Micro-computed tomography and histology to explore internal morphology in decapod larvae

Traditionally, the internal morphology of crustacean larvae has been studied using destructive techniques such as dissection and microscopy. The present study combines advances in microcomputed tomography (micro-CT) and histology to study the internal morphology of decapod larvae, using the common spider crab (Maja brachydactyla Balss, 1922) as a model and resolving the individual limitations of these techniques. The synergy of micro-CT and histology allows the organs to be easily identified, revealing simultaneously the gross morphology (shape, size, and location) and histological organization (tissue arrangement and cell identification). Micro-CT shows mainly the exoskeleton, musculature, digestive and nervous systems, and secondarily the circulatory and respiratory systems, while histology distinguishes several cell types and confirms the organ identity. Micro-CT resolves a discrepancy in the literature regarding the nervous system of crab larvae. The major changes occur in the metamorphosis to the megalopa stage, specifically the formation of the gastric mill, the shortening of the abdominal nerve cord, the curving of the abdomen beneath the cephalothorax, and the development of functional pereiopods, pleopods, and lamellate gills. The combination of micro-CT and histology provides better results than either one alone.Financial support was provided by the Spanish Ministry of Economy and Competitiveness through the INIA project (grant number RTA2011-00004-00-00) to G.G. and a pre-doctoral fellowship to D.C. (FPI-INIA)

Cardiac multi-scale investigation of the right and left ventricle ex vivo: a review

The heart is a complex multi-scale system composed of components integrated at the subcellular, cellular, tissue and organ levels. The myocytes, the contractile elements of the heart, form a complex three-dimensional (3D) network which enables propagation of the electrical signal that triggers the contraction to efficiently pump blood towards the whole body. Cardiovascular diseases (CVDs), a major cause of mortality in developed countries, often lead to cardiovascular remodeling affecting cardiac structure and function at all scales, from myocytes and their surrounding collagen matrix to the 3D organization of the whole heart. As yet, there is no consensus as to how the myocytes are arranged and packed within their connective tissue matrix, nor how best to image them at multiple scales. Cardiovascular imaging is routinely used to investigate cardiac structure and function as well as for the evaluation of cardiac remodeling in CVDs. For a complete understanding of the relationship between structural remodeling and cardiac dysfunction in CVDs, multi-scale imaging approaches are necessary to achieve a detailed description of ventricular architecture along with cardiac function. In this context, ventricular architecture has been extensively studied using a wide variety of imaging techniques: ultrasound (US), optical coherence tomography (OCT), microscopy (confocal, episcopic, light sheet, polarized light), magnetic resonance imaging (MRI), micro-computed tomography (micro-CT) and, more recently, synchrotron X-ray phase contrast imaging (SR X-PCI). Each of these techniques have their own set of strengths and weaknesses, relating to sample size, preparation, resolution, 2D/3D capabilities, use of contrast agents and possibility of performing together with in vivo studies. Therefore, the combination of different imaging techniques to investigate the same sample, thus taking advantage of the strengths of each method, could help us to extract the maximum information about ventricular architecture and function. In this review, we provide an overview of available and emerging cardiovascular imaging techniques for assessing myocardial architecture ex vivo and discuss their utility in being able to quantify cardiac remodeling, in CVDs, from myocyte to whole organ

Resolving the True Ventricular Mural Architecture.

The precise nature of packing together of the cardiomyocytes within the ventricular walls has still to be determined. The spiraling nature of the chains of interconnected cardiomyocytes has long been recognized. As long ago as the end of the nineteenth century, Pettigrew had emphasized that the ventricular cone was not arranged on the basis of skeletal muscle. Despite this guidance, subsequent anatomists described entities such as “bulbo-spiral muscles”, with this notion of subunits culminating in the suggestion that the ventricular cone could be unwrapped so as to produce a “ventricular myocardial band”. Others, in contrast, had suggested that the ventricular walls were arranged on the basis of “sheets”, or more recently “sheetlets”, with investigators seeking to establishing the angulation of these entities using techniques such as magnetic resonance imaging. Our own investigations, in contrast, have shown that the cardiomyocytes are aggregated together within the supporting fibrous matrix so as to produce a three-dimensional myocardial mesh. In this review, we summarize the previous accounts, and provide the anatomical evidence we have thus far accumulated to support the model of the myocardial mesh. We show how these anatomic findings underscore the concept of the myocardial mesh functioning in antagonistic fashion. They lend evidence to support the notion that the ventricular myocardium works as a muscular hydrostat

