Study of the Murine Cardiac Mechanical Function Using Magnetic Resonance Imaging : The Current Status , Challenges , and Future Perspectives

© 2012 Constantinides, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Study of the Murine Cardiac Mechanical Function Using Magnetic Resonance Imaging: The Current Status, Challenges, and Future Perspectives


Introduction
While cardiac mechanical function studies initially focused on large mammals and the human, the mouse emerged as the preferred animal species for such research in recent years [Collins 2003]. Despite the fact that evidence supports that bio-energetically and hemodynamically the mouse scales in a linear fashion with larger mammals and humans [Dobson 1995, Nielsen 1958], nevertheless, important physiological questions still remain , Balaban 2001] on whether such a model is the most appropriate for extrapolation of conclusions to man [Schaper 1998, Balaban 2012]. With the complete characterization of the mouse and human genomes (a National Institutes of Health initiative) in 2002 and 2003 respectively [Collins 2003, Gregory 2002], a plethora of mouse studies emerged targeting the cardiovascular system in animals with genetic modifications [James 1998, Hoit 2001, Gehmann 2000, Ehmke 2003], marking the onset of the molecular physiology, proteomics, and (structural and functional) genomics era. Collectively, these studies [Milano 1994, Barbee 1994, MacGowan 2001 initially targeted six important areas of cardiac function including the: (a) excitation-contraction cascade; (b) the beta-adrenergic system; (c) the cytosolic/structural system and the cytoskeleton; (d) the extracellular matrix and its coupling to important cytosolic elements that assist the mechanical force generation or propagation; (e) molecules that determine spatial-temporal mechanical changes (due to differential gene expression, phosphorylation, or recruitment of fetal development gene programs); and (f) the energetic-metabolic status of the muscle. Equally important in most of these studies was the non-invasive imaging of such animals for phenotypic and genotypic screening, often conducted under inhalational anesthesia [Erhart 1984, Hart 2001, Price 1980, Kober 2005, Constantinides_ILAR 2011].

MRI of the mouse: Challenges for cardiac image-based mouse phenotyping 2.1. The mouse as a research model
The mouse emerged as an attractive animal research model following the rapid advances in experimental molecular biology techniques that allowed targeted mutagenesis in single genes [Capecchi 1989], in addition to the tremendous success for extraction, manipulation, and use of embryonic stem cells [Koller 1989]. Practical and ethical advantages were also associated with mice, such as their stable genetic lines, immune system, short gestation periods, low cost, and ease of use. Initial research strides were supported by US National initiatives including the Human and Mouse Genome projects administered through the National Institutes of Health (NIH) for cloning and mapping the entire human and mouse genomes [Collins 2003, Gregory 2002, efforts that were successfully completed in 2003.

Genetic background and gene homology
Through various structural genomics attempts to map and compare human and mouse genomes [Schaper 1998, Gregory 2002, an overall homology of more than 85% and an identity of more than 80% were recorded [Schaper 1998], suggestive of the increased conservation during differentiation of the two species. Based on such findings, it is shown that the homeobox (hox) genes (transcription factors responsible for development), other transcription factors that bind to promoters encoding acute phase genes, and heat shock proteins, exhibit increased identity with humans. Based on Schaper et al. [Schaper 1998], however, structural genomics shows that (except primates) all other species (including mouse, rat, horse, bovine, etc) are equidistant from man, and evidence is thus inconclusive for the choice of the best species (based only on genetic homology and protein identity) for assessment of cardiovascular function and dysfunction.

Developmental and morphological differences between mouse and human Cardiac dimensions
Indicatively, the human heart weighs around 250-300 g (left and right ventricular [RV, LV] and atrial chambers), and has an intrinsic rhythm of around 60-70 beats per minute (bpm), while the murine heart has a weight of 0.2-0.3 g and beats at a rate of 600-700 bpm. Additionally, the most evident external morphological difference between the adult mouse heart and the human heart is its shape and size ( Figure 2). Developmental differences also exist; human development starts during the 3 rd week of gestation and lasts approximately 7 months to complete, while mouse cardiac development spans a little more than 2 weeks. At human birth, most of the cardiac organ development has been completed while in the infant mouse cardiac development may still be in progress.

