the System A family of amino acid transporters, Slc38a2/SNAT2. Thus, this adaptability in placental phenotype provides a functional reserve capacity for maximizing fetal growth during late gestation when placental growth is compromised.