Robert S. Stephenson

Micro-computed tomography with iodine staining resolves the arrangement of muscle fibres

We illustrate here microCT images in which contrast between muscle and connective tissue has been achieved by means of staining with iodine. Enhancement is shown to be dependent on the concentration of iodine solution (I(2)KI), time in solution and specimen size. Histological examination confirms that the arrangement of individual muscle fibres can be visualised on the enhanced microCT images, and that the iodine accumulates in the muscle fibres in preference to the surrounding connective tissues. We explore the application of this technique to describe the fibrous structure of skeletal muscle, and conclude that it has the potential to become a non-destructive and cost-effective method for investigating muscle fascicle architecture, particularly in comparative morphological studies.

Application of Micro-Computed Tomography With Iodine Staining to Cardiac Imaging, Segmentation, and Computational Model Development

Micro-computed tomography (micro-CT) has been widely used to generate high-resolution 3-D tissue images from small animals nondestructively, especially for mineralized skeletal tissues. However, its application to the analysis of soft cardiovascular tissues has been limited by poor inter-tissue contrast. Recent ex vivo studies have shown that contrast between muscular and connective tissue in micro-CT images can be enhanced by staining with iodine. In the present study, we apply this novel technique for imaging of cardiovascular structures in canine hearts. We optimize the method to obtain high-resolution X-ray micro-CT images of the canine atria and its distinctive regions-including the Bachmanns bundle, atrioventricular node, pulmonary arteries and veins-with clear inter-tissue contrast. The imaging results are used to reconstruct and segment the detailed 3-D geometry of the atria. Structure tensor analysis shows that the arrangement of atrial fibers can also be characterized using the enhanced micro-CT images, as iodine preferentially accumulates within the muscular fibers rather than in connective tissues. This novel technique can be particularly useful in nondestructive imaging of 3-D cardiac architectures from large animals and humans, due to the combination of relatively high speed ( ~ 1 h/per scan of the large canine heart) and high voxel resolution (36 μm) provided. In summary, contrast micro-CT facilitates fast and nondestructive imaging and segmenting of detailed 3-D cardiovascular geometries, as well as measuring fiber orientation, which are crucial in constructing biophysically detailed computational cardiac models.

Contrast enhanced micro-computed tomography resolves the 3-dimensional morphology of the cardiac conduction system in mammalian hearts.

The general anatomy of the cardiac conduction system (CCS) has been known for 100 years, but its complex and irregular three-dimensional (3D) geometry is not so well understood. This is largely because the conducting tissue is not distinct from the surrounding tissue by dissection. The best descriptions of its anatomy come from studies based on serial sectioning of samples taken from the appropriate areas of the heart. Low X-ray attenuation has formerly ruled out micro-computed tomography (micro-CT) as a modality to resolve internal structures of soft tissue, but incorporation of iodine, which has a high molecular weight, into those tissues enhances the differential attenuation of X-rays and allows visualisation of fine detail in embryos and skeletal muscle. Here, with the use of a iodine based contrast agent (I2KI), we present contrast enhanced micro-CT images of cardiac tissue from rat and rabbit in which the three major subdivisions of the CCS can be differentiated from the surrounding contractile myocardium and visualised in 3D. Structures identified include the sinoatrial node (SAN) and the atrioventricular conduction axis: the penetrating bundle, His bundle, the bundle branches and the Purkinje network. Although the current findings are consistent with existing anatomical representations, the representations shown here offer superior resolution and are the first 3D representations of the CCS within a single intact mammalian heart.