Cardiac anatomy
No major differences exist in the outer morphology, ventricular structure, or valves. Noted differences exist, however, in atrial and venous parts. Specifically, the left superior caval vein drains directly into the right atrium in the mouse, whereas the pulmonary vein has an opening in the left atrium [Doevendans 1998], and a secondary atrial septum is lacking [Webb 1996]. While availability of data on the cardiomyofiber structure is scarce, a prior publication [McLean 1992] indicates some fiber orientation differences, primarily on the middle layer of the myocardium. Prominent differences also exist for the conduction system and coronary anatomy. In contrast to the human where the sino-atrial (SA) node is distinctly located on the right atrium, in the mouse it lies on the superior vena cava (SVC), at the juncture of the SVC with the right atrium. Coronary anatomy exhibits branching of a septal coronary artery from the left coronary system supplying the left anterior ventricular wall, while the right coronary artery branches into a right coronary and a circumflex vessel supplying the posterior ventricular wall [Doevendans 1998].

Cellular size and content
Cardiomyocytes are reported to have lengths of approximately 80-100 μm along their major axis, and lengths of 10-20 μm along their minor axis [Doevendans 1998, Smaill 1991. Eightyfive percent (85%) of the total number of cardiac cells (amounting to 7-10 million approximately) are interstitial while the remaining 15% that represent cardiomyocytes occupy 90% of the available total ventricular tissue volume [Doevendans 1998]. Also important is an increased capillary to fiber ratio (of the order of 1.4) in the mouse, a direct necessity for the higher energetic and metabolic demands compared to rats, canine, or humans, (with a fiber ratio of the order of 1-1.1) [Sabbah 1995, Przylenk 1983, Olivetti 1989, Doevendans 1998]. The duration of the cell cycle (once G1 is entered) is reported to be almost the same in mouse and man [Schaper 1998].

Functional physiological differences Cardiovascular metabolism
Energetic requirements in biological organisms are often assessed by their oxidative capacity, myosin ATPase and SERCA activities [Blank 1989] with a commensurate increased demand in small animals, such as rodents. In compensation of such increased energetic demand, an increased volume density of mitochondria is reported by Doevendans et al. [Doevendans 1998] in mice (37.9%) compared to humans (25.3%), with similar myofibril density (approximately 50%). However, recent findings by Phillips et al. [Phillips 2012] argue in favor of a constant density of mitochondria across species (approximately 21%), yet an increased enzymatic activity of mitochondrial enzymes in mice and a smaller dynamic range of metabolism is reported. Also of interest are the relative patterns of cardiac myosin chain isoforms (αα, ββ, αβ), a mouse αα-isoform compared to a human-ββ isoform dominance [Sheuer 1979], and a developmental switch in mice compared to humans, resulting in higher myosin ATPase activity. Noteworthy and more important is the increased basal activity of metabolic enzymes (Complex V) [Phillips 2012] and the smaller energetic reserve in mice compared to humans [Blank 1989, Phillips 2012], raising concerns for the appropriateness of the mouse as a proper model for comparative pharmacologic (doputamine, dipyridamole) stress studies in ischemia-infarction-reperfusion models, compared to human disease [van Rugge 1992, Wiesmann_Circ_Res 2001, Williams 2001].

Perfusion, angiogenesis-collateral flow, coronary reserve
Based on the extensive number of literature publications that focused on the elucidation of the basic principles of cardiovascular physiology (during the early and latter half of the 20 th century), including the autoregulation of blood flow in rats and canine [Feng 2001, Sandgaard 2002], important differences exist in mouse and man with regard to perfusion, capillary density [Stoker 1982, Rakusan 1994, total blood volume (2.5 ml in the mouse compared to 400-500 ml in humans), relative distribution of blood flow in the capillary bed and redistribution capability subject to stimuli (temperature, anesthesia) [Barbee 1992, Rosenblum 1997, Sarin 1990, angiogenetic capacity and formation of collateral vessels, as well as the resting coronary reserve, as factors that primarily project to ischemia-reperfusion studies (or other cardiovascular pathology models) and comparative analyses between pigs, canine, and man.

