tissue-preparation-techniques-for-contrast-enhanced-micro-computed

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

Univ. Bordeaux

Fondation Bordeaux Univ.

Cardiovascular magnetic resonance (CMR) can through the two methods 3D FLASH and diffusion tensor imaging (DTI) give complementary information on the local orientations of cardiomyocytes and their laminar arrays.

Comparison of diffusion tensor imaging by cardiovascular magnetic resonance and gadolinium enhanced 3D image intensity approaches to investigation of structural anisotropy in explanted rat hearts.

To provide a model close to the human heart, and to study intrinsic cardiac function at the same time as electromechanical coupling, we developed a magnetic resonance (MR)-compatible setup of isolated working perfused pig hearts. Hearts from pigs (40 kg, n = 20) and sheep (n = 1) were blood perfused ex vivo in the working mode with and without loaded right ventricle (RV), for 80 min. Cardiac function was assessed by measuring left intraventricular pressure and left ventricular (LV) ejection fraction (LVEF), aortic and mitral valve dynamics, and native T1 mapping with MR imaging (1.5 Tesla). Potential myocardial alterations were assessed at the end of ex vivo perfusion from late-Gadolinium enhancement T1 mapping. The ex vivo cardiac function was stable across the 80 min of perfusion. Aortic flow and LV-dP/dtmin were significantly higher (P < 0.05) in hearts perfused with loaded RV, without differences for heart rate, maximal and minimal LV pressure, LV-dP/dtmax, LVEF, and kinetics of aortic and mitral valves. T1 mapping analysis showed a spatially homogeneous distribution over the LV. Simultaneous recording of hemodynamics, LVEF, and local cardiac electrophysiological signals were then successfully performed at baseline and during electrical pacing protocols without inducing alteration of MR images. Finally, (31)P nuclear MR spectroscopy (9.4 T) was also performed in two pig hearts, showing phosphocreatine-to-ATP ratio in accordance with data previously reported in vivo. We demonstrate the feasibility to perfuse isolated pig hearts in the working mode, inside an MR environment, allowing simultaneous assessment of cardiac structure, mechanics, and electrophysiology, illustrating examples of potential applications.

Magnetic resonance-compatible model of isolated working heart from large animal for multimodal assessment of cardiac function, electrophysiology, and metabolism.

Currently, no non-invasive cardiac pacing device acceptable for prolonged use in conscious patients exists. High Intensity Focused Ultrasound (HIFU) can be used to perform remote pacing using reversibility of electromechanical coupling of cardiomyocytes. Here we described an extracorporeal cardiac stimulation device and study its efficacy and safety. We conducted experiments ex vivo and in vivo in a large animal model (pig) to evaluate clinical potential of such a technique. The stimulation threshold was determined in 10 different ex vivo hearts and different clinically relevant electrical effects such as consecutive stimulations of different heart chambers with a single ultrasonic probe, continuous pacing or the inducibility of ventricular tachycardia were shown. Using ultrasonic contrast agent, consistent cardiac stimulation was achievable in vivo for up to 1 hour sessions in 4 different animals. No damage was observed in inversion-recovery MR sequences performed in vivo in the 4 animals. Histological analysis revealed no differences between stimulated and control regions, for all ex vivo and in vivo cases.

Non-invasive cardiac pacing with image-guided focused ultrasound.

Thrombotic risk constitutes a major complication of atrial fibrillation (AF). Platelets and microparticles (MPs) are important for hemostasis and thrombosis, however their participation during AF is not well known. The aim of this study was to characterize platelet function and MPs procoagulant and fibrinolytic activity in AF patients and to determine the effects of an acute-AF episode.

Platelet function and microparticle levels in atrial fibrillation: Changes during the acute episode.

Sudden cardiac death because of ventricular fibrillation (VF) is commonly unexplained in younger victims. Detailed electrophysiological mapping in such patients has not been reported.

Localized Structural Alterations Underlying a Subset of Unexplained Sudden Cardiac Death.

