MRI overestimates articular cartilage thickness and volume compared to synchrotron radiation phase-contrast imaging.

Accurate evaluation of morphological changes in articular cartilage are necessary for early detection of osteoarthritis (OA). 3T magnetic resonance imaging (MRI) has highly sensitive contrast resolution and is widely used clinically to detect OA. However, synchrotron radiation phase-contrast imaging computed tomography (SR-PCI) can also provide contrast to tissue interfaces that do not have sufficient absorption differences, with the added benefit of very high spatial resolution. Here, MRI was compared with SR-PCI for quantitative evaluation of human articular cartilage. Medial tibial condyles were harvested from non-OA donors and from OA patients receiving knee replacement surgery. Both imaging methods revealed that average cartilage thickness and cartilage volume were significantly reduced in the OA group, compared to the non-OA group. When comparing modalities, the superior resolution of SR-PCI enabled more precise mapping of the cartilage surface relative to MRI. As a result, MRI showed significantly higher average cartilage thickness and cartilage volume, compared to SR-PCI. These data highlight the potential for high-resolution imaging of articular cartilage using SR-PCI as a solution for early OA diagnosis. Recognizing current limitations of using a synchrotron for clinical imaging, we discuss its nascent utility for preclinical models, particularly longitudinal studies of live animal models of OA.

Daily administration of parathyroid hormone slows the progression of basic multicellular units in the cortical bone of the rabbit distal tibia.

Basic Multicellular Units (BMUs) conduct bone remodeling, a critical process of tissue turnover which, if imbalanced, can lead to disease, including osteoporosis. Parathyroid hormone (PTH 1-34; Teriparatide) is an osteoanabolic treatment for osteoporosis; however, it elevates the rate of intra-cortical remodeling (activation frequency) leading, at least transiently, to increased porosity. The purpose of this study was to test the hypothesis that PTH not only increases the rate at which cortical BMUs are initiated but also increases their progression (Longitudinal Erosion Rate; LER). Two groups (n = 7 each) of six-month old female New Zealand white rabbits were both administered 30 µg/kg of PTH once daily for a period of two weeks to induce remodeling. Their distal right tibiae were then imaged in vivo by in-line phase contrast micro-CT at the Canadian Light Source synchrotron. Over the following two weeks the first group (PTH) received continued daily PTH while the second withdrawal group (PTHW) was administrated 0.9 % saline. At four weeks all animals were euthanized, their distal tibiae were imaged by conventional micro-CT ex vivo and histomorphometry was performed. Matching micro-CT datasets (in vivo and ex vivo) were co-registered in 3D and LER was measured from 612 BMUs. Counter to our hypothesis, mean LER was lower (p < 0.001) in the PTH group (30.19 ± 3.01 µm/day) versus the PTHW group (37.20 ± 2.77 µm/day). Despite the difference in LER, osteonal mineral apposition rate (On.MAR) did not differ between groups indicating the anabolic effect of PTH was sustained after withdrawal. The slowing of BMU progression by PTH warrants further investigation; slowed resorption combined with elevated bone formation rate, may play an important role in how PTH enhances coupling between resorption and formation within the BMU. Finally, the prolonged anabolic response following withdrawal may have utility in terms of optimizing clinical dosing regimens.

Direct Assessment of Rabbit Cortical Bone Basic Multicellular Unit Longitudinal Erosion Rate: A 4D Synchrotron-Based Approach.

Cortical bone remodeling is carried out by basic multicellular units (BMUs), which couple resorption to formation. Although fluorochrome labeling has facilitated study of BMU formative parameters since the 1960s, some resorptive parameters, including the longitudinal erosion rate (LER), have remained beyond reach of direct measurement. Indeed, our only insights into this spatiotemporal parameter of BMU behavior come from classical studies that indirectly inferred LER. Here, we demonstrate a 4D in vivo method to directly measure LER through in-line phase contrast synchrotron imaging. The tibias of rabbits (n = 15) dosed daily with parathyroid hormone were first imaged in vivo (synchrotron micro-CT; day 15) and then ex vivo 14 days later (conventional micro-CT; day 29). Mean LER assessed by landmarking the co-registered scans was 23.69 ± 1.73 µm/d. This novel approach holds great promise for the direct study of the spatiotemporal coordination of bone remodeling, its role in diseases such as osteoporosis, as well as related treatments. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Wide field imaging energy dispersive X-ray absorption spectroscopy.

