Synchrotron CT of an equine digit at the Australian Synchrotron Imaging and Medical Beamline.

Laminitis is an extremely painful and debilitating condition of horses that can affect their athletic ability and even quality of life. The current gold standard for assessment of laminar tissue is histology, which is the only modality that enables detailed visualization of the lamina. Histology requires dissection of the hoof and therefore can only represent one specific time point. The superior spatial and contrast resolution of synchrotron computed tomography (sCT), when compared with readily available imaging modalities, such as radiographs and conventional CT, provides an opportunity for detailed studies of the lamina without the need for hoof dissection and histological assessment. If the resolution of histology can be matched or even approached, dynamic events, such as laminar blood flow, could also be studied on the microscopic tissue level. To investigate this possible application of sCT further, two objectives are presented: (i) to develop a protocol for sCT of an equine digit using cadaver limbs and (ii) to apply the imaging protocol established during (i) for sCT imaging of the vasculature within the foot using an ex vivo perfusion system to deliver the vascular contrast. The hypotheses were that sCT would allow sufficient resolution for detailed visualization to the level of the secondary lamellae and associated capillaries within the equine digit. Synchrotron CT enabled good visualization of the primary lamellae (average length 3.6 mm) and the ex vivo perfusion system was able to deliver vascular contrast agent to the vessels of the lamina. The individual secondary lamellae (average length 0.142 mm) could not be seen in detail, although differentiation between primary and secondary lamellae was achieved. This approaches, but does not yet reach, the current gold standard, histology, for assessment of the lamellae; however, with further refinement of this imaging technique, improved resolution may be accomplished in future studies.

Screening of anti-mycobacterial compounds in a naturally infected zebrafish larvae model.

OBJECTIVES: Mycobacterium tuberculosis is a deadly human pathogen that causes the lung disease TB. M. tuberculosis latently infects a third of the world's population, resulting in ∼1.5 million deaths per year. Due to the difficulties and expense of carrying out animal drug trials using M. tuberculosis and rodents, infections of the zebrafish Danio rerio with Mycobacterium marinum have become a useful surrogate. However, the infection methods described to date require specialized equipment and a high level of operator expertise. METHODS: We investigated whether zebrafish larvae could be naturally infected with bioluminescently labelled M. marinum by immersion, and whether infected larvae could be used for rapid screening of anti-mycobacterial compounds using bioluminescence. We used rifampicin and a variety of nitroimidazole-based next-generation and experimental anti-mycobacterial drugs, selected for their wide range of potencies against M. tuberculosis, to validate this model for anti-mycobacterial drug discovery. RESULTS: We observed that five of the six treatments (rifampicin, pretomanid, delamanid, SN30488 and SN30527) significantly reduced the bioluminescent signal from M. marinum within naturally infected zebrafish larvae. Importantly, these same five treatments also retarded the growth of M. tuberculosis in vitro. In contrast, only three of the six treatments tested (rifampicin, delamanid and SN30527) retarded the growth of M. marinum in vitro. CONCLUSIONS: We have demonstrated that zebrafish larvae naturally infected with bioluminescent M. marinum M can be used for the rapid screening of anti-mycobacterial compounds with readily available equipment and limited expertise. The result is an assay that can be carried out by a wide variety of laboratories for minimal cost and without high levels of zebrafish expertise.

Imaging Breast Microcalcifications Using Dark-Field Signal in Propagation-Based Phase-Contrast Tomography.

Breast microcalcifications are an important primary radiological indicator of breast cancer. However, microcalcification classification and diagnosis may be still challenging for radiologists due to limitations of the standard 2D mammography technique, including spatial and contrast resolution. In this study, we propose an approach to improve the detection of microcalcifications in propagation-based phase-contrast X-ray computed tomography of breast tissues. Five fresh mastectomies containing microcalcifications were scanned at different X-ray energies and radiation doses using synchrotron radiation. Both bright-field (i.e. conventional phase-retrieved images) and dark-field images were extracted from the same data sets using different image processing methods. A quantitative analysis was performed in terms of visibility and contrast-to-noise ratio of microcalcifications. The results show that while the signal-to-noise and the contrast-to-noise ratios are lower, the visibility of the microcalcifications is more than two times higher in the dark-field images compared to the bright-field images. Dark-field images have also provided more accurate information about the size and shape of the microcalcifications.