 Non-invasive electrocardiographic imaging (ECGI) is a promising tool to provide high-resolution panoramic imaging of cardiac electrical activity noninvasively from body surface potential measurements. Current experimental methods for ECGI validation are limited to comparison with unipolar electrograms and the relatively low spatial resolution of cardiac mapping arrays. We aim to develop a novel experimental set up combining a human shaped torso tank with high-resolution optical mapping allowing the validation of ECGI reconstructions.  Langendorff-perfused pig hearts ( = 3) were suspended in a human torso-shaped tank, with the left anterior descending artery (LAD) cannulated on a separate perfusion. Electrical signals were recorded from an 108-electrode epicardial sock and 128 electrodes embedded in the tank surface. Simultaneously, optical mapping of the heart was performed through the anterior surface of the tank. Recordings were made in sinus rhythm and ventricular pacing ( = 55), with activation and repolarization heterogeneities induced by perfusion of hot and cold solutions as well as Sotalol through the LAD. Fluoroscopy provided 3D cardiac and electrode geometries in the tank that were transformed to the 2D optical mapping window using an optimization algorithm. Epicardial unipolar electrograms were reconstructed from torso potentials using ECGI and validated using optical activation and repolarization maps.  The transformation and alignment of the 3D geometries onto the 2D optical mapping window was good with an average correlation of 0.87 ± 0.10 and error of 7.7 ± 3.1 ms with activation derived from the sock. The difference in repolarization times were more substantial (error = 17.4 ± 3.7 ms) although the sock and optical repolarization patterns themselves were very similar (correlation = 0.83 ± 0.13). Validation of ECGI reconstructions revealed ECGI accurately captures the pattern of activation (correlation = 0.79 ± 0.11) and identified regions of late and/or early repolarization during different perfusions through LAD. ECGI also correctly demonstrated gradients in both activation and repolarization, although in some cases these were under or over-estimated or shifted slightly in space.  A novel experimental setup has been developed, combining a human-shaped torso tank with optical mapping, which can be effectively used in the validation of ECGI techniques; including the reconstruction of activation and repolarization patterns and gradients.

Optical Imaging of Ventricular Action Potentials in a Torso Tank: A New Platform for Non-Invasive Electrocardiographic Imaging Validation.

Idiopathic ventricular fibrillation and J-wave syndromes are causes of sudden cardiac death (SCD) without any identified structural cardiac disease after extensive investigations. Recent data show that high-density electrophysiological mapping may ultimately offer diagnoses of subclinical diseases in most patients including those termed "unexplained" SCD. Three major conditions can underlie the occurrence of SCD: (1) localized depolarization abnormalities (due to microstructural myocardial alteration), (2) Purkinje abnormalities manifesting as triggering ectopy and inducible reentry; or (3) repolarization heterogeneities. Each condition may result from a spectrum of pathophysiologic processes with implications for individual therapy.

The Spectrum of Idiopathic Ventricular Fibrillation and J-Wave Syndromes: Novel Mapping Insights.

To develop a reproducible and stable closed chest model of ischemic cardiogenic shock in sheep, with high survival rate and potential insight into human pathology. We established a protocol for multi-step myocardial alcoholisation of the left anterior descending coronary artery by percutaneous ethanol injection. A thorough hemodynamic assessment was obtained by invasive and non-invasive monitoring devices. Repeated blood samples were obtained to determine haemoglobin and alcohol concentration, electrolytes, blood gas parameters and cardiac troponin I. After sacrifice, tissue was excised for quantification of infarction and histology. Cardiogenic shock was characterized by a significant decrease in mean arterial pressure (- 33%), cardiac output (- 29%), dP/dt (- 28%), carotid blood flow (- 22%), left ventricular fractional shortening (- 28%), and left ventricle end-systolic pressure-volume relationship (- 51%). Lactate and cardiac troponin I levels increased from 1.4 ± 0.2 to 4.9 ± 0.7 mmol/L (p = 0.001) and from 0.05 ± 0.02 to 14.74 ± 2.59 µg/L (p = 0.001), respectively. All haemodynamic changes were stable over a three-hour period with a 71% survival rate. The necrotic volume (n = 5) represented 24.0 ± 1.9% of total ventricular mass. No sham exhibited any variation under general anaesthesia. We described and characterized, for the first time, a stable, reproducible sheep model of cardiogenic shock obtained by percutaneous intracoronary ethanol administration.

A total closed chest sheep model of cardiogenic shock by percutaneous intracoronary ethanol injection.