A new energy dispersive X-ray absorption spectroscopy (EDXAS) method is presented for simultaneous wide-field imaging and transmission X-ray absorption spectroscopy (XAS) to enable rapid imaging and speciation of elements. Based on spectral K-Edge Subtraction imaging (sKES), a bent Laue imaging system diffracting in the vertical plane was developed on a bend magnet beamline for selenium speciation. The high flux and small vertical focus, forming a wide horizontal line beam for projection imaging and computed tomography applications, is achieved by precise matching of lattice plane orientation and crystal surface (asymmetry angle). The condition generating a small vertical focus for imaging also provides good energy dispersion. Details for achieving sufficient energy and spatial resolution are demonstrated for both full field imaging and computed tomography in quantifying selenium chemical species. While this system has lower sensitivity as it uses transmission and may lack the flux and spatial resolution of a dedicated focused beamline system, it has significant potential in rapid screening of heterogeneous biomedical or environmental systems to correlate metal speciation with function.

Nebulized hypertonic saline triggers nervous system-mediated active liquid secretion in cystic fibrosis swine trachea.

Inhaled hypertonic saline (HTS) treatment is used to improve lung health in patients with cystic fibrosis (CF). The current consensus is that the treatment generates an osmotic gradient that draws water into the airways and increases airway surface liquid (ASL) volume. However, there is evidence that HTS may also stimulate active secretion of ASL by airway epithelia through the activation of sensory neurons. We tested the contribution of the nervous system and airway epithelia on HTS-stimulated ASL height increase in CF and wild-type swine airway. We used synchrotron-based imaging to investigate whether airway neurons and epithelia are involved in HTS treatment-triggered ASL secretion in CFTR-/- and wild-type swine. We showed that blocking parasympathetic and sensory neurons in airway resulted in ~50% reduction of the effect of HTS treatment on ASL volume in vivo. Incubating tracheal preparations with inhibitors of epithelial ion transport across airway decreased secretory responses to HTS treatment. CFTR-/- swine ex-vivo tracheal preparations showed substantially decreased secretory response to HTS treatment after blockage of neuronal activity. Our results indicated that HTS-triggered ASL secretion is partially mediated by the stimulation of airway neurons and the subsequent activation of active epithelia secretion; osmosis accounts for only ~50% of the effect.

Biodistribution of strontium and barium in the developing and mature skeleton of rats.

Bone acts as a reservoir for many trace elements. Understanding the extent and pattern of elemental accumulation in the skeleton is important from diagnostic, therapeutic, and toxicological perspectives. Some elements are simply adsorbed to bone surfaces by electric force and are buried under bone mineral, while others can replace calcium atoms in the hydroxyapatite structure. In this article, we investigated the extent and pattern of skeletal uptake of barium and strontium in two different age groups, growing, and skeletally mature, in healthy rats. Animals were dosed orally for 4 weeks with either strontium chloride or barium chloride or combined. The distribution of trace elements was imaged in 3D using synchrotron K-edge subtraction micro-CT at 13.5 µm resolution and 2D electron probe microanalysis (EPMA). Bulk concentration of the elements in serum and bone (tibiae) was also measured by mass spectrometry to study the extent of uptake. Toxicological evaluation did not show any cardiotoxicity or nephrotoxicity. Both elements were primarily deposited in the areas of active bone turnover such as growth plates and trabecular bone. Barium and strontium concentration in the bones of juvenile rats was 2.3 times higher, while serum levels were 1.4 and 1.5 times lower than adults. In all treatment and age groups, strontium was preferred to barium even though equal molar concentrations were dosed. This study displayed spatial co-localization of barium and strontium in bone for the first time. Barium and strontium can be used as surrogates for calcium to study the pathological changes in animal models of bone disease and to study the effects of pharmaceutical compounds on bone micro-architecture and bone remodeling in high spatial sensitivity and precision.

High-power-load DCLM monochromator for a computed tomography program at BMIT at energies of 25-150 keV.