Assessment of the Helical Ventricular Myocardial Band Using Standard Echocardiography

In the discussion of their recent article, Hayabuchi and his colleagues acknowledge that the “helical myocardial band” remains controversial. In the accompanying editorial, Buckberg harbored no such doubts. Are the limited echocardiographic findings illustrated truly sufficient for Hayabuchi and his colleagues to conclude that there is a “helical ventricular myocardial band”? They refer to a model that Torrent-Guasp had carved out of the ventricular muscular mass by disrupting myriads of myocardial branches, suggesting moreover that this band is freely moveable on itself. The histological studies produced by Hort and Feneis, however, provided evidence that the ventricular cone does not have discrete origins and insertions of the cardiomyocytes as found in skeletal muscle. Pettigrew had demonstrated more than a century ago the multiple interleaving sheets of cardiomyocytes to be found within the cone. Lev and Simkins, cited by Buckberg, also had emphasized that the cone can be dissected at the whim of the prosector, as achieved by Torrent-Guasp when subjectively producing the preparations now modeled by Buckberg. Our investigations, cited by Hayabuchi and colleagues, endorse the works of Feneis and Hort. The histological findings show no obvious anatomical substrate, other than the obvious change in alignment of the aggregated chains of cardiomyocytes, to explain the echocardiographic feature emphasized by the Japanese workers. They certainly provide none that represent a substantial proportion of the width of the septum, as the echocardiograms seem to suggest. The echogenic band is seen in the equatorial and basal regions of each of the walls of the left ventricle when viewed from the apex. No such band is seen when the ventricular mass is viewed using the parasternal window. We suggest that the echogenic band represents an area of distinct myocyte orientation within the continuous mesh of the septum, where the reflected ultrasound is perpendicular to the dominant orientation of the cardiomyocytes, thus giving maximum intensity compared with the surrounding tissue. The echogenic band, when viewed from the apex, therefore, is likely to represent no more than the chains of cardiomyocytes located within the mid-wall of the ventricular cone which are aligned circumferentially. The concept of the helical ventricular myocardial band does not model the circumferential orientation in this region. There are further problems, however, with the concepts advanced by Buckberg, His inferences are based on imaging systems that measure only strain, as opposed to assessing the local development of force. The onset of shortening is not identical with the onset of contraction, so it is his mistake to interpret late shortening as delayed contraction. We have shown that within the ventricular cone, there are extended zones in which the myocardium contracts auxotonically, that is, the force increases during systole. The features of such auxotonic contraction are delayed onset, restricted shortening, and delayed termination.

Insights from echocardiography, magnetic resonance imaging, and microcomputed tomography relative to the mid-myocardial left ventricular echogenic zone.

The anatomical substrate for the mid‐mural ventricular hyperechogenic zone remains uncertain, but it may represent no more than ultrasound reflected from cardiomyocytes orientated orthogonally to the ultrasonic beam. We sought to ascertain the relationship between the echogenic zone and the orientation of the cardiomyocytes.

High resolution 3-Dimensional imaging of the human cardiac conduction system from microanatomy to mathematical modeling

Cardiac arrhythmias and conduction disturbances are accompanied by structural remodelling of the specialised cardiomyocytes known collectively as the cardiac conduction system. Here, using contrast enhanced micro-computed tomography, we present, in attitudinally appropriate fashion, the first 3-dimensional representations of the cardiac conduction system within the intact human heart. We show that cardiomyocyte orientation can be extracted from these datasets at spatial resolutions approaching the single cell. These data show that commonly accepted anatomical representations are oversimplified. We have incorporated the high-resolution anatomical data into mathematical simulations of cardiac electrical depolarisation. The data presented should have multidisciplinary impact. Since the rate of depolarisation is dictated by cardiac microstructure, and the precise orientation of the cardiomyocytes, our data should improve the fidelity of mathematical models. By showing the precise 3-dimensional relationships between the cardiac conduction system and surrounding structures, we provide new insights relevant to valvar replacement surgery and ablation therapies. We also offer a practical method for investigation of remodelling in disease, and thus, virtual pathology and archiving. Such data presented as 3D images or 3D printed models, will inform discussions between medical teams and their patients, and aid the education of medical and surgical trainees.

The functional architecture of skeletal compared to cardiac musculature: Myocyte orientation, lamellar unit morphology, and the helical ventricular myocardial band.