Innate immune cells and bacterial infection in zebrafish.

The physical attributes of the zebrafish, including optical transparency during embryogenesis, large clutch sizes, external development, and rapid organogenesis were features that initially attracted developmental biologists to use this vertebrate as an experimental model system. With the progressive development of an extensive genetic "tool kit" and an ever-growing number of transgenic reporter lines, the zebrafish model has evolved into an informative system in which to mimic and study aspects of human disease, including those associated with bacterial infections. This chapter provides detailed protocols for microinjection of bacterial strains into zebrafish larvae and subsequent experiments to investigate single-larva bacterial burdens, live imaging of specific neutrophil and macrophage bactericidal functions, and how these protocols may be applied to drug discovery approaches to uncover novel immunomodulatory drugs.

Effects of anti-phencyclidine and anti-(+)-methamphetamine monoclonal antibodies alone and in combination on the discrimination of phencyclidine and (+)-methamphetamine by pigeons.

RATIONALE: Drug-specific monoclonal antibodies against phencyclidine (PCP) and (+)-methamphetamine [(+)-METH] should bind to these drugs to block their discriminative stimulus effects. OBJECTIVES: To determine if mouse monoclonal antibodies against PCP and (+)-METH can block the discriminative stimulus effects of the drugs in pigeons. MATERIALS AND METHODS: Pigeons were trained to discriminate among intramuscular injections of saline, 1 mg/kg PCP, and 2 mg/kg (+)-METH. After responding stabilized, cumulative dose-response curves were obtained for PCP and (+)-METH. Doses of an anti-PCP antibody at 620 mg/kg (anti-PCP mAb6B5) with a K (D) of 1.3 nM for PCP and no measurable affinity for (+)-METH and 1,000 mg/kg doses of anti-(+)-METH antibody (anti-METH mAb6H7) with a K (D) of 41 nM for (+)-METH and no measurable affinity for PCP were subsequently administered, first alone and later in combination after which the dose-response curves were redetermined. RESULTS: When the antibodies were given alone, the anti-PCP antibody blocked the discriminative stimulus effects of PCP, but not those of (+)-METH, and the anti-(+)-METH antibody blocked the discriminative stimulus effects of (+)-METH, but not those of PCP. The anti-PCP antibody shifted the PCP dose-response curve further to the right and for a longer time than the anti-(+)-METH antibody shifted the dose response curve for (+)-METH. When the anti-PCP and anti-(+)-METH antibodies were administered on the same day, the discriminative stimulus effects of both drugs were completely blocked 1 day after antibody administration. CONCLUSIONS: These experiments demonstrate the high specificity of the antibodies for the drugs to which they bind and show that monoclonal antibodies can be combined to antagonize the effects of more than one drug.

Model fitting in two dimensions to small angle diffraction patterns from soft tissue.

In our research programme small angle x-ray scattering (SAXS) is used to provide information on the axial arrangement of collagen molecules as well as data about the state of other components of the extra cellular matrix (ECM) in human tissues. Derivation of parameters to describe and simplify the data is required for much of the SAXS patterns analysis. A method is presented here to achieve function fitting to collagen diffraction peaks along with a representation of the underlying diffuse scatter. A simple model was used which proved reliable in fitting a variety of 2D diffraction patterns. The logarithm of the scatter intensity over the area of the scatter image was taken to reduce the range and improve fitting accuracy. Our model was then used to fit the log data. The model consisted of a radial exponential diffuse scatter component added to a specified number of Gaussian peaks. In 2D the peak model is toroidal, each component being rotated about a common specified centre. Initial search parameters from a 1D averaged sector were supplied to the iterative 2D fitting routine. With the aid of data weighting and basic wavelet filtering, successful and reliable fitting of a specified 2D model to real data is achievable. The process is easily automated. Multiple SAXS patterns can be fitted without operator intervention. As described the model is simple enough to converge rapidly and yet allows image data to be parameterized to a form suitable for extracting the requisite information. The fitting method is flexible enough to be extended to achieve a more comprehensive and complex pattern fitting in two dimensions if this turns out to be necessary. It is our intention to implement orientation distribution functions in the near future by including an angular scaling factor.