In vivo cardiac function
While direct comparative studies between mouse and man are still few, nevertheless published results favor similar hemodynamic and global cardiac functional indices, including blood pressure , inotropic (ejection fraction [EF], maximum developed ventricular pressure rates -dP/dtmax, stroke work (SW), preload adjusted maximum power (PAMP), Preload recruitable stroke work (PRSW), end-systolic elastance (ES), and the end-systolic pressure volume relationship) and lusitropic (minimum developed ventricular pressure rates -dP/dtmin, Weiss and Glantz relaxation constants, and the end-diastolic pressure-volume relationship), left ventricular contractile indices [Constantinides_ABME 2011, Doevendans 1998]. Recent MRI findings also support similar torsional patterns, as reported by normalized (to left ventricular lengths) twist and torsional angles [Henson 2000, Zhou 2003, Liu 2006, Zhong 2010, however, limited and less steep responses in the force-frequency relationship-response of the mouse compared to humans [Stull 2002] refers to altered calcium kinetics [Stuyvers 1997] and handling under stress.

Integrative physiological control
In consideration of the various morphological and anatomical comparisons listed, the murine cardiovascular system (under normal physiological conditions) resembles relatively closely evoked responses in larger mammals. As mentioned, numerous cardiovascular indices in mice match corresponding values in rats and humans, predicted by allometric scaling laws.
At the integrative physiological level, the major blood pressure regulating systems, namely the baroreceptor reflex [Ma 2002] and the renin-angiotensin system (regulating electrolyte balance) [Cholewa 2001] seem to resemble closely those in larger mammals.

Allometric scaling of function, energetics, and metabolism
Overall, under physiological conditions, evidence supports that bioenergetically and hemodynamically the mouse scales linearly with larger mammals and humans [Dobson 1995, Nielsen 1958, Phillips 2012, exhibiting a similar maximal aerobic capacity across species [Phillips 2012]. Preliminary evidence for allometric scaling to heart size in mechanical kinematic performance has also been presented [Popovic 2005, Zhong 2010 supported by preliminary comparative mouse-human results in this present work.
Given all considerations, extrapolations of inferences from mice to man is appropriate under physiological conditions, however, similar attempts in pathological models or states may be indeed risky.

The impact of anesthesia for mouse studies
Even if advances in telemetric techniques have allowed the pursuit of mouse studies under conscious conditions to a large extent, most of the research (terminal, invasive, or noninvasive imaging) studies utilize anesthetics. Anesthetics, however, are known to cause severe cardio-depression [Hart 2001] with adverse physiological effects on hormonal release, centrally to the heart and peripherally to the vasculature [Price 1980, Ohnishi 1974 at the cellular level, affecting calcium entry through L-type Ca 2+ channels, the calcium binding sensitivity of the contractile proteins to calcium, on conduction and excitability, and possibly on other sarcoplasmic reticular sites [Price 1980]. Also prominent are effects on the central and peripheral nervous system, but most notable are effects on the metabolism (through mitochondrial vasomotor changes in coronary circulation and perfusion, vasodilation, and blood flow changes in the microvasculature [Kober 2005], possibly synergistic to Nitric Oxide), and the decoupling of oxidative phosphorylation manifested through myocardial oxygen consumption changes.

Mechanisms of anesthesia action
Linus Pauling [Pauling 1952] was one of the first scientists to attempt to explain the molecular and cellular mechanisms of action of anesthetics. Nevertheless, his proposed theory on clathrate formation post-anesthesia administration, proved inaccurate. With recent advances in molecular biology, new published evidence justifies the prominent role of the cellular chlorine channel as a mediator for anesthesia induction and maintenance [Brunson 2008, Maze 2008]. Specifically, hyperpolarizing chlorine channel currents lead to inhibition of cellular excitability and hypnotic action. It appears that one of the major hypnotic action centers is the locus coeruleus in the central nervous system, with mediatory action referred to the α2-adrenergic receptors via cyclic-AMP-mediated transduction pathways.
At the integrative physiological level, the effects of anesthesia target multiple cellular sites ( Figure 3) and thereby often lead to cardio-depression. Major effects include their potency in inducing vasodilation of both the cerebral and coronary vasculature [Toyama 2004] leading to increased perfusion. A rapid hyperglycemic effect is often expressed (primarily via the sympathetic nervous system innervating the liver) causing immediate hormonal (catecholamine) release from the adrenal medulla [Durand 2009]. Glucose metabolic rates are also down-regulated (via inhibition of ATP synthesis in mitochondria), eventually leading to impairment of glucose tolerance (mediated via enzymatic protein activity in the liver). Also observed is an eventual oxidative phosphorylation decoupling.

