Jurgen E. Schneider

Multiparametric magnetic resonance for the non-invasive diagnosis of liver disease

Background & Aims With the increasing prevalence of liver disease worldwide, there is an urgent clinical need for reliable methods to diagnose and stage liver pathology. Liver biopsy, the current gold standard, is invasive and limited by sampling and observer dependent variability. In this study, we aimed to assess the diagnostic accuracy of a novel magnetic resonance protocol for liver tissue characterisation. Methods We conducted a prospective study comparing our magnetic resonance technique against liver biopsy. The individual components of the scanning protocol were T1 mapping, proton spectroscopy and T2⁎ mapping, which quantified liver fibrosis, steatosis and haemosiderosis, respectively. Unselected adult patients referred for liver biopsy as part of their routine care were recruited. Scans performed prior to liver biopsy were analysed by physicians blinded to the histology results. The associations between magnetic resonance and histology variables were assessed. Receiver-operating characteristic analyses were also carried out. Results Paired magnetic resonance and biopsy data were obtained in 79 patients. Magnetic resonance measures correlated strongly with histology (rs = 0.68 p <0.0001 for fibrosis; rs = 0.89 p <0.001 for steatosis; rs = −0.69 p <0.0001 for haemosiderosis). The area under the receiver operating characteristic curve was 0.94, 0.93, and 0.94 for the diagnosis of any degree of fibrosis, steatosis and haemosiderosis respectively. Conclusion The novel scanning method described here provides high diagnostic accuracy for the assessment of liver fibrosis, steatosis and haemosiderosis and could potentially replace liver biopsy for many indications. This is the first demonstration of a non-invasive test to differentiate early stages of fibrosis from normal liver.

Assessment of motion gating strategies for mouse magnetic resonance at high magnetic fields.

To assess the performance of motion gating strategies for mouse cardiac magnetic resonance (MR) at high magnetic fields by quantifying the levels of motion artifact observed in images and spectra in vivo.

Endothelial Cell-Specific ROS Production Increases Susceptibility to Aortic Dissection

Background— Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. Methods and Results— A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II–mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45+ inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II–induced vascular smooth muscle cell ROS production. Conclusions— These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II–mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.

Endothelial Cell–Specific Reactive Oxygen Species Production Increases Susceptibility to Aortic Dissection

Background— Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. Methods and Results— A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II–mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45+ inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II–induced vascular smooth muscle cell ROS production. Conclusions— These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II–mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.

Images as drivers of progress in cardiac computational modelling

Computational models have become a fundamental tool in cardiac research. Models are evolving to cover multiple scales and physical mechanisms. They are moving towards mechanistic descriptions of personalised structure and function, including effects of natural variability. These developments are underpinned to a large extent by advances in imaging technologies. This article reviews how novel imaging technologies, or the innovative use and extension of established ones, integrate with computational models and drive novel insights into cardiac biophysics. In terms of structural characterization, we discuss how imaging is allowing a wide range of scales to be considered, from cellular levels to whole organs. We analyse how the evolution from structural to functional imaging is opening new avenues for computational models, and in this respect we review methods for measurement of electrical activity, mechanics and flow. Finally, we consider ways in which combined imaging and modelling research is likely to continue advancing cardiac research, and identify some of the main challenges that remain to be solved.

Quantitative Study of the Effect of Tissue Microstructure on Contraction in a Computational Model of Rat Left Ventricle

Tissue microstructure, in particular the alignment of myocytes (fibre direction) and their lateral organisation into sheets, is fundamental to cardiac function. We studied the effect of microstructure on contraction in a computational model of rat left ventricular electromechanics. Different fibre models, globally rule-based or locally optimised to DT-MRI data, were compared, in order to understand whether a subject-specific fibre model would enhance the predictive power of our model with respect to the global ones. We also studied the impact of sheets on ventricular deformation by comparing: (a) a transversely isotropic versus an orthotropic material law and (b) a linear model with a bimodal model of sheet transmural variation. We estimated ejection fraction, wall thickening and base-to-apex shortening and compared them with measures from cine-MRI. We also evaluated Lagrangian strains as local metrics of cardiac deformation. Our results show that the subject-specific fibre model provides little improvement in the metric predictions with respect to global fibre models while material orthotropy allows closer agreement with measures than transverse isotropy. Nonetheless, the impact of sheets in our model is smaller than that of fibres. We conclude that further investigation of the modelling of sheet dynamics is necessary to fully understand the impact of tissue structure on cardiac deformation.

