Time-resolved velocity mapping at high magnetic fields: A preclinical comparison between stack‐of‐stars and cartesian 4D-Flow

Purpose: Prospectively-gated Cartesian 4D-flow (referred to as Cartesian-4D-flow) imaging suffers from long TE and intensified flow-related intravoxel-dephasing especially in preclinical ultra-high field MRI. The ultra-short-echo (UTE) 4D-flow technique can resolve the signal loss in higher-order blood flows; however, the long scan time of the high resolution UTE-4D-flow is considered as a disadvantage for preclinical imaging. To compensate for prolonged acquisitions, an accelerated k0-navigated golden-angle center-out stack-of-stars 4D-flow sequence (referred to as SoS-4D-flow) was implemented at 9.4T and the results were compared to conventional Cartesian-4D-flow mapping in-vitro and in-vivo. Methods: The study was conducted in three steps (A) In-vitro evaluation in a static phantom: to quantify the background velocity bias. (B) In-vitro evaluation in a flowing water phantom: to investigate the effects of polar undersampling (US) on the measured velocities and to compare the spatial velocity profiles between both sequences. (C) In-vivo evaluations: 24 C57BL/6 mice were measured by SoS-4D-flow (n = 14) and Cartesian-4D-flow (n = 10). The peak systolic velocity in the ascending aorta and the background velocity in the anterior chest wall were analyzed for both techniques and were compared to each other. Results: According to the in-vitro analysis, the background velocity bias was significantly lower in SoS-4D-flow than in Cartesian-4D-flow (p < 0.05). Polar US in SoS-4D-flow influenced neither the measured velocity values nor the spatial velocity profiles in comparison to Cartesian-4D-flow. The in-vivo analysis showed significantly higher diastolic velocities in Cartesian-4D-flow than in SoS-4D-flow (p < 0.05). A systemic background bias was observed in the Cartesian velocity maps which influenced their streamline directions and magnitudes. Conclusion: The results of our study showed that at 9.4T SoS-4D-flow provided higher accuracy in slow flow imaging than Cartesian-4D-flow, while the same measurement time could be achieved

Deficiency of the BMP Type I receptor ALK3 partly protects mice from anemia of inflammation

Background: Inflammatory stimuli induce the hepatic iron regulatory hormone hepcidin, which contributes to anaemia of inflammation (AI). Hepcidin expression is regulated by the bone morphogenetic protein (BMP) and the interleukin-6 (IL-6) signalling pathways. Prior results indicate that the BMP type I receptor ALK3 is mainly involved in the acute inflammatory hepcidin induction four and 72 h after IL-6 administration. In this study, the role of ALK3 in a chronic model of inflammation was investigated. The intact, heat-killed bacterium Brucella abortus (BA) was used to analyse its effect on the development of inflammation and hypoferremia in mice with hepatocyte-specific Alk3-deficiency (Alk3fl/fl; Alb-Cre) compared to control (Alk3fl/fl) mice. Results: An iron restricted diet prevented development of the iron overload phenotype in mice with hepatocyte-specific Alk3 deficiency. Regular diet leads to iron overload and increased haemoglobin levels in these mice, which protects from the development of AI per se. Fourteen days after BA injection Alk3fl/fl; Alb-Cre mice presented milder anaemia (Hb 16.7 g/dl to 11.6 g/dl) compared to Alk3fl/fl control mice (Hb 14.9 g/dl to 8.6 g/dl). BA injection led to an intact inflammatory response in all groups of mice. In Alk3fl/fl; Alb-Cre mice, SMAD1/5/8 phosphorylation was reduced after BA as well as after infection with Staphylococcus aureus. The reduction of the SMAD1/5/8 signalling pathway due to hepatocyte-specific Alk3 deficiency partly suppressed the induction of STAT3 signalling. Conclusion: The results reveal in vivo, that 1) hepatocyte-specific Alk3 deficiency partly protects from AI, 2) the development of hypoferremia is partly dependent on ALK3, and 3) the ALK3/BMP/hepcidin axis may serve as a possible therapeutic target to attenuate AI

Characterization and prediction of the mechanism of action of antibiotics through NMR metabolomics