Types of anesthesia
Widely used anesthetics for animal research are categorized into injectable (such as Ketamine/Xylazine, propofol, lidocaine, nembutal) and inhalational (such as isoflurane, halothane, and sevoflurane). Hedlund et al. [Hedlund 2008] and Brunson et al. [Brunson 2008] have published excellent summaries of the different types of anesthetics used for rodents to which the reader is referred.

Basal physiology and maintenance
While anesthesia usage causes physiological changes [Hildebrandt 2008], proper protocols and administration methodologies (type, dose) can achieve optimal conditions of animal study, maintain animal stability and homeostasis, and minimize time-induced accumulated effects, especially for prolonged imaging studies often exceeding 45 minutes in duration. Apart from physiological indices of hormonal release, respiration and metabolism, carefully controlled cardiac indices (such as heart rate, ejection fraction, arterial pressure, and heart variability) ensure proper conditions of study of the cardiovascular system avoiding detrimental hypotension-induced blood volume changes, metabolic and contractile downregulation, and arrhythmogenicity. Heart variability (HRV) analyses have also been applied for phenotypic screening of transgenic mice, study of heart rhythm mediators through signaling pathways as well as the effects of pharmacologic intervention on intrinsic heart rhythm and arrythmogenesis [Thireau 1997, Bernston 1997, Gehrmann 2000]. Standardization of HRV analyses tools for mice have, however, been limited due to the numerous data acquisition types and analysis techniques employed. Most importantly, variability of HRV is dependent on the type of anesthesia being used, that may also be further influenced by anesthetic balancing agents such as nitrous oxide (N2O), medical air, and oxygen. Interpretation of HRV results has also been difficult [Hoit 2004], due to their dependence on a number of factors including aging, posture, circadian variability, and the duration of the sampling periods used, with recent evidence supporting the fact that the primary contribution of autonomic activity in the mouse is due to the sympathetic tone, in contrast to the parasympathetic vagus contribution , Janssen 2000, Constantinides_IEEE 2010].   Despite such previous work, the extent of the contribution of sympathetic and parasympathetic tone to heart rate (HR) and HRV in the mouse is still a matter of debate. Recent, carefully controlled studies based on the level of anesthesia and additional factors that relate to animal preparation indicate that overall, HRV is expected to be generally lower during anesthesia in comparison to the conscious state. Indicative in HRV is an SDNN reduction under FiO2 conditions at 50% but also a noted increase in the standard deviation of averages of all normal R-R intervals (SDNN) and the square root of mean squared differences between adjacent normal R-R intervals (RMSSD) as a result of N2O administration. Variation of FiO2 seems to result in prominent effects only at specific levels (50, 100%). Since HRV indices are determined by an inter-play between both mechanical and neural factors, the exact mechanisms responsible for this effect are still unknown. Overall, the major practical benefit is that the protocol described for physiological studies of mice under anesthesia has the potential for high reproducibility in diagnostic modalities including MRI, microCT, ultrasound, and microPET. Elicited results show that the optimal ISO anesthetic regimen for mice is a dose of approximately

RMSSD [ms]
N2O=25% N2O=50% and 75-50% N 2 O. However, despite the optimization of murine physiological conditions under anesthesia, it is yet not possible from this study to determine whether the mechanism of action involves transient sympathetic activation, steroid release, a direct effect of ISO, or a combination of such effects. The overall effects of ISO may be the result of opposing vasodilatory and vasoconstrictive effects either directly (vasodilation) or secondary to an anesthesia-induced decrease of the heart's metabolic demand. Surely, the major limitation of all studies under anesthesia is that a noted cardio-depressive effect on basic cardiovascular function exists, compared to the conscious state.