The research program at the biomedical imaging facility requires a high-flux hard-X-ray monochromator that can also provide a wide beam. A wide energy range is needed for standard radiography, phase-contrast imaging, K-edge subtraction imaging and monochromatic beam therapy modalities. The double-crystal Laue monochromator, developed for the BioMedical Imaging and Therapy facility, is optimized for the imaging of medium- and large-scale samples at high energies with the resolution reaching 4 µm. A pair of 2 mm-thick Si(111) bent Laue-type crystals were used in fixed-exit beam mode with a 16 mm vertical beam offset and the first crystal water-cooled. The monochromator operates at energies from 25 to 150 keV, and the measured size of the beam is 189 mm (H) × 8.6 mm (V) at 55 m from the source. This paper presents our approach in developing a complete focusing model of the monochromator. The model uses mechanical properties of crystals and benders to obtain a finite-element analysis of the complete assembly. The modeling results are compared and calibrated with experimental measurements. Using the developed analysis, a rough estimate of the bending radius and virtual focus (image) position of the first crystal can be made, which is also the real source for the second crystal. On the other hand, by measuring the beam height in several points in the SOE-1 hutch, the virtual focus of the second crystal can be estimated. The focusing model was then calibrated with measured mechanical properties, the values for the force and torque applied to the crystals were corrected, and the actual operating parameters of the monochromator for fine-tuning were provided.

Cystic fibrosis swine fail to secrete airway surface liquid in response to inhalation of pathogens.

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) channel, which can result in chronic lung disease. The sequence of events leading to lung disease is not fully understood but recent data show that the critical pathogenic event is the loss of the ability to clear bacteria due to abnormal airway surface liquid secretion (ASL). However, whether the inhalation of bacteria triggers ASL secretion and whether this is abnormal in cystic fibrosis has never been tested. Here we show, using a novel synchrotron-based in vivo imaging technique, that wild-type pigs display both a basal and a Toll-like receptor-mediated ASL secretory response to the inhalation of cystic fibrosis relevant bacteria. Both mechanisms fail in CFTR-/- swine, suggesting that cystic fibrosis airways do not respond to inhaled pathogens, thus favoring infection and inflammation that may eventually lead to tissue remodeling and respiratory disease.Cystic fibrosis is caused by mutations in the CFTR chloride channel, leading to reduced airway surface liquid secretion. Here the authors show that exposure to bacteria triggers secretion in wild-type but not in pig models of cystic fibrosis, suggesting an impaired response to pathogens contributes to infection.

Spectral K-edge subtraction imaging of experimental non-radioactive barium uptake in bone.

PURPOSE: To evaluate the feasibility of using non-radioactive barium as a bone tracer for detection with synchrotron spectral K-edge subtraction (SKES) technique. METHODS: Male rats of 1-month old (i.e., developing skeleton) and 8-month old (i.e., skeletally mature) were orally dosed with low dose of barium chloride (33mg/kg/day Ba2+) for 4weeks. The fore and hind limbs were dissected for imaging in projection and computed tomography modes at 100µm and 52µm pixel sizes. The SKES method utilizes a single bent Laue monochromator to prepare a 550eV energy spectrum to encompass the K-edge of barium (37.441keV), for collecting both 'above' and 'below' the K-edge data sets in a single scan. RESULTS: The SKES has a very good focal size, thus limits the 'crossover' and motion artifacts. In juvenile rats, barium was mostly incorporated in the areas of high bone turnover such as at the growth plate and the trabecular surfaces, but also in the cortical bone as the animals were growing at the time of tracer administration. However, the adults incorporated approximately half the concentration and mainly in the areas where bone remodeling was predominant and occasionally in the periosteal and endosteal layers of the diaphyseal cortical bone. CONCLUSIONS: The presented methodology is simple to implement and provides both structural and functional information, after labeling with barium, on bone micro-architecture and thus has great potential for in vivo imaging of pre-clinical animal models of musculoskeletal diseases to better understand their mechanisms and to evaluate the efficacy of pharmaceuticals.

Design of a mouse restraint for synchrotron-based computed tomography imaging.

High-resolution computed tomography (CT) imaging of a live animal within a lead-lined synchrotron light hutch presents several unique challenges. In order to confirm that the animal is under a stable plane of anaesthesia, several physiological parameters (e.g. heart rate, arterial oxygen saturation, core body temperature and respiratory rate) must be remotely monitored from outside the imaging hutch. In addition, to properly scan the thoracic region using CT, the animal needs to be held in a vertical position perpendicular to the fixed angle of the X-ray beam and free to rotate 180°-360°. A new X-ray transparent mouse restraint designed and fabricated using computer-aided design software and three-dimensional rapid prototype printing has been successfully tested at the Biomedical Imaging and Therapy bending-magnet (BMIT-BM) beamline at the Canadian Light Source.

