Quinapril treatment curtails decline of global longitudinal strain and mechanical function in hypertensive rats.

BACKGROUND: Left ventricular (LV) global longitudinal strain (GLS) has been proposed as an early imaging biomarker of cardiac mechanical dysfunction. OBJECTIVE: To assess the impact of angiotensin-converting enzyme (ACE) inhibitor treatment of hypertensive heart disease on LV GLS and mechanical function. METHODS: The spontaneously hypertensive rat (SHR) model of hypertensive heart disease ( n  = 38) was studied. A subset of SHRs received quinapril (TSHR, n  = 16) from 3 months (mo). Wistar Kyoto rats (WKY, n  = 13) were used as controls. Tagged cardiac MRI was performed using a 4.7 T Varian preclinical scanner. RESULTS: The SHRs had significantly lower LV ejection fraction (EF) than the WKYs at 3 mo (53.0 ±â€Š1.7% vs. 69.6 ±â€Š2.1%, P  < 0.05), 14 mo (57.0 ±â€Š2.5% vs. 74.4 ±â€Š2.9%, P  < 0.05) and 24 mo (50.1 ±â€Š2.4% vs. 67.0 ±â€Š2.0%, P  < 0.01). At 24 mo, ACE inhibitor treatment was associated with significantly greater LV EF in TSHRs compared to untreated SHRs (64.2 ±â€Š3.4% vs. 50.1 ±â€Š2.4%, P  < 0.01). Peak GLS magnitude was significantly lower in SHRs compared with WKYs at 14 months (7.5% ±â€Š0.4% vs. 9.9 ±â€Š0.8%, P  < 0.05). At 24 months, Peak GLS magnitude was significantly lower in SHRs compared with both WKYs (6.5 ±â€Š0.4% vs. 9.7 ±â€Š1.0%, P  < 0.01) and TSHRs (6.5 ±â€Š0.4% vs. 9.6 ±â€Š0.6%, P  < 0.05). CONCLUSIONS: ACE inhibitor treatment curtails the decline in global longitudinal strain in hypertensive rats, with the treatment group exhibiting significantly greater LV EF and GLS magnitude at 24 mo compared with untreated SHRs.

Multimodal imaging shows fibrosis architecture and action potential dispersion are predictors of arrhythmic risk in spontaneous hypertensive rats.

Hypertensive heart disease (HHD) increases risk of ventricular tachycardia (VT) and ventricular fibrillation (VF). The roles of structural vs. electrophysiological remodelling and age vs. disease progression are not fully understood. This cross-sectional study of cardiac alterations through HHD investigates mechanistic contributions to VT/VF risk. Risk was electrically assessed in Langendorff-perfused, spontaneously hypertensive rat hearts at 6, 12 and 18 months, and paced optical membrane voltage maps were acquired from the left ventricular (LV) free wall epicardium. Distributions of LV patchy fibrosis and 3D cellular architecture in representative anterior LV mid-wall regions were quantified from macroscopic and microscopic fluorescence images of optically cleared tissue. Imaging showed increased fibrosis from 6 months, particularly in the inner LV free wall. Myocyte cross-section increased at 12 months, while inter-myocyte connections reduced markedly with fibrosis. Conduction velocity decreased from 12 months, especially transverse to the myofibre direction, with rate-dependent anisotropy at 12 and 18 months, but not earlier. Action potential duration (APD) increased when clustered by age, as did APD dispersion at 12 and 18 months. Among 10 structural, functional and age variables, the most reliably linked were VT/VF risk, general LV fibrosis, a measure quantifying patchy fibrosis, and non-age clustered APD dispersion. VT/VF risk related to a quantified measure of patchy fibrosis, but age did not factor strongly. The findings are consistent with the notion that VT/VF risk is associated with rate-dependent repolarization heterogeneity caused by structural remodelling and reduced lateral electrical coupling between LV myocytes, providing a substrate for heterogeneous intramural activation as HHD progresses. KEY POINTS: There is heightened arrhythmic risk with progression of hypertensive heart disease. Risk is related to increasing left ventricular fibrosis, but the nature of this relationship has not been quantified. This study is a novel systematic characterization of changes in active electrical properties and fibrotic remodelling during progression of hypertensive heart disease in a well-established animal disease model. Arrhythmic risk is predicted by several left ventricular measures, in particular fibrosis quantity and structure, and epicardial action potential duration dispersion. Age alone is not a good predictor of risk. An improved understanding of links between arrhythmic risk and fibrotic architectures in progressive hypertensive heart disease aids better interpretation of late gadolinium-enhanced cardiac magnetic resonance imaging and electrical mapping signals.