MRI cardiac imaging: The current status and future perspectives
Mouse cardiac MRI emerged as a logical consequence to the human and mouse genome mapping initiatives and parallel developments and advances in human cardiac MRI in the late 1980's and early 1990's, focusing on establishing MRI as the 'one-stop-shop' in clinical practice. Correspondingly, technological advances and developments of novel hardware and pulse sequences were fairly limited, merely revolving on the scalability of existing technology to match smaller field-of-view acquisitions for mouse and rat imaging. Manning et al. [Manning 1990] and Shapiro [Shapiro 1994] first reported cardiac MRI-based LV mass estimations, and Siri et al. [Siri 1997] the first mouse cardiac imaging results from a 9.4 T system. Subsequent to such early studies were the first quantitative studies of function ] and myocardial mass in diseased mice post-hypertrophy induction using the βadrenergic agonist isoproterenol (ISO) [Slawson 1998]. Despite attempts early on to utilize conventional 1.5T clinical systems to conduct mouse cardiac MRI [Franco 1998], it was soon realized that dedicated, high field, high resolution mouse systems were necessary for imaging, a realization that drove technological evolution and migration to field strengths of 4.7-11.7 T systems ].
Collectively, efforts over the last 14 years targeted the study of global   [Osman 1999, Kuijer 2001]. This section discusses some of the fundamental aspects of mouse preparation, positioning, physiology and its maintenance during MRI, advances in hardware and pulse sequence acquisitions, image processing techniques, and global and regional cardiac phenotyping, as these are complemented with recent findings from our group.

Mouse physiology -Maintenance and stability during MRI
Even if physiological protocols are easier to optimize on the bench, murine cardiac MRI imposes additional stringent challenges that relate to the animal's heart rate, pressure monitoring, and thermoregulation , Hedlund 2008, Constantinides_ILAR 2011. While most research sites are equipped with commercial mouse imaging systems, a number of adjustments often need to be implemented to ensure proper physiological maintenance (including, but not limited to, computer controlled ECG, breathing, temperature monitoring systems and MR-compatible [fiber-optic and other] devices) [ Figure  6]. Often specially designed negative-feedback air-flow systems are interfaced with closedbore scanner systems to facilitate fast and efficient bore air-heating, compensating for the lower bore temperatures due to the cryogenic environment of the magnet and gradient coils.
Proper mouse positioning almost always requires specially designed cradles to fit imaging probes (often constructed on specially designed casings that fit in fast-switching, high-slew rate gradient inserts) and scanner high-performance RF coil and gradient inserts.
Mouse cradle designs have evolved in complexity over the past 14 years and include those custom-made and commercially available types. Most of them are customized to allow mouse placement in prone and supine positions, placement of non-magnetic metallic or carbon-fiber ECG electrodes on the front paws and limbs, a rectal probe and an inflated air-bellow, for temperature and respiration monitoring [ Figure 6]. Also of importance is the administration of anesthesia gases, often accomplished via a nose cone (with mice being obligatory nostril breathers), with tubing that connects to a vaporized-flowmeter device system (freely breathing [Schwartz 2000

High field magnets
While gradient and RF coil technology spans numerous decades of history (as early as Paul Lauterbur's ground-breaking inception of zeugmatography [Lauterbur 1973]), the stringent technical requirements imposed by the mouse physiology for cardiac imaging have led to optimization of prior technology or to recent introductions of new technologies, as reviewed and discussed briefly in this section. Interestingly, large-bore high-field systems emerged during the past decade at field strengths spanning 4.7, 7.1, 9.4, and 11.7T maintaining, however, the same design technology as originally developed for human superconducting systems in the late 1980's and 1990's.

Gradient coil technology
The requirements for increased spatial resolution acquisitions (of orders matching cellular size of approximately 100-200 μm 3 ) led to fast switching, high-amplitude (up to 1000 mT/m), high-slew rate (up to 11250 mT/s) gradients (exhibiting linearity of better than ±3-5%/mm) for ex-vivo constructions of atlases [Johnson 2002] or in-vivo high-resolution isotropic cardiac imaging [Bucholz 2008, Perperidis 2011]. Often associated with extra inserts (of approximately 6-20 cm in inner diameter), such gradients impose further spatial restrictions in animal placement and monitoring during cardiac imaging. However modern, commercially available systems are often equipped with actuator-controlled manual or computerized cradle positioning systems.