Diffraction-Enhanced Computed Tomographic Imaging of Growing Piglet Joints by Using a Synchrotron Light Source.

The objective of this project was to develop and test a new technology for imaging growing joints by means of diffraction-enhanced imaging (DEI) combined with CT and using a synchrotron radiation source. DEI-CT images of an explanted 4-wk-old piglet stifle joint were acquired by using a 40-keV beam. The series of scanned slices was later 'stitched' together, forming a 3D dataset. High-resolution DEI-CT images demonstrated fine detail within all joint structures and tissues. Striking detail of vasculature traversing between bone and cartilage, a characteristic of growing but not mature joints, was demonstrated. This report documents for the first time that DEI combined with CT and a synchrotron radiation source can generate more detailed images of intact, growing joints than can currently available conventional imaging modalities.

Design and implementation of a compact low-dose diffraction enhanced medical imaging system.

RATIONALE AND OBJECTIVES: Diffraction-enhanced imaging (DEI) is a new x-ray imaging modality that differs from conventional radiography in its use of three physical mechanisms to generate contrast. DEI is able to generate contrast from x-ray absorption, refraction, and ultra-small-angle scatter rejection (extinction) to produce high-contrast images with a much lower radiation dose compared to conventional radiography. MATERIALS AND METHODS: A prototype DEI system was constructed using a 1-kW tungsten x-ray tube and a single silicon monochromator and analyzer crystal. The monochromator crystal was aligned to reflect the combined Kalpha1 (59.32 keV) and Kalpha2 (57.98 keV) characteristic emission lines of tungsten using a tube voltage of 160 kV. System performance and demonstration of contrast were evaluated using a nylon monofilament refraction phantom, full-thickness breast specimens, a human thumb, and a live mouse. RESULTS: Images acquired using this system successfully demonstrated all three DEI contrast mechanisms. Flux measurements acquired using this 1-kW prototype system demonstrated that this design can be scaled to use a more powerful 60-kW x-ray tube to generate similar images with an image time of approximately 30 seconds. This single-crystal pair design can be further modified to allow for an array of crystals to reduce clinical image times to <3 seconds. CONCLUSIONS: This paper describes the design, construction, and performance of a new DEI system using a commercially available tungsten anode x-ray tube and includes the first high-quality low-dose diffraction-enhanced images of full-thickness human tissue specimens.

Synchrotron X-ray fluorescence reveals abnormal metal distributions in brain and spinal cord in spinocerebellar ataxia: a case report.

For the first time, synchrotron rapid-scanning X-ray fluorescence (RS-XRF) was used to simultaneously localize and quantify iron, copper, and zinc in spinal cord and brain in a case of spinocerebellar ataxia (SCA). In the normal medulla, a previously undescribed copper enrichment was seen associated with spinocerebellar fibers and amiculum olivae. This region was virtually devoid of all metals in the SCA case. Regions with neuronal loss and gliosis in the cerebellar cortex, inferior olivary, and dentate nuclei and areas showing loss of myelinated fibers were also low in all metals in SCA compared to control. In contrast, the ventral columns of the spinal cord that exhibited only moderate myelin pallor had increased metal levels. Iron and zinc were also elevated in the globus pallidus pars externa in SCA relative to control. We hypothesize that metals increase as part of the initial neurodegenerative process, but once degeneration is advanced, the metal levels drop. This implies a role for multiple metals in SCA neurodegeneration, but further study is required to establish a causative role. We suggest that if these findings are generally true of at least some cases of SCA, not only iron but also copper and zinc should be considered as possible therapeutic targets.

Mapping metals in Parkinson's and normal brain using rapid-scanning x-ray fluorescence.

Rapid-scanning x-ray fluorescence (RS-XRF) is a synchrotron technology that maps multiple metals in tissues by employing unique hardware and software to increase scanning speed. RS-XRF was validated by mapping and quantifying iron, zinc and copper in brain slices from Parkinson's disease (PD) and unaffected subjects. Regions and structures in the brain were readily identified by their metal complement and each metal had a unique distribution. Many zinc-rich brain regions were low in iron and vice versa. The location and amount of iron in brain regions known to be affected in PD agreed with analyses using other methods. Sample preparation is simple and standard formalin-fixed autopsy slices are suitable. RS-XRF can simultaneously and non-destructively map and quantify multiple metals and holds great promise to reveal metal pathologies associated with PD and other neurodegenerative diseases as well as diseases of metal metabolism.