Interrogation of living myocardium in multiple static deformation states with diffusion tensor and diffusion spectrum imaging

Diffusion tensor magnetic resonance imaging (MRI) reveals valuable insights into tissue histo-anatomy and microstructure, and has steadily gained traction in the cardiac community. Its wider use in small animal cardiac imaging in vivo has been constrained by its extreme sensitivity to motion, exaggerated by the high heart rates usually seen in rodents. Imaging of the isolated heart eliminates respiratory motion and, if conducted on arrested hearts, cardiac pulsation. This serves as an important intermediate step for basic and translational studies. However, investigating the micro-structural basis of cardiac deformation in the same heart requires observations in different deformation states. Here, we illustrate the imaging of isolated rat hearts in three mechanical states mimicking diastole (cardioplegic arrest), left-ventricular (LV) volume overload (cardioplegic arrest plus LV balloon inflation), and peak systole (lithium-induced contracture). An optimised MRI-compatible Langendorff perfusion setup with the radio-frequency (RF) coil integrated into the wet chamber was developed for use in a 9.4T horizontal bore scanner. Signal-to-noise ratio improved significantly, by 75% compared to a previous design with external RF coil, and stability tests showed no significant changes in mean T1, T2 or LV wall thickness over a 170 min period. In contracture, we observed a significant reduction in mean fractional anisotropy from 0.32 ± 0.02 to 0.28 ± 0.02, as well as a significant rightward shift in helix angles with a decrease in the proportion of left-handed fibres, as referring to the locally prevailing cell orientation in the heart, from 24.9% to 23.3%, and an increase in the proportion of right-handed fibres from 25.5% to 28.4%. LV overload, in contrast, gave rise to a decrease in the proportion of left-handed fibres from 24.9% to 21.4% and an increase in the proportion of right-handed fibres from 25.5% to 26.0%. The modified perfusion and coil setup offers better performance and control over cardiac contraction states. We subsequently performed high-resolution diffusion spectrum imaging (DSI) and 3D whole heart fibre tracking in fixed ex vivo rat hearts in slack state and contracture. As a model-free method, DSI augmented the measurements of water diffusion by also informing on multiple intra-voxel diffusion orientations and non-Gaussian diffusion. This enabled us to identify the transition from right- to left-handed fibres from the subendocardium to the subepicardium, as well as voxels in apical regions that were traversed by multiple fibres. We observed that both the mean generalised fractional anisotropy and mean kurtosis were lower in hearts in contracture compared to the slack state, by 23% and 9.3%, respectively. While its heavy acquisition burden currently limits the application of DSI in vivo, ongoing work in acceleration techniques may enable its use in live animals and patients. This would provide access to the as yet unexplored dimension of non-Gaussian diffusion that could serve as a highly sensitive marker of cardiac micro-structural integrity.

Three-dimensional histology: tools and application to quantitative assessment of cell-type distribution in rabbit heart.