RF coils
In a similar fashion to the evolution and migration of magnet and gradient technology from human to mouse applications, advances were noted in RF coils. Of all three hardware components (magnets, gradients, transmit/receive coils), RF coils received most of the Traditionally, RF surface coils reduce the spatial coverage (and hence the field-of-view) compared to volume coils, while maintaining higher local signal-to-ratio (SNR). The limitation for expansion of the spatial region of surface coil coverage (while maintaining or improving SNR), yielded to phased array designs, often in combination with circularlypolarized volume transmit coils. Introduction of alternative coils to phased array designs, such as the one shown presents one of the first attempts to ameliorate such a surface coil limitation, in consideration of the small spatial scales, and the complexity and cost of phased arrays. Additionally, the use of such a surface coil is not prohibitive in terms of its concurrent use with dedicated and specially designed transmit (or receive coil arrays).
Specifically, imaging results showed improved performance of the cylindrical spiral coil in comparison to the flat counterpart. The cylindrical coil has increased field of penetration that allows visualization of the entire lateral and inferior myocardial walls with adequate relative SNR (rSNR). Its performance compares well and outperforms its flat equivalent in septal, inferior, anterior, and lateral myocardial areas (rSNR improvement between 27 and 167%), despite its non-optimal placement and positioning on the mouse (anteriorly and laterally), in this particular case. Its design and response, that clearly exploits the additive effect of the transverse B1-field component, also compares favorably with a commercially available birdcage coil within the region where the mouse heart resides. However, the birdcage coil exhibits the best performance associated with three to five times higher rSNR values over the entire left ventricular myocardial regions. Promising are anticipated uses and applications of new hyperpolarized cryostat volume (birdcage) coils that have recently become commercially available [Kovacs 2005].
The fact that the constructed coils are transceiver surface resonators, certainly presents a conceptual and practical limitation for their wide-spread use, in the context of inhomogeneous excitation and their ability to allow quantitative cardiac mouse imaging. Direct use of such coils for high field mouse cardiac imaging is likely to result in increased Study of the Murine Cardiac Mechanical Function Using Magnetic Resonance Imaging: The Current Status, Challenges, and Future Perspectives 359 loading effects between the RF and gradient coil inserts, imposing the need for use of specially constructed isolation shields. The inhomogeneous response of such coils, obviously translates to issues associated with artifacts, imperfect application of magnetization preparation pulses, or biased quantitative measures in cardiac functional and perfusion imaging. Furthermore, flip-angle sensitive techniques, such as fat saturation in cardiac MRI cannot be applied, due to the inherent variations in the flip angle spatial distribution, thereby limiting their potential applications.

Conventional and new imaging methodologies for mouse cardiac imaging
Similar to hardware developments numerous pulse acquisition schemes migrated from human cardiac applications to the mouse. Nevertheless, a number of new techniques emerged over the years that include cartesian (rectilinear) and radial (spiral, twisted projection, other) data acquisition schemes for fast mouse cardiac imaging. A brief review and analysis of the most-successful imaging and reconstruction practices (fast Fourier and non-uniform data regridding-Fourier transformations) is attempted below.

Cartesian k-space pulse sequence acquisitions
Two were the major types of Cartesian imaging acquisition techniques that migrated from prior human MRI efforts, extensively used for mouse cardiac imaging. They are both gradient echo sequences and adhere to lexicographic k-space or rectilinear sampling and are based on spin-warp imaging. The first, a steady state incoherent pulse sequence, was developed by Haase [Haase 1986] and became known as Fast Low Angle Shot and was dubbed with the acronym FLASH (equivalent to Spoiled Gradient Recalled Imaging, later developed by General Electric). The second type, a steady state coherent sequence known as Steady State Free Precession (SSFP) was developed early on by Carr [Carr 1958], employing ultra-short repetition times (TR) for imaging later adopted in an equivalent sequence with gradients by Hinshaw [Hinshaw 1976 . Navigator echoes may also be used prior to the gating pulse with a number of preparation pulses (pre-pulses) often executed immediately after the trigger pulse ( Figure 8). Such optional pulses may include fat suppression and spectral-spatial pulses for black-blood imaging and/or fat suppression, as well as other prep-pulses including inversion, DENSE, spin-tagging and others.