Recent developments in clearing and microscopy enable 3D imaging with cellular resolution up to the whole organ level. These methods have been used extensively in neurobiology, but their uptake in other fields has been much more limited. Application of this approach to the human heart and effective use of the data acquired present challenges of scale and complexity. Four interlinked issues need to be addressed: 1) efficient clearing and labelling of heart tissue, 2) fast microscopic imaging of human-scale samples, 3) handling and processing of multi-terabyte 3D images, and 4) extraction of structural information in computationally tractable structure-based models of cardiac function. Preliminary studies show that each of these requirements can be achieved with the appropriate application and development of existing technologies.

It's clearly the heart! Optical transparency, cardiac tissue imaging, and computer modelling.

The pig is commonly used as an experimental model of human heart disease, including for the study of mechanisms of arrhythmia. However, there exist differences between human and porcine cellular electrophysiology: The pig action potential (AP) has a deeper phase-1 notch, a longer duration at 50% repolarization, and higher plateau potentials than human. Ionic differences underlying the AP include larger rapid delayed-rectifier and smaller inward-rectifier K+-currents (IKr and IK1 respectively) in humans. AP steady-state rate-dependence and restitution is steeper in pigs. Porcine Ca2+ transients can have two components, unlike human. Although a reliable computational model for human ventricular myocytes exists, one for pigs is lacking. This hampers translation from results obtained in pigs to human myocardium. Here, we developed a computational model of the pig ventricular cardiomyocyte AP using experimental datasets of the relevant ionic currents, Ca2+-handling, AP shape, AP duration restitution, and inducibility of triggered activity and alternans. To properly capture porcine Ca2+ transients, we introduced a two-step process with a faster release in the t-tubular region, followed by a slower diffusion-induced release from a non t-tubular subcellular region. The pig model behavior was compared with that of a human ventricular cardiomyocyte (O'Hara-Rudy) model. The pig, but not the human model, developed early afterdepolarizations (EADs) under block of IK1, while IKr block led to EADs in the human but not in the pig model. At fast rates (pacing cycle length = 400 ms), the human cell model was more susceptible to spontaneous Ca2+ release-mediated delayed afterdepolarizations (DADs) and triggered activity than pig. Fast pacing led to alternans in human but not pig. Developing species-specific models incorporating electrophysiology and Ca2+-handling provides a tool to aid translating antiarrhythmic and arrhythmogenic assessment from the bench to the clinic.

A computational model of pig ventricular cardiomyocyte electrophysiology and calcium handling: Translation from pig to human electrophysiology.

The Brugada pattern manifests as a spontaneous variability of the electrocardiographic marker, suggesting a variability of the underlying electrical substrate.

Article	Year
Comparison of diffusion tensor imaging by cardiovascular magnetic resonance and gadolinium enhanced 3D image intensity approaches to investigation of structural anisotropy in explanted rat h... Journal of cardiovascular magnetic resonance : official journal of the Society for Cardiovascular Magnetic Resonance\| PubMed ID: 25926126	2015
Magnetic resonance-compatible model of isolated working heart from large animal for multimodal assessment of cardiac function, electrophysiology, and metabolism. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 26968545	2016
Non-invasive cardiac pacing with image-guided focused ultrasound. Scientific reports\| PubMed ID: 27827415	2016
Platelet function and microparticle levels in atrial fibrillation: Changes during the acute episode. International journal of cardiology\| PubMed ID: 28747025	2017
Localized Structural Alterations Underlying a Subset of Unexplained Sudden Cardiac Death. Circulation. Arrhythmia and electrophysiology\| PubMed ID: 30002064	2018
Optical Imaging of Ventricular Action Potentials in a Torso Tank: A New Platform for Non-Invasive Electrocardiographic Imaging Validation. Frontiers in physiology\| PubMed ID: 30863318	2019
The Spectrum of Idiopathic Ventricular Fibrillation and J-Wave Syndromes: Novel Mapping Insights. Cardiac electrophysiology clinics\| PubMed ID: 31706476	2019
A total closed chest sheep model of cardiogenic shock by percutaneous intracoronary ethanol injection. Scientific reports\| PubMed ID: 32709984	2020
It's clearly the heart! Optical transparency, cardiac tissue imaging, and computer modelling. Progress in biophysics and molecular biology\| PubMed ID: 34126113	2022
A computational model of pig ventricular cardiomyocyte electrophysiology and calcium handling: Translation from pig to human electrophysiology. PLoS computational biology\| PubMed ID: 34191797	2021
Multisite conduction block in the epicardial substrate of Brugada syndrome. Heart rhythm\| PubMed ID: 34737095	2021

Olivier Bernus

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