How the cardiomyocytes are aggregated within the heart walls remains contentious. We still do not fully understand how the end‐to‐end longitudinal myocytic chains are arranged, nor the true extent and shape of the lamellar units they aggregate to form. In this article, we show that an understanding of the complex arrangement of cardiac musculature requires knowledge of three‐dimensional myocyte orientation (helical and intrusion angle), and appreciation of myocyte packing within the connective tissue matrix. We show how visualization and segmentation of high‐resolution three‐dimensional image data can accurately identify the morphology and orientation of the myocytic chains, and the lamellar units. Some maintain that the ventricles can be unwrapped in the form of a “helical ventricular myocardial band,” that is, as a compartmentalized band with selective regional innervation and deformation, and a defined origin and insertion like most skeletal muscles. In contrast to the simpler interpretation of the helical ventricular myocardial band, we provide insight as to how the complex myocytic chains, the heterogeneous lamellar units, and connective tissue matrix form an interconnected meshwork, which facilitates the complex internal deformations of the ventricular wall. We highlight the dangers of disregarding the intruding cardiomyocytes. Preparation of the band destroys intruding myocytic chains, and thus disregards the functional implications of the antagonistic auxotonic forces they produce. We conclude that the ventricular myocardium is not analogous to skeletal muscle, but is a complex three‐dimensional meshwork, with a heterogeneous branching lamellar architecture. Clin. Anat. 29:316–332, 2016.

Studying the Microanatomy of the Heart in Three Dimensions: A Practical Update

The structure and function of the heart needs to be understood in three dimensions. We give a brief historical summary of the methods by which such an understanding has been sought, and some practical details of the relatively new technique of micro-CT with iodine contrast enhancement in samples from rat and rabbit. We discuss how the improved anatomical detail available in fixed cadaveric hearts will enhance our ability to model and to understand the integrated function of the cardiomyocytes, conducting tissues, and fibrous supporting structures that generate the pumping function of the heart.

Congestive Heart Failure Leads to Prolongation of the PR Interval and Atrioventricular Junction Enlargement and Ion Channel Remodelling in the Rabbit

Heart failure is a major killer worldwide. Atrioventricular conduction block is common in heart failure; it is associated with worse outcomes and can lead to syncope and bradycardic death. We examine the effect of heart failure on anatomical and ion channel remodelling in the rabbit atrioventricular junction (AVJ). Heart failure was induced in New Zealand rabbits by disruption of the aortic valve and banding of the abdominal aorta resulting in volume and pressure overload. Laser micro-dissection and real-time polymerase chain reaction (RT-PCR) were employed to investigate the effects of heart failure on ion channel remodelling in four regions of the rabbit AVJ and in septal tissues. Investigation of the AVJ anatomy was performed using micro-computed tomography (micro-CT). Heart failure animals developed first degree heart block. Heart failure caused ventricular myocardial volume increase with a 35% elongation of the AVJ. There was downregulation of HCN1 and Cx43 mRNA transcripts across all regions and downregulation of Cav1.3 in the transitional tissue. Cx40 mRNA was significantly downregulated in the atrial septum and AVJ tissues but not in the ventricular septum. mRNA abundance for ANP, CLCN2 and Navβ1 was increased with heart failure; Nav1.1 was increased in the inferior nodal extension/compact node area. Heart failure in the rabbit leads to prolongation of the PR interval and this is accompanied by downregulation of HCN1, Cav1.3, Cx40 and Cx43 mRNAs and anatomical enlargement of the entire heart and AVJ.

The end of the unique myocardial band: Part II. Clinical and functional considerations

Two of the leading concepts of mural ventricular architecture are the unique myocardial band and the myocardial mesh model. We have described, in an accompanying article published in this journal, how the anatomical, histological and high-resolution computed tomographic studies strongly favour the latter concept. We now extend the argument to describe the linkage between mural architecture and ventricular function in both health and disease. We show that clinical imaging by echocardiography and magnetic resonance imaging, and electrophysiological studies, all support the myocardial mesh model. We also provide evidence that the unique myocardial band model is not compatible with much of scientific research.