Eigenspectra optoacoustic tomography achieves quantitative blood oxygenation imaging deep in tissues

Light propagating in tissue attains a spectrum that varies with location due to wavelength-dependent fluence attenuation by tissue optical properties, an effect that causes spectral corruption. Predictions of the spectral variations of light fluence in tissue are challenging since the spatial distribution of optical properties in tissue cannot be resolved in high resolution or with high accuracy by current methods. Spectral corruption has fundamentally limited the quantification accuracy of optical and optoacoustic methods and impeded the long sought-after goal of imaging blood oxygen saturation (sO2) deep in tissues; a critical but still unattainable target for the assessment of oxygenation in physiological processes and disease. We discover a new principle underlying light fluence in tissues, which describes the wavelength dependence of light fluence as an affine function of a few reference base spectra, independently of the specific distribution of tissue optical properties. This finding enables the introduction of a previously undocumented concept termed eigenspectra Multispectral Optoacoustic Tomography (eMSOT) that can effectively account for wavelength dependent light attenuation without explicit knowledge of the tissue optical properties. We validate eMSOT in more than 2000 simulations and with phantom and animal measurements. We find that eMSOT can quantitatively image tissue sO2 reaching in many occasions a better than 10-fold improved accuracy over conventional spectral optoacoustic methods. Then, we show that eMSOT can spatially resolve sO2 in muscle and tumor; revealing so far unattainable tissue physiology patterns. Last, we related eMSOT readings to cancer hypoxia and found congruence between eMSOT tumor sO2 images and tissue perfusion and hypoxia maps obtained by correlative histological analysis

Mapping Myocardial Fiber Orientation Using Echocardiography-Based Shear Wave Imaging

The assessment of disrupted myocardial fiber arrangement may help to understand and diagnose hypertrophic or ischemic cardiomyopathy. We hereby proposed and developed shear wave imaging (SWI), which is an echocardiography-based, noninvasive, real-time, and easy-to-use technique, to map myofiber orientation. Five in vitro porcine and three in vivo open-chest ovine hearts were studied. Known in physics, shear wave propagates faster along than across the fiber direction. SWI is a technique that can generate shear waves travelling in different directions with respect to each myocardial layer. SWI further analyzed the shear wave velocity across the entire left-ventricular (LV) myocardial thickness, ranging between 10 (diastole) and 25 mm (systole), with a resolution of 0.2 mm in the middle segment of the LV anterior wall region. The fiber angle at each myocardial layer was thus estimated by finding the maximum shear wave speed. In the in vitro porcine myocardium (n=5), the SWI-estimated fiber angles gradually changed from +80° ± 7° (endocardium) to +30° ± 13° (midwall) and-40° ± 10° (epicardium) with 0° aligning with the circumference of the heart. This transmural fiber orientation was well correlated with histology findings (r2=0.91± 0.02, p<0.0001). SWI further succeeded in mapping the transmural fiber orientation in three beating ovine hearts in vivo. At midsystole, the average fiber orientation exhibited 71° ± 13° (endocardium), 27° ± 8° (midwall), and-26° ± 30° (epicardium). We demonstrated the capability of SWI in mapping myocardial fiber orientation in vitro and in vivo. SWI may serve as a new tool for the noninvasive characterization of myocardial fiber structure. © 2012 IEEE.published_or_final_versio

High-Resolution Magnetic Resonance Imaging of the Regenerating Adult Zebrafish Heart