Assessment of biocompatibility of 3D printed photopolymers using zebrafish embryo toxicity assays.

3D printing has emerged as a rapid and cost-efficient manufacturing technique to enable the fabrication of bespoke, complex prototypes. If the technology is to have a significant impact in biomedical applications, such as drug discovery and molecular diagnostics, the devices produced must be biologically compatible to enable their use with established reference assays and protocols. In this work we demonstrate that we can adapt the Fish Embryo Test (FET) as a new method to quantify the toxicity of 3D printed microfluidic devices. We assessed the biocompatibility of four commercially available 3D printing polymers (VisiJetCrystal EX200, Watershed 11122XC, Fototec SLA 7150 Clear and ABSplus P-430), through the observation of key developmental markers in the developing zebrafish embryos. Results show all of the photopolymers to be highly toxic to the embryos, resulting in fatality, although we do demonstrate that post-printing treatment of Fototec 7150 makes it suitable for zebrafish culture within the FET.

A preliminary study of breast cancer diagnosis using laboratory based small angle x-ray scattering.

Breast tissue collected from tumour samples and normal tissue from bi-lateral mastectomy procedures were examined using small angle x-ray scattering. Previous work has indicated that breast tissue disease diagnosis could be performed using small angle x-ray scattering (SAXS) from a synchrotron radiation source. The technique would be more useful to health services if it could be made to work using a conventional x-ray source. Consistent and reliable differences in x-ray scatter distributions were observed between samples from normal and tumour tissue samples using the laboratory based 'SAXSess' system. Albeit from a small number of samples, a sensitivity of 100% was obtained. This result encourages us to pursue the implementation of SAXS as a laboratory based diagnosis technique.

Dynamic imaging of the lungs using x-ray phase contrast.

High quality real-time imaging of lungs in vivo presents considerable challenges. We demonstrate here that phase contrast x-ray imaging is capable of dynamically imaging the lungs. It retains many of the advantages of simple x-ray imaging, whilst also being able to map weakly absorbing soft tissues based on refractive index differences. Preliminary results reported herein show that this novel imaging technique can identify and locate airway liquid and allows lung aeration in newborn rabbit pups to be dynamically visualized.

Phase contrast X-ray imaging of mice and rabbit lungs: a comparative study.

The significant degree of X-ray phase contrast created by air-tissue interfaces, coupled with the poor radiographic contrast of conventional chest radiographs, makes the inflated lung an ideal candidate for investigating the potential diagnostic improvement afforded by phase contrast X-ray imaging. In small animals these methods highlight the lung airways and lobe boundaries and reveal the lung tissue as a speckled intensity pattern not seen in other soft tissues. We have compared analyser-based and propagation-based phase contrast imaging modalities, together with conventional radiographic imaging, to ascertain which technique shows the greatest image enhancement for various lung sizes. The conventional radiographic image of a mouse was obtained on a Siemens Nova 3000 mammography system, whilst phase contrast images of mice and rabbit chests were acquired at the medical imaging beamline (20B2) at the SPring-8 synchrotron radiation research facility in Japan. For mice aged 1 day, 1 week and 1 month old it was determined that analyser-based imaging showed the greatest overall image contrast, however, for an adult rabbit both techniques yielded excellent contrast. The success of these methods in creating high quality images for rabbit lungs raises the possibility of improving human lung imaging using phase contrast techniques.

Metabolism of [14C]testosterone by human foetal and adult brain tissue.

The metabolism of [14C]testosterone in vitro by various areas of the human foetal brain has been studied and compared with that of adult brain. The predominant metabolites were 5alpha-dihydrotestosterone and 5alpha-androstane-3alpha, 17beta-diol, and also androstenedione, and all areas of the foetal brain showed similar activity. In the foetal pituitary gland, the activity of 5alpha-reductase was less prominent than that of 17beta-hydroxysteroid-dehydrogenase. Small quantities of oestradiol-17beta were produced from testosterone by the hypothalamus, temporal lobe and amygdala only, and no aromatization could be detected in the pituitary gland. 5alpha-Reductase activity was much lower in adult brain tissues and no oestradiol was identified in adult temporal lobe tissue.