Spiral or non-cartesian k-space pulse sequences
The ultrafast mouse heart rates, even under anesthesia, necessitate fast imaging acquisitions (within tRR=100-120 ms). While Cartesian sampling schemes cover k-space adequately nevertheless, non-rectilinear and spiral k-space acquisitions have gained tremendous interest recently as efficient and fast sampling schemes. Radial acquisition variants by Bucholz et al. [Bucholz 2008], and STEAM-based DENSE encoding followed by interleaved spiral acquisitions proposed by Zhong [Zhong 2010] (Figure 9) are also adopted for mouse cardiac functional imaging.   In summary, numerous practical benefits are associated with mouse cardiac MR imaging, including the non-invasive nature of the technique, the inherent capability to map cardiac morphology and function, for both LV and RV chambers, and their motional patterns. High spatial and temporal resolution imaging can thus be achieved, through execution of highthroughput protocols, yielding direct, accurate estimates of global and regional indices of cardiac function, avoiding any assumptions whatsoever or model-based derivation approaches endorsed by other imaging techniques such as ultrasound.
Despite the extensive use of cartesian imaging with adequate SNR performance and spatial resolution (using FLASH, SSFP, or FISP), 3D acquisition studies maybe more efficiently completed using radial or spiral imaging sequences, especially for dynamic cardiac imaging (with pharmacologic interventions or contrast agent infusions). Nevertheless important and critical drawbacks are associated with such sequences, including the necessity to maintain data density as sampling extends to outer k-space regions, the convoluted and complex reconstructions (often associated with data re-gridding, kernel de-convolution, filtering, and inverse fourier transformation), and inherently lower SNR performance than rectilinear imaging. Thus, the choice between cartesian and radial imaging reduces to a tradeoff between SNR, spatial resolution, and efficiency of data sampling for 3D coverage.  Stuckey 2012]. Critical to successful phenotypic screening of mouse models of the cardiovascular system using MRI are highly efficient four-dimensional (4D) acquisition ex-vivo and [Zamyadi 2010] in-vivo protocols [Bucholz 2010]. Such protocols ought to span the scales of the embryo to the adult, fully-developed mouse, and ought to lead to the reduction of the computational image processing complexity for accurate quantification of motion, global and regional cardiac function, strain, elasticity, and others.   Correspondingly, estimated cardiac volumes can easily be computed using standard image processing tools and converted to absolute volume units using the voxel dimension and the myocardial tissue density. Hemodynamic indices such as end-diastolic (EDV) and end-B A systolic volumes (ESV), stroke volume (SV), cardiac output (CO), and ejection fractions (EF) can now be routinely calculated according to standard cardiac mechanical functional relations [Constantinides_SBI 2009], based on CINE-MRI ( Figure 11). CINE based local contractile function, including estimation of wall thickness, motion and fractional shortening, indicators of systolic function and long-term prognostic biomarkers in dysfunction remain the gold-standard for assessment of motional patterns and cardiac function in small animals due to the excellent soft tissue contrast of MRI and its high spatial and temporal resolution (Figure 12).

Interstrain morphological and 4D motional variability -Statistical altases
The advances in high-field, high-resolution cardiac imaging techniques, have also allowed the development of atlas-based approaches for the description of anatomical structures and their function [Ali 2005, Sharief 2008] in normal , Bucholz 1998, Zamyadi 2010] and transgenic mice [Chien 2000, Epstein 2007]. Completion of the sequencing of the mouse genome has led to increased requirements for identifying the specific phenotype Study of the Murine Cardiac Mechanical Function Using Magnetic Resonance Imaging: The Current Status, Challenges, and Future Perspectives 365 elicited post-transgenic modifications and the genetic basis of pathology [Henkelman 2010]. Therefore mouse imaging, combined with probabilistic, statistical atlas constructions and morphometric methods [Zamayadi 2010], is envisaged to play an increasing role in imagebased phenotyping and gene expression mapping of genetically altered mice [Ng 2010] in the future. Construction of probabilistic and statistical atlases [Perperidis 2011] can potentially enable the study of murine, global cardiac structure and function with increased quantitative accuracy, identifying modal components of shape variability (from embryogenesis to adulthood), and disseminating components of global mechanical motion. Similar to existing cardiac atlases [Perperidis 1995, Hoogendoorn 2007], these can be population-based instead of single-subject. Prior efforts have focused on modeling cardiac anatomy in humans [Helm 2006] but only limited attempts have been made to construct accurate, high-spatial and hightemporal resolution computerized atlases for mice [Perperidis 2011]. Despite multiple prior