The adult zebrafish is a well-established model for studying heart regeneration, but due to its tissue opaqueness, repair has been primarily assessed using destructive histology, precluding repeated investigations of the same animal. We present a high-resolution, non-invasive in vivo magnetic resonance imaging (MRI) method incorporating a miniature respiratory and anaesthetic perfusion set-up for live adult zebrafish, allowing for visualization of scar formation and heart regeneration in the same animal over time at an isotropic 31 µm voxel resolution. To test the method, we compared well and poorly healing cardiac ventricles using a transgenic fish model that exhibits heat-shock (HS) inducible impaired heart regeneration. HS-treated groups revealed persistent scar tissue for 10 weeks, while control groups were healed after 4 weeks. Application of the advanced MRI technique allowed clear discrimination of levels of repair following cryo- and resection injury for several months. It further provides a novel tool for in vivo time-lapse imaging of adult fish for non-cardiac studies, as the method can be readily applied to image wound healing in other injured or diseased tissues, or to monitor tissue changes over time, thus expanding the range of questions that can be addressed in adult zebrafish and other small aquatic species

Assessing myocardial architecture:the challenges and controversies

In recent decades, investigators have strived to describe and quantify the orientation of the cardiac myocytes in an attempt to classify their arrangement in healthy and diseased hearts. There are, however, striking differences between the investigations from both a technical and methodological standpoint, thus limiting their comparability and impeding the drawing of appropriate physiological conclusions from the structural assessments. This review aims to elucidate these differences, and to propose guidance to establish methodological consensus in the field. The review outlines the theory behind myocyte orientation analysis, and importantly has identified pronounced differences in the definitions of otherwise widely accepted concepts of myocytic orientation. Based on the findings, recommendations are made for the future design of studies in the field of myocardial morphology. It is emphasised that projection of myocyte orientations, before quantification of their angulation, introduces considerable bias, and that angles should be assessed relative to the epicardial curvature. The transmural orientation of the cardiomyocytes should also not be neglected, as it is an important determinant of cardiac function. Finally, there is considerable disagreement in the literature as to how the orientation of myocardial aggregates should be assessed, but to do so in a mathematically meaningful way, the normal vector of the aggregate plane should be utilised

Detailed quantification of cardiac ventricular myocardial architecture in the embryonic and fetal mouse heart by application of structure tensor analysis to high resolution episcopic microscopic data

The mammalian heart, which is one of the first organs to form and function during embryogenesis, develops from a simple tube into a complex organ able to efficiently pump blood towards the rest of the body. The progressive growth of the compact myocardium during embryonic development is accompanied by changes in its structural complexity and organisation. However, how myocardial myoarchitecture develops during embryogenesis remain poorly understood. To date, analysis of heart development has focused mainly on qualitative descriptions using selected 2D histological sections. High resolution episcopic microscopy (HREM) is a novel microscopic imaging technique that enables to obtain high-resolution three-dimensional images of the heart and perform detailed quantitative analyses of heart development. In this work, we performed a detailed characterization of the development of myocardial architecture in wildtype mice, from E14.5 to E18.5, by means of structure tensor analysis applied to HREM images of the heart. Our results shows that even at E14.5, myocytes are already aligned, showing a gradual change in their helical angle from positive angulation in the endocardium towards negative angulation in the epicardium. Moreover, there is gradual increase in the degree of myocardial organisation concomitant with myocardial growth. However, the development of the myoarchitecture is heterogeneous showing regional differences between ventricles, ventricular walls as well as between myocardial layers, with different growth patterning between the endocardium and epicardium. We also found that the percentage of circumferentially arranged myocytes within the LV significantly increases with gestational age. Finally, we found that fractional anisotropy (FA) within the LV gradually increases with gestational age, while the FA within RV remains unchanged

Will the real ventricular architecture please stand up?

Ventricular twisting, essential for cardiac function, is attributed to the contraction of myocardial helical fibers. The exact relationship between ventricular anatomy and function remains to be determined, but one commonly used explanatory model is the helical ventricular myocardial band (HVMB) model of Torrent-Guasp. This model has been successful in explaining many aspects of ventricular function, (Torrent-Guasp et al. Eur. J. Cardiothorac. Surg., 25, 376, 2004; Buckberg et al. Eur. J. Cardiothorac. Surg., 47, 587, 2015; Buckberg et al. Eur. J. Cardiothorac. Surg. 47, 778, 2015) but the model ignores important aspects of ventricular anatomy and should probably be replaced. The purpose of this review is to compare the HVMB model with a different model (nested layers). A complication when interpreting experimental observations that relate anatomy to function is that, in the myocardium, shortening does not always imply activation and lengthening does not always imply inactivation