Non-Contact Intracardiac Potential Mapping Using Mesh-Based and Meshless Inverse Solvers.

Atrial fibrillation (AF) is the most common cardiac dysrhythmia and percutaneous catheter ablation is widely used to treat it. Panoramic mapping with multi-electrode catheters has been used to identify ablation targets in persistent AF but is limited by poor contact and inadequate coverage of the left atrial cavity. In this paper, we investigate the accuracy with which atrial endocardial surface potentials can be reconstructed from electrograms recorded with non-contact catheters. An in-silico approach was employed in which "ground-truth" surface potentials from experimental contact mapping studies and computer models were compared with inverse potential maps constructed by sampling the corresponding intracardiac field using virtual basket catheters. We demonstrate that it is possible to 1) specify the mixed boundary conditions required for mesh-based formulations of the potential inverse problem fully, and 2) reconstruct accurate inverse potential maps from recordings made with appropriately designed catheters. Accuracy improved when catheter dimensions were increased but was relatively stable when the catheter occupied >30% of atrial cavity volume. Independent of this, the capacity of non-contact catheters to resolve the complex atrial potential fields seen in reentrant atrial arrhythmia depended on the spatial distribution of electrodes on the surface bounding the catheter. Finally, we have shown that reliable inverse potential mapping is possible in near real-time with meshless methods that use the Method of Fundamental Solutions.

Intracardiac Inverse Potential Mapping Using the Method of Fundamental Solutions.

Introduction: Atrial fibrillation (AF) is the most prevalent cardiac dysrhythmia and percutaneous catheter ablation is widely used to treat it. Panoramic mapping with multi-electrode catheters can identify ablation targets in persistent AF, but is limited by poor contact and inadequate coverage. Objective: To investigate the accuracy of inverse mapping of endocardial surface potentials from electrograms sampled with noncontact basket catheters. Methods: Our group has developed a computationally efficient inverse 3D mapping technique using a meshless method that employs the Method of Fundamental Solutions (MFS). An in-silico test bed was used to compare ground-truth surface potentials with corresponding inverse maps reconstructed from noncontact potentials sampled with virtual catheters. Ground-truth surface potentials were derived from high-density clinical contact mapping data and computer models. Results: Solutions of the intracardiac potential inverse problem with the MFS are robust, fast and accurate. Endocardial surface potentials can be faithfully reconstructed from noncontact recordings in real-time if the geometry of cardiac surface and the location of electrodes relative to it are known. Larger catheters with appropriate electrode density are needed to resolve complex reentrant atrial rhythms. Conclusion: Real-time panoramic potential mapping is feasible with noncontact intracardiac catheters using the MFS. Significance: Accurate endocardial potential maps can be reconstructed in AF with appropriately designed noncontact multi-electrode catheters.

Myocardial mesostructure and mesofunction.