Effect of gonadal hormones on the incorporation of [3H]lysine into proteins of human fetal brain.

Samples were obtained from the hypothalamus, temporal lobe and frontal cortex of brains from human fetuses aged 14-22 weeks. Slices were prepared and incubated with estradiol, testosterone or dihydrotestosterone, and the incorporation of [3H]lysine into protein was compared with that of untreated control tissue. Cytosol and crude nuclear subcellular fractions from each area were made and resolution of specific proteins was carried out using a polyacrylamide gel system. Steroid hormones had no detectable effect on total protein synthesis, but incorporation of [3H]lysine into certain low molecular weight proteins of the hypothalamus increased significantly in the presence of estradiol and testosterone. Dihydrotestosterone was inactive. The temporal lobe and frontal cortex were unaffected by the steroid hormones.

X-ray refraction effects: application to the imaging of biological tissues.

The purpose of this study was to explore the potential of refraction contrast X-ray imaging of biological tissues. Images of dissected mouse lungs, heart, liver and legs were produced using the medical beamline at the Elettra Synchrotron at Trieste, Italy. The technique used was diffraction enhanced imaging. This utilizes a silicon crystal positioned between the tissue sample and the detector to separate refracted X-rays from transmitted and scattered radiation by Bragg diffraction. The contrast in the images produced is related to changes in the X-ray refractive index of the tissues, resulting in remarkable clarity compared with conventional X-ray images based on absorption effects. These changes were greatest at the boundaries between different tissues, giving a marked edge enhancement effect and three-dimensional appearance to the images. The technique provides a way of imaging a property of biological tissues not yet exploited, and further studies are planned to identify specific applications in medical imaging.

Effect of timing of urea feeding on the yield and quality of embryos in lactating dairy cows.

High protein diets, which lead to excess production of nonprotein nitrogen such as ammonia and urea, have been associated with reduced fertility in dairy cows. In this study we test the hypothesis that diets containing high levels of quickly degradable urea nitrogen (QDN) compromise embryo development. Lactating dairy cows were fed mixed silage and concentrates twice daily. At 60 days postpartum, a synchronized estrus was induced and the cows were subsequently superovulated and inseminated using a standard protocol. On Day 7 after insemination, the uteri were flushed and embryos retrieved. At the start of treatment, cows were randomly allocated into three nutritional groups: control (CONT, n = 8), long (L-) QDN (n = 8) and short (S-) QDN (n = 9). The L-QDN cows were fed a supplement of urea from 10 days before insemination, and the S-QDN cows were fed the supplement from insemination until embryo collection. Both L- and S-QDN diets produced significant increases in plasma ammonia and urea 3 h post-feeding. The S-QDN but not the L-QDN diet was associated with a significant reduction in embryo yield. Embryo quality was also significantly reduced in the S-QDN cows. This study indicates that there is no deleterious effect on the yield and quality of embryos recovered 7 days after breeding when QDN feeding is initiated during the previous midluteal phase. However, introduction of a similar diet 10 days later, at the time of insemination, was deleterious. We suggest that QDN is toxic to embryos but cows can adjust within 10 days.

The effects of early orthodontic force application on pulp test responses.

The effects of orthodontic tooth movement on pulpal responses are of interest to the clinician. Alterations to pulpal physiology may result in altered responses to external stimuli. This pilot project tested a small group of orthodontic patients during the early stages of treatment. Heat, cold and electrical stimuli were applied prior to treatment, after the placement of fixed appliances and after one month of force application. A smaller group was tested after two months of force application. One month after force application there was a lack of response to electrical stimulation but there was continued response to thermal stimulation. The lack of response to electrical stimulation continued in the smaller group up to two months. The results suggest that the outcome of electric pulp testing during orthodontic treatment should be interpreted cautiously. Thermal testing will offer more reliable data.