Background: The emergence of antibiotic resistant pathogenic bacteria has reduced our ability to combat infectious diseases. At the same time the numbers of new antibiotics reaching the market have decreased. This situation has created an urgent need to discover novel antibiotic scaffolds. Recently, the application of pattern recognition techniques to identify molecular fingerprints in ‘omics’ studies, has emerged as an important tool in biomedical research and laboratory medicine to identify pathogens, to monitor therapeutic treatments or to develop drugs with improved metabolic stability, toxicological profile and efficacy. Here, we hypothesize that a combination of metabolic intracellular fingerprints and extracellular footprints would provide a more comprehensive picture about the mechanism of action of novel antibiotics in drug discovery programs. Results: In an attempt to integrate the metabolomics approach as a classification tool in the drug discovery processes, we have used quantitative 1H NMR spectroscopy to study the metabolic response of Escherichia coli cultures to different antibiotics. Within the frame of our study the effects of five different and well-known antibiotic classes on the bacterial metabolome were investigated both by intracellular fingerprint and extracellular footprint analysis. The metabolic fingerprints and footprints of bacterial cultures were affected in a distinct manner and provided complementary information regarding intracellular and extracellular targets such as protein synthesis, DNA and cell wall. While cell cultures affected by antibiotics that act on intracellular targets showed class-specific fingerprints, the metabolic footprints differed significantly only when antibiotics that target the cell wall were applied. In addition, using a training set of E. coli fingerprints extracted after treatment with different antibiotic classes, the mode of action of streptomycin, tetracycline and carbenicillin could be correctly predicted. Conclusion: The metabolic profiles of E. coli treated with antibiotics with intracellular and extracellular targets could be separated in fingerprint and footprint analysis, respectively and provided complementary information. Based on the specific fingerprints obtained for different classes of antibiotics, the mode of action of several antibiotics could be predicted. The same classification approach should be applicable to studies of other pathogenic bacteria

Isolating Crucial Steps in Induction of Infective Endocarditis With Preclinical Modeling of Host Pathogen Interaction

Animal models of Staphylococcus aureus infective endocarditis (IE), especially in rodents, are commonly used to investigate the underlying pathogenesis, disease progression, potential diagnostic approaches, and therapeutic treatment. All these models are based on surgical interventions, and imply valve trauma by placing a polyurethane catheter at the aortic root. While the influence of endothelial damage and inflammation on the induction of IE has been studied intensively, the role of the catheter, as permanent source of bacteremia, and the interplay with bacterial virulence factors during the formation of IE is poorly understood. In our study, we aimed at identifying which set of preconditions is required for induction and formation of IE: (1) tissue injury, (2) permanent presence of bacteria, and (3) presence of the full bacterial repertoire of adhesion proteins. We investigated the manifestation of the disease in different modifications of the animal model, considering different degrees of endothelial damage and the presence or absence of the catheter. In four infection models the induction of IE was assessed by using two bacterial strains with different expression patterns of virulence factors – S. aureus 6850 and Newman. In vivo magnetic resonance imaging showed conspicuous morphological structures on the aortic valves, when an endothelial damage and a continuous bacterial source were present simultaneously. Cellular and inflammatory pathophysiology were characterized additionally by histology, real-time quantitative polymerase chain reaction analysis, and bacterial counts, revealing strain-specific pathogenesis and manifestation of IE, crucially influenced by bacterial adherence and toxicity. The severity of IE was dependent on the degree of endothelial irritation. However, even severe endothelial damage in the absence of a permanent bacterial source resulted in reduced valve infection. The spread of bacteria to other organs was also dependent on the pathogenic profile of the infectious agent

MRI visualization of Staphyloccocus aureus-induced infective endocarditis in mice.

Infective endocarditis (IE) is a severe and often fatal disease, lacking a fast and reliable diagnostic procedure. The purpose of this study was to establish a mouse model of Staphylococcus aureus-induced IE and to develop a MRI technology to characterize and diagnose IE. To establish the mouse model of hematogenous IE, aortic valve damage was induced by placing a permanent catheter into right carotid artery. 24 h after surgery, mice were injected intravenously with either iron particle-labeled or unlabeled S. aureus (strain 6850). To distinguish the effect of IE from mere tissue injury or recruited macrophages, subgroups of mice received sham surgery prior to infection (n = 17), received surgery without infection (n = 8), or obtained additionally injection of free iron particles to label macrophages (n = 17). Cardiac MRI was performed 48 h after surgery using a self-gated ultra-short echo time (UTE) sequence (TR/TE, 5/0.31 ms; in-plane/slice, 0.125/1 mm; duration, 12∶08 min) to obtain high-resolution, artifact-free cinematographic images of the valves. After MRI, valves were either homogenized and plated on blood agar plates for determination of bacterial titers, or sectioned and stained for histology. In the animal model, both severity of the disease and mortality increased with bacterial numbers. Infection with 105 S. aureus bacteria reliably caused endocarditis with vegetations on the valves. Cinematographic UTE MRI visualised the aortic valve over the cardiac cycle and allowed for detection of bacterial vegetations, while mere tissue trauma or labeled macrophages were not detected. Iron labeling of S. aureus was not required for detection. MRI results were consistent with histology and microbial assessment. These data showed that S. aureus-induced IE in mice can be detected by MRI. The established mouse model allows for investigation of the pathophysiology of IE, testing of novel drugs and may serve for the development of a clinical diagnostic strategy

Schematic diagram of the endocarditis model.

<p>The experimental procedure involved four stages. First, valve trauma was induced by placing a 32-G catheter (red) at the aortic root via the right carotid artery. Second, <i>S. aureus</i> bacteria were injected via the tail vain 24 h after surgery. Third, MRI was performed 48 h after surgery. Fourth, animals were euthanized after MRI, hearts and organs were removed, inspected macroscopically and processed for microbial assessment or histology. Visual inspection showed clear, translucent valves (white arrow) for non-infected hearts (top) and yellow, thickened valves (white arrow) for colonized hearts (bottom).</p