The complex and highly organized structural arrangement of some five billion cardiomyocytes directs the coordinated electrical activity and mechanical contraction of the human heart. The characteristic transmural change in cardiomyocyte orientation underlies base-to-apex shortening, circumferential shortening, and left ventricular torsion during contraction. Individual cardiomyocytes shorten ∼15% and increase in diameter ∼8%. Remarkably, however, the left ventricular wall thickens by up to 30-40%. To accommodate this, the myocardium must undergo significant structural rearrangement during contraction. At the mesoscale, collections of cardiomyocytes are organized into sheetlets, and sheetlet shear is the fundamental mechanism of rearrangement that produces wall thickening. Herein, we review the histological and physiological studies of myocardial mesostructure that have established the sheetlet shear model of wall thickening. Recent developments in tissue clearing techniques allow for imaging of whole hearts at the cellular scale, whereas magnetic resonance imaging (MRI) and computed tomography (CT) can image the myocardium at the mesoscale (100 µm to 1 mm) to resolve cardiomyocyte orientation and organization. Through histology, cardiac diffusion tensor imaging (DTI), and other modalities, mesostructural sheetlets have been confirmed in both animal and human hearts. Recent in vivo cardiac DTI methods have measured reorientation of sheetlets during the cardiac cycle. We also examine the role of pathological cardiac remodeling on sheetlet organization and reorientation, and the impact this has on ventricular function and dysfunction. We also review the unresolved mesostructural questions and challenges that may direct future work in the field.

A novel system for mapping regional electrical properties and characterizing arrhythmia in isolated intact rat atria.

Detailed global maps of atrial electrical activity are needed to understand mechanisms of atrial rhythm disturbance in small animal models of heart disease. To date, optical mapping systems have not provided enough spatial resolution across sufficiently extensive regions of intact atrial preparations to achieve this goal. The aim of this study was to develop an integrated platform for quantifying regional electrical properties and analyzing reentrant arrhythmia in a biatrial preparation. Intact atria from 6/7-mo-old female spontaneously hypertensive rats (SHRs; n = 6) were isolated and secured in a constant flow superfusion chamber at 37°C. Optical mapping was performed with the membrane-voltage dye di-4-ANEPPS using LED excitation and a scientific complementary metal-oxide semiconductor (sCMOS) camera. Programmed stimulus trains were applied from right atrial (RA) and left atrial (LA) sites to assess rate-dependent electrical behavior and to induce atrial arrhythmia. Signal-to-noise ratio was improved by sequential processing steps that included spatial smoothing, temporal filtering, and, in stable rhythms, ensemble-averaging. Activation time, repolarization time, and action potential duration (APD) maps were constructed at high spatial resolution for a wide range of coupling intervals. These data were highly consistent within and between experiments. They confirmed preferential atrial conduction pathways and demonstrated distinct medial-to-lateral APD gradients. We also showed that reentrant arrhythmias induced in this preparation were explained by the spatial variation of these electrical properties. Our new methodology provides a robust means of 1) quantifying regional electrical properties in the intact rat atria at higher spatiotemporal resolution than previously reported, and 2) characterizing reentrant arrhythmia and analyzing mechanisms that give rise to it.NEW & NOTEWORTHY Despite wide-ranging optical mapping studies, detailed information on regional atrial electrical properties in small animal models of heart disease and how these contribute to reentrant arrhythmia remains limited. We have developed a novel experimental platform that enables both to be achieved in a geometrically intact isolated rat bi-atrial preparation.

High-resolution 3D imaging and topological mapping of the lymph node conduit system.

The conduit network is a hallmark of lymph node microanatomy, but lack of suitable imaging technology has prevented comprehensive investigation of its topology. We employed an extended-volume imaging system to capture the conduit network of an entire murine lymph node (comprising over 280,000 segments). The extensive 3D images provide a comprehensive overview of the regions supplied by conduits, including perivascular sleeves and distinctive "follicular reservoirs" within B cell follicles, surrounding follicular dendritic cells. A 3D topology map of conduits within the T-cell zone showed homogeneous branching, but conduit density was significantly higher in the superficial T-cell zone compared with the deep zone, where distances between segments are sufficient for T cells to lose contact with fibroblastic reticular cells. This topological mapping of the conduit anatomy can now aid modeling of its roles in lymph node function, as we demonstrate by simulating T-cell motility in the different T-cell zones.

Impaired Baroreflex Function in an Ovine Model of Chronic Heart Failure Induced by Multiple Coronary Microembolizations.

Testing new therapies in heart failure (HF) requires a chronic stable model of HF in large animals. Microembolization of the coronary arteries has been used to model HF previously; however, neural control has not been previously explored in this model. Thus the aim of this study was to further characterize neural control in this model of HF. HF was induced by infusion of microspheres (45 micron; 1.3 ml) into the proximal left coronary artery or left descending coronary arteries, with three sequential embolizations over 3 weeks. Twelve to 14 weeks after the final embolization, and when ejection fraction had decreased below 45%, animals were instrumented to record blood pressure and heart rate. Baroreflex control of heart rate was investigated in conscious animals. Additionally, pressure-volume loops were constructed under anesthesia. Embolization-induced HF was associated with a decrease in mean arterial pressure (67 ± 2 vs. 85 ± 4 mmHg, p < 0.05), an increase in heart rate (108 ± 4 vs. 94 ± 4 bpm, p < 0.05), and a significant increase in left ventricular end-diastolic pressure (11.4 ± 2 vs. 6.2 ± 1 mmHg, p < 0.01). Under conscious conditions, there was a significant decrease in the gain (-8.2 ± 2 vs. -4.1 ± 1 beats/min/mmHg, p < 0.05) as well as the lower plateau of the baroreflex in HF compared to control animals. HF was also associated with significantly increased respiratory rate (107 ± 4 vs. 87 ± 4 breaths/min, p < 0.01) and incidence of apneas (520 ± 24 vs. 191 ± 8 apnea periods >4 s, p < 0.05), compared to control sheep. The microembolization model of heart failure is associated with an increase in left ventricular end-diastolic pressure, impaired cardiac function, and altered baroreflex control of the heart. These findings suggest this chronic model of HF is appropriate to use for investigating interventions aimed at improving neural control in HF.

Microstructurally Motivated Constitutive Modeling of Heart Failure Mechanics.

Heart failure (HF) is one of the leading causes of death worldwide. HF is associated with substantial microstructural remodeling, which is linked to changes in left ventricular geometry and impaired cardiac function. The role of myocardial remodeling in altering the mechanics of failing hearts remains unclear. Structurally based constitutive modeling provides an approach to improve understanding of the relationship between biomechanical function and tissue organization in cardiac muscle during HF. In this study, we used cardiac magnetic resonance imaging and extended-volume confocal microscopy to quantify the remodeling of left ventricular geometry and myocardial microstructure of healthy and spontaneously hypertensive rat hearts at the ages of 12 and 24 months. Passive cardiac mechanical function was characterized using left ventricular pressure-volume compliance measurements. We have developed a, to our knowledge, new structurally based biomechanical constitutive equation built on parameters quantified directly from collagen distributions observed in confocal images of the myocardium. Three-dimensional left ventricular finite element models were constructed from subject-specific in vivo magnetic resonance imaging data. The structurally based constitutive equation was integrated into geometrically subject-specific finite element models of the hearts and used to investigate the underlying mechanisms of ventricular dysfunction during HF. Using a single pair of material parameters for all hearts, we were able to produce compliance curves that reproduced all of the experimental compliance measurements. The value of this study is not limited to reproducing the mechanical behavior of healthy and diseased hearts, but it also provides important insights into the structure-function relationship of diseased myocardium that will help pave the way toward more effective treatments for HF.

Cardiac intramural electrical mapping reveals focal delays but no conduction velocity slowing in the peri-infarct region.