Haemochromatosis: a time for guidelines?

Hereditary haemochromatosis is an autosomal recessive metabolic abnormality which causes excessive absorption of dietary iron. Iron accumulation leads to potentially fatal damage to organs such as the heart, liver and pancreas. Normal life expectancy can be restored simply by therapeutic venesection. Discovery of the gene, HFE, has rekindled interest in pathogenesis, management and screening strategies.

Absolute x-ray dosimetry on a synchrotron medical beam line with a graphite calorimeter.

PURPOSE: The absolute dose rate of the Imaging and Medical Beamline (IMBL) on the Australian Synchrotron was measured with a graphite calorimeter. The calorimetry results were compared to measurements from the existing free-air chamber, to provide a robust determination of the absolute dose in the synchrotron beam and provide confidence in the first implementation of a graphite calorimeter on a synchrotron medical beam line. METHODS: The graphite calorimeter has a core which rises in temperature when irradiated by the beam. A collimated x-ray beam from the synchrotron with well-defined edges was used to partially irradiate the core. Two filtration sets were used, one corresponding to an average beam energy of about 80 keV, with dose rate about 50 Gy/s, and the second filtration set corresponding to average beam energy of 90 keV, with dose rate about 20 Gy/s. The temperature rise from this beam was measured by a calibrated thermistor embedded in the core which was then converted to absorbed dose to graphite by multiplying the rise in temperature by the specific heat capacity for graphite and the ratio of cross-sectional areas of the core and beam. Conversion of the measured absorbed dose to graphite to absorbed dose to water was achieved using Monte Carlo calculations with the EGSnrc code. The air kerma measurements from the free-air chamber were converted to absorbed dose to water using the AAPM TG-61 protocol. RESULTS: Absolute measurements of the IMBL dose rate were made using the graphite calorimeter and compared to measurements with the free-air chamber. The measurements were at three different depths in graphite and two different filtrations. The calorimetry measurements at depths in graphite show agreement within 1% with free-air chamber measurements, when converted to absorbed dose to water. The calorimetry at the surface and free-air chamber results show agreement of order 3% when converted to absorbed dose to water. The combined standard uncertainty is 3.9%. CONCLUSIONS: The good agreement of the graphite calorimeter and free-air chamber results indicates that both devices are performing as expected. Further investigations at higher dose rates than 50 Gy/s are planned. At higher dose rates, recombination effects for the free-air chamber are much higher and expected to lead to much larger uncertainties. Since the graphite calorimeter does not have problems associated with dose rate, it is an appropriate primary standard detector for the synchrotron IMBL x rays and is the more accurate dosimeter for the higher dose rates expected in radiotherapy applications.

Can post-acute care programmes for older people reduce overall costs in the health system? A case study using the Australian Transition Care Programme.

There is an increasing demand for acute care services due in part to rising proportions of older people and increasing rates of chronic diseases. To reduce pressure and costs in the hospital system, community-based post-acute care discharge services for older people have evolved as one method of reducing length of stay in hospital and preventing readmissions. However, it is unclear whether they reduce overall episode cost or expenditure in the health system at a more general level. In this paper, we review the current evidence on the likely costs and benefits of these services and consider whether they are potentially cost-effective from a health services perspective, using the Australian Transition Care Programme as a case study. Evaluations of community-based post-acute services have demonstrated that they reduce length of stay, prevent some re-hospitalisations and defer nursing home placement. There is also evidence that they convey some additional health benefits to older people. An economic model was developed to identify the maximum potential benefits and the likely cost savings from reduced use of health services from earlier discharge from hospital, accelerated recovery, reduced likelihood of readmission to hospital and delayed entry into permanent institutional care for participants of the Transition Care Programme. Assuming the best case scenario, the Transition Care Programme is still unlikely to be cost saving to a healthcare system. Hence for this service to be justified, additional health benefits such as quality of life improvements need to be taken into account. If it can be demonstrated that this service also conveys additional quality of life improvements, community-based programmes such as Transition Care could be considered to be cost-effective when compared with other healthcare programmes.