Memory and survival after microbeam radiation therapy.

BACKGROUND: Disturbances of memory function are frequently observed in patients with malignant brain tumours and as adverse effects after radiotherapy to the brain. Experiments in small animal models of malignant brain tumour using synchrotron-based microbeam radiation therapy (MRT) have shown a promising prolongation of survival times. MATERIALS AND METHODS: Two animal models of malignant brain tumour were used to study survival and memory development after MRT. Thirteen days after implantation of tumour cells, animals were submitted to MRT either with or without adjuvant therapy (buthionine-SR-sulfoximine=BSO or glutamine). We used two orthogonal 1-cm wide arrays of 50 microplanar quasiparallel microbeams of 25 microm width and a center-to-center distance of about 200 microm, created by a multislit collimator, with a skin entrance dose of 350 Gy for each direction. Object recognition tests were performed at day 13 after tumour cell implantation and in monthly intervals up to 1 year after tumour cell implantation. RESULTS: In both animal models, MRT with and without adjuvant therapy significantly increased survival times. BSO had detrimental effects on memory function early after therapy, while administration of glutamine resulted in improved memory.

Diffraction-enhanced imaging of a porcine eye.

BACKGROUND: Diffraction-enhanced imaging (DEI) is a synchrotron-based x-ray imaging technique that has dramatically improved contrast over standard x-ray imaging techniques. It is possible to acquire images that analyze the x-ray refraction and the apparent absorption (elimination of small-angle scattering) of the object. METHODS: Three formalin-fixed porcine eyes were studied at the National Synchrotron Light Source using DEI. Conventional absorption-type radiography was conducted for comparison. RESULTS: Conventional absorption radiography did not yield significant detail of the eye anatomy. DEI showed excellent characterization of many ocular structures. The cornea, iris, lens, retina, optic nerve, as well as choroidal vasculature and the ampullae of the vortex veins could all be visualized. INTERPRETATION: DEI represents a novel, high-resolution imaging technique that has excellent characterization of ocular anatomy. Further application of this imaging modality will be undertaken to study cataract and choroidal tumors and to examine ocular surface structures, such as the extraocular muscle insertions, more closely.

Diffraction-enhanced imaging of the rat spine.

INTRODUCTION: Diffraction-enhanced imaging (DEI) uses monochromatic synchrotron X-rays to image tissue. This technique has been shown to produce superior bony and soft tissue characterization when compared with conventional absorption radiography. Application of this imaging modality is under investigation, and this study represents the first DEI analysis of the vertebral column. METHODS: Four male Wistar rats were studied. Spine muscle blocks were imaged in 3 of the rats after thoracic laminectomy (n = 1), after lumbar laminectomy (n = 1), and in a control condition (n = 1). The fourth rat was imaged as a whole animal control. Conventional radiography and synchrotron-supported DEI at 40 keV were performed on all specimens. We compared images side by side, using a nonvalidated subjective assessment technique. RESULTS: DEI produced superior visualization of the vertebral anatomy, compared with conventional absorption radiography for all specimens. Greater bony and soft tissue detail was noted, with improved image contrast. In addition to imaging the anatomical structures, DEI showed the polyglactin suture material used for fascial closure in the 2 animals that underwent surgery. Artifact from air bubbles was present on DEI images but not on plain radiographs. CONCLUSIONS: This represents the first use of DEI, a novel imaging modality, to image the vertebral column. It provides excellent anatomic detail with superior contrast and visualization of both bone and soft tissue when compared with conventional radiography. Future applications of this investigational technique may include analysis of spinal fusion as well as degenerative and neoplastic conditions of the spine.

Radiography of rabbit articular cartilage with diffraction-enhanced imaging.

Articular cartilage of synovial joints is not visible with conventional X-ray imaging. Hence, the gradual degeneration and destruction of articular cartilage, which is characteristic of degenerative joint diseases, is only detected at a late stage when the cartilage is lost and the joint space that it once occupied narrows. The development of an X-ray imaging technique that could detect both the degenerative cartilage and bone features of joint diseases is of special interest. Here we show, for the first time, that a high-contrast imaging technique, diffraction-enhanced X-ray imaging (DEI), allows the visualization of articular cartilage of both disarticulated and articulated rabbit knee joints. Furthermore, a single cartilage lesion can be visualized within an intact joint. The results suggest that DEI has the potential to be of use in the study of cartilage degeneration.