Altered electrical behavior alongside healed myocardial infarcts (MIs) is associated with increased risk of sudden cardiac death. However, the multidimensional mechanisms are poorly understood and described. This study characterizes, for the first time, the intramural spread of electrical activation in the peri-infarct region of chronic reperfusion MIs. Four sheep were studied 13 wk after antero-apical reperfusion infarction. Extracellular potentials (ECPs) were recorded in a ~20 × 20-mm2 region adjacent to the infarct boundary (25 plunge needles <0.5-mm diameter with 15 electrodes at 1-mm centers) during multisite stimulation. Infarct geometry and electrode locations were reconstructed from magnetic resonance images. Three-dimensional activation spread was characterized by local activation times and interpolated ECP fields (n = 191 records). Control data were acquired in 4 non-infarcted sheep (n = 96 records). Electrodes were distributed uniformly around 15 ± 5% of the intramural infarct boundary. There were marked changes in pacing success and ECP morphology across a functional border zone (BZ) ±2 mm from the boundary. Stimulation adjacent to the infarct boundary was associated with low-amplitude electrical activity within the BZ and delayed activation of surrounding myocardium. Bulk tissue depolarization occurred 3.5-14.6 mm from the pacing site for 39% of stimuli with delays of 4-37 ms, both significantly greater than control (P < 0.0001). Conduction velocity (CV) adjacent to the infarct was not reduced compared with control, consistent with structure-only computer model results. Insignificant CV slowing, irregular stimulus-site specific activation delays, and obvious indirect activation pathways strongly suggest that the substrate for conduction abnormalities in chronic MI is predominantly structural in nature.NEW & NOTEWORTHY Intramural in vivo measurements of peri-infarct electrical activity were not available before this study. We use pace-mapping in a three-dimensional electrode array to show that a subset of stimuli in the peri-infarct region initiates coordinated myocardial activation some distance from the stimulus site with substantial associated time delays. This is site dependent and heterogeneous and occurs for <50% of ectopic stimuli in the border zone. Furthermore, once coordinated activation is initiated, conduction velocity adjacent to the infarct boundary is not significantly different from control. These results give new insights to peri-infarct electrical activity and do not support the widespread view of uniform electrical remodeling in the border zone of chronic myocardial infarcts, with depressed conduction velocity throughout.

Shift of leading pacemaker site during reflex vagal stimulation and altered electrical source-to-sink balance.

KEY POINTS: Vagal reflexes slow heart rate and can change where the heartbeat originates within the sinoatrial node (SAN). The mechanisms responsible for this process - termed leading pacemaker (LP) shift - have not been investigated fully. We used optical mapping to measure the effects of baroreflex, chemoreflex and carbachol on pacemaker entrainment and electrical conduction across the SAN. All methods of stimulation triggered shifts in LP site from the central SAN to one or two caudal pacemaker regions. These shifts were associated with reduced current generation capacity centrally and increased electrical load caudally. Previous studies suggest LP shift is a rate-dependent phenomenon whereby acetylcholine slows central pacemaker rate disproportionately, enabling caudal cells that are less acetylcholine sensitive to assume control. However, our findings indicate the LP region is defined by both pacemaker rate and capacity to drive activation. Shifts in LP site provide an important homeostatic mechanism for rapid switches in heart rate. ABSTRACT: Reflex vagal activity causes abrupt heart rate slowing with concomitant caudal shifts of the leading pacemaker (LP) site within the sinoatrial node (SAN). However, neither the mechanisms responsible nor their dynamics have been investigated fully. Therefore, the objective of this study was to elucidate the mechanisms driving cholinergic LP shift. Optical maps of right atrial activation were acquired in a rat working heart-brainstem preparation during baroreflex and chemoreflex stimulation or with carbachol. All methods of stimulation triggered shifts in LP site from the central SAN to caudal pacemaker regions, which were positive for HCN4 and received uniform cholinergic innervation. During baroreflex onset, the capacity of the central region to drive activation declined with a decrease in amplitude and gradient of optical action potentials (OAPs) in the surrounding myocardium. Accompanying this decline, there was altered entrainment in the caudal SAN as shown by decreased conduction velocity, OAP amplitude, gradient and activation time. Atropine abolished these responses. Chemoreflex stimulation produced similar effects but central capacity to drive activation was preserved before the LP shift. In contrast, carbachol produced a prolonged period of reduced capacity to drive and altered entrainment. Previous studies suggest LP shift is a rate-dependent phenomenon whereby acetylcholine slows central pacemaker rate disproportionately, enabling caudal cells that are less acetylcholine sensitive to assume control. Our findings indicate that cholinergic LP shifts are also determined by altered electrical source-to-sink balance in the SAN. We conclude that the LP region is defined by both rate and capacity to drive atrial activation.

Reconstruction of coronary circulation networks: A review of methods.

Building anatomically accurate models of the coronary vascular system enables potentially deeper understandings of coronary circulation. To achieve this, (a) images at different levels of vascular network-arteries, arterioles, capillaries, venules, and veins-need to be obtained through suitable imaging modalities; and (b) from images, morphological and topological information needs to be extracted using image processing techniques. While there are several modalities that enable the imaging of large vessels, microcirculation imaging-capturing vessels having diameter lesser than 100 µm-has to date been typically confined to small regions of the heart. This spatially limited microcirculatory information has often been used within cardiac models, with the potentially erroneous assumption that it is representative of the whole organ. However, with the recent advancements in imaging and image processing, it is rapidly becoming feasible to acquire, process, and quantify microcirculation data at the scale of whole organ. In this review, we summarize the progress toward this goal followed through a presentation of the current state-of-the-art imaging and image processing techniques in the context of coronary microcirculation extraction, prominently but not exclusively, from small animals.

How Accurate Is Inverse Electrocardiographic Mapping? A Systematic In Vivo Evaluation.

BACKGROUND: Inverse electrocardiographic mapping reconstructs cardiac electrical activity from recorded body surface potentials. This noninvasive technique has been used to identify potential ablation targets. Despite this, there has been little systematic evaluation of its reliability. METHODS: Torso and ventricular epicardial potentials were recorded simultaneously in anesthetized, closed-chest pigs (n=5), during sinus rhythm, epicardial, and endocardial ventricular pacing (70 records in total). Body surface and cardiac electrode positions were determined and registered using magnetic resonance imaging. Epicardial potentials were reconstructed during ventricular activation using experiment-specific magnetic resonance imaging-based thorax models, with homogeneous or inhomogeneous (lungs, skeletal muscle, fat) electrical properties. Coupled finite/boundary element methods and a meshless approach based on the method of fundamental solutions were compared. Inverse mapping underestimated epicardial potentials >2-fold (P<0.0001). RESULTS: Mean correlation coefficients for reconstructed epicardial potential distributions ranged from 0.60±0.08 to 0.64±0.07 across all methods. Epicardial electrograms were recovered with reasonable fidelity at ≈50% of sites (median correlation coefficient, 0.69-0.72), but variation was substantial. General activation spread was reproduced (median correlation coefficient, 0.72-0.78 for activation time maps after spatio-temporal smoothing). Epicardial foci were identified with a median location error ≈16 mm (interquartile range, 9-29 mm). Inverse mapping with meshless method of fundamental solutions was better than with finite/boundary element methods, and the latter were not improved by inclusion of inhomogeneous torso electrical properties. CONCLUSIONS: Inverse potential mapping provides useful information on the origin and spread of epicardial activation. However the spatio-temporal variability of recovered electrograms limit resolution and must constrain the accuracy with which arrhythmia circuits can be identified independently using this approach.

Development of 3-D Intramural and Surface Potentials in the LV: Microstructural Basis of Preferential Transmural Conduction.

INTRODUCTION: Extracellular potentials measured on the heart surfaces are used to infer events that originate deep within the heart wall. We have reconstructed intramural potentials in three dimensions for the first time, and compare these with epicardial and endocardial surface potentials and cardiac microstructure. METHODS AND RESULTS: Extracellular potentials from intramural point stimulation were measured from a high density 3-D electrode array in the in vivo pig LV. MR and extended volume imaging were used to register electrode locations and characterize fiber and laminar orientations throughout the recording volume. Measured potentials were compared with predictions of tissue-specific bidomain computer activation models. Positive potentials recorded in the LV wall preceded the depolarization wavefront as it spread in the fiber direction. Transverse to this, passive and active potentials spread preferentially in the laminar direction (anisotropy ratio ∼1.6:1). Epicardial surface potentials reflect initial intramural propagation at the stimulus location, but endocardial potentials do not, particularly adjacent to papillary muscles. Measured 3-D potentials were consistently better captured by computer models that incorporate three distinct conductivities aligned with local microstructural axes, but the preferential spread of potentials in the laminar direction was not fully predicted. CONCLUSIONS: This study provides evidence for preferential transmural conduction and raises questions about the extent to which intramural electrical events can be inferred from endocardial potentials.

Increased cardiac work provides a link between systemic hypertension and heart failure.

The spontaneously hypertensive rat (SHR) is an established model of human hypertensive heart disease transitioning into heart failure. The study of the progression to heart failure in these animals has been limited by the lack of longitudinal data. We used MRI to quantify left ventricular mass, volume, and cardiac work in SHRs at age 3 to 21 month and compared these indices to data from Wistar-Kyoto (WKY) controls. SHR had lower ejection fraction compared with WKY at all ages, but there was no difference in cardiac output at any age. At 21 month the SHR had significantly elevated stroke work (51 ± 3 mL.mmHg SHR vs. 24 ± 2 mL.mmHg WKY; n = 8, 4; P < 0.001) and cardiac minute work (14.2 ± 1.2 L.mmHg/min SHR vs. 6.2 ± 0.8 L.mmHg/min WKY; n = 8, 4; P < 0.001) compared to control, in addition to significantly larger left ventricular mass to body mass ratio (3.61 ± 0.15 mg/g SHR vs. 2.11 ± 0.008 mg/g WKY; n = 8, 6; P < 0.001). SHRs showed impaired systolic function, but developed hypertrophy to compensate and successfully maintained cardiac output. However, this was associated with an increase in cardiac work at age 21 month, which has previously demonstrated fibrosis and cell death. The interplay between these factors may be the mechanism for progression to failure in this animal model.

Organ-wide 3D-imaging and topological analysis of the continuous microvascular network in a murine lymph node.

Understanding of the microvasculature has previously been limited by the lack of methods capable of capturing and modelling complete vascular networks. We used novel imaging and computational techniques to establish the topology of the entire blood vessel network of a murine lymph node, combining 63,706 confocal images at 2 µm pixel resolution to cover a volume of 3.88 mm(3). Detailed measurements including the distribution of vessel diameters, branch counts, and identification of voids were subsequently re-visualised in 3D revealing regional specialisation within the network. By focussing on critical immune microenvironments we quantified differences in their vascular topology. We further developed a morphology-based approach to identify High Endothelial Venules, key sites for lymphocyte extravasation. These data represent a comprehensive and continuous blood vessel network of an entire organ and provide benchmark measurements that will inform modelling of blood vessel networks as well as enable comparison of vascular topology in different organs.

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.

BACKGROUND: 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. METHODS: Eight explanted rat hearts were perfused with Gd-DTPA contrast agent and fixative and imaged in a 9.4T magnet by two types of acquisition: 3D fast low angle shot (FLASH) imaging, voxels 50 × 50 × 50 µm, and 3D spin echo DTI with monopolar diffusion gradients of 3.6 ms duration at 11.5 ms separation, voxels 200 × 200 × 200 µm. The sensitivity of each approach to imaging parameters was explored. RESULTS: The FLASH data showed laminar alignments of voxels with high signal, in keeping with the presumed predominance of contrast in the interstices between sheetlets. It was analysed, using structure-tensor (ST) analysis, to determine the most (v1(ST)), intermediate (v2(ST)) and least (v3(ST)) extended orthogonal directions of signal continuity. The DTI data was analysed to determine the most (e1(DTI)), intermediate (e2(DTI)) and least (e3(DTI)) orthogonal eigenvectors of extent of diffusion. The correspondence between the FLASH and DTI methods was measured and appraised. The most extended direction of FLASH signal (v1(ST)) agreed well with that of diffusion (e1(DTI)) throughout the left ventricle (representative discrepancy in the septum of 13.3 ± 6.7°: median ± absolute deviation) and both were in keeping with the expected local orientations of the long-axis of cardiomyocytes. However, the orientation of the least directions of FLASH signal continuity (v3(ST)) and diffusion (e3(ST)) showed greater discrepancies of up to 27.9 ± 17.4°. Both FLASH (v3(ST)) and DTI (e3(DTI)) where compared to directly measured laminar arrays in the FLASH images. For FLASH the discrepancy between the structure-tensor calculated v3(ST) and the directly measured FLASH laminar array normal was of 9 ± 7° for the lateral wall and 7 ± 9° for the septum (median ± inter quartile range), and for DTI the discrepancy between the calculated v3(DTI) and the directly measured FLASH laminar array normal was 22 ± 14° and 61 ± 53.4°. DTI was relatively insensitive to the number of diffusion directions and to time up to 72 hours post fixation, but was moderately affected by b-value (which was scaled by modifying diffusion gradient pulse strength with fixed gradient pulse separation). Optimal DTI parameters were b = 1000 mm/s(2) and 12 diffusion directions. FLASH acquisitions were relatively insensitive to the image processing parameters explored. CONCLUSIONS: We show that ST analysis of FLASH is a useful and accurate tool in the measurement of cardiac microstructure. While both FLASH and the DTI approaches appear promising for mapping of the alignments of myocytes throughout myocardium, marked discrepancies between the cross myocyte anisotropies deduced from each method call for consideration of their respective limitations.

Forward problem of electrocardiography: is it solved?

BACKGROUND: The relationship between epicardial and body surface potentials defines the forward problem of electrocardiography. A robust formulation of the forward problem is instrumental to solving the inverse problem, in which epicardial potentials are computed from known body surface potentials. Here, the accuracy of different forward models has been evaluated experimentally. METHODS AND RESULTS: Body surface and epicardial potentials were recorded simultaneously in anesthetized closed-chest pigs (n=5) during sinus rhythm, and epicardial and endocardial ventricular pacing (65 records in total). Body surface potentials were simulated from epicardial recordings using experiment-specific volume conductor models constructed from magnetic resonance imaging. Results for homogeneous (isotropic electric properties) and inhomogeneous (incorporating lungs, anisotropic skeletal muscle, and subcutaneous fat) forward models were compared with measured body surface potentials. Correlation coefficients were 0.85±0.08 across all animals and activation sequences with no significant difference between homogeneous and inhomogeneous solutions (P=0.85). Despite this, there was considerable variance between simulated and measured body surface potential distributions. Differences between the body surface potential extrema predicted with homogeneous forward models were 55% to 78% greater than observed (P<0.05) and attenuation of potentials adjacent to extrema were 10% to 171% greater (P<0.03). The length and orientation of the vector between potential extrema were also significantly different. Inclusion of inhomogeneous electric properties in the forward model reduced, but did not eliminate these differences. CONCLUSIONS: These results demonstrate that homogeneous volume conductor models introduce substantial spatial inaccuracies in forward problem solutions. This probably affects the precision of inverse reconstructions of cardiac potentials, in which this assumption is made.

Image-based motion correction for optical mapping of cardiac electrical activity.

Optical mapping, with membrane-bound, voltage-sensitive dyes, is widely used for in vitro recording of cardiac electrical activity. The spatial registration of such maps is lost when the heart moves with respect to a fixed photodetector array and contraction can generate substantial artifact if background fluorescence is not uniformly distributed. While motion artifact is commonly suppressed with electromechanical uncoupling agents, there are circumstances where these are undesirable. This study outlines a novel image-based approach for retrospective motion artifact correction. Isolated Langendorff-supported rat hearts (n = 8), stained with di-4-ANEPPS, were illuminated at 516 ± 14 nm and fluorescent emission (>565 ± 10 nm) was acquired with a charge multiplying CCD camera. Background fluorescence was segmented in successive frames and stabilized using a non-rigid image registration algorithm. The resultant image deformation was used to estimate material point movement on the heart surface, so that total fluorescence could be mapped frame-by-frame to appropriate reference pixels. Finally, residual motion artifact was identified and removed. The effectiveness of this correction method was evaluated over 18 experimental datasets. Signal-to-noise ratio was increased more than fourfold, and activation time and action potential duration (APD) could be estimated at 24% more pixels than in the raw data. The variability of all APD measures was substantially reduced (i.e. APD50 estimated as 83.8 ± 45.8 ms before correction was 52.1 ± 4.7 ms afterward). This approach provides a robust means of recovering optical action potentials in the presence of substantial motion artifact.