Aims Cardiac histo-anatomical organization is a major determinant of function. Changes in tissue structure are a relevant factor in normal and disease development, and form targets of therapeutic interventions. The purpose of this study was to test tools aimed to allow quantitative assessment of cell-type distribution from large histology and magnetic resonance imaging- (MRI) based datasets. Methods and results Rabbit heart fixation during cardioplegic arrest and MRI were followed by serial sectioning of the whole heart and light-microscopic imaging of trichrome-stained tissue. Segmentation techniques developed specifically for this project were applied to segment myocardial tissue in the MRI and histology datasets. In addition, histology slices were segmented into myocytes, connective tissue, and undefined. A bounding surface, containing the whole heart, was established for both MRI and histology. Volumes contained in the bounding surface (called ‘anatomical volume’), as well as that identified as containing any of the above tissue categories (called ‘morphological volume’), were calculated. The anatomical volume was 7.8 cm3 in MRI, and this reduced to 4.9 cm3 after histological processing, representing an ‘anatomical’ shrinkage by 37.2%. The morphological volume decreased by 48% between MRI and histology, highlighting the presence of additional tissue-level shrinkage (e.g. an increase in interstitial cleft space). The ratio of pixels classified as containing myocytes to pixels identified as non-myocytes was roughly 6:1 (61.6 vs. 9.8%; the remaining fraction of 28.6% was ‘undefined’). Conclusion Qualitative and quantitative differentiation between myocytes and connective tissue, using state-of-the-art high-resolution serial histology techniques, allows identification of cell-type distribution in whole-heart datasets. Comparison with MRI illustrates a pronounced reduction in anatomical and morphological volumes during histology processing.

Myocardial creatine levels do not influence response to acute oxidative stress in isolated perfused heart

Background Multiple studies suggest creatine mediates anti-oxidant activity in addition to its established role in cellular energy metabolism. The functional significance for the heart has yet to be established, but antioxidant activity could contribute to the cardioprotective effect of creatine in ischaemia/reperfusion injury. Objectives To determine whether intracellular creatine levels influence responses to acute reactive oxygen species (ROS) exposure in the intact beating heart. We hypothesised that mice with elevated creatine due to over-expression of the creatine transporter (CrT-OE) would be relatively protected, while mice with creatine-deficiency (GAMT KO) would fare worse. Methods and Results CrT-OE mice were pre-selected for creatine levels 20–100% above wild-type using in vivo 1H–MRS. Hearts were perfused in isovolumic Langendorff mode and cardiac function monitored throughout. After 20 min equilibration, hearts were perfused with either H2O2 0.5 µM (30 min), or the anti-neoplastic drug doxorubicin 15 µM (100 min). Protein carbonylation, creatine kinase isoenzyme activities and phospho-PKCδ expression were quantified in perfused hearts as markers of oxidative damage and apoptotic signalling. Wild-type hearts responded to ROS challenge with a profound decline in contractile function that was ameliorated by co-administration of catalase or dexrazoxane as positive controls. In contrast, the functional deterioration in CrT-OE and GAMT KO hearts was indistinguishable from wild-type controls, as was the extent of oxidative damage and apoptosis. Exogenous creatine supplementation also failed to protect hearts from doxorubicin-induced dysfunction. Conclusions Intracellular creatine levels do not influence the response to acute ROS challenge in the intact beating heart, arguing against creatine exerting (patho-)physiologically relevant anti-oxidant activity.

A computational pipeline for quantification of mouse myocardial stiffness parameters

The mouse is an important model for theoretical-experimental cardiac research, and biophysically based whole organ models of the mouse heart are now within reach. However, the passive material properties of mouse myocardium have not been much studied. We present an experimental setup and associated computational pipeline to quantify these stiffness properties. A mouse heart was excised and the left ventricle experimentally inflated from 0 to 1.44kPa in eleven steps, and the resulting deformation was estimated by echocardiography and speckle tracking. An in silico counterpart to this experiment was built using finite element methods and data on ventricular tissue microstructure from diffusion tensor MRI. This model assumed a hyperelastic, transversely isotropic material law to describe the force-deformation relationship, and was simulated for many parameter scenarios, covering the relevant range of parameter space. To identify well-fitting parameter scenarios, we compared experimental and simulated outcomes across the whole range of pressures, based partly on gross phenotypes (volume, elastic energy, and short- and long-axis diameter), and partly on node positions in the geometrical mesh. This identified a narrow region of experimentally compatible values of the material parameters. Estimation turned out to be more precise when based on changes in gross phenotypes, compared to the prevailing practice of using displacements of the material points. We conclude that the presented experimental setup and computational pipeline is a viable method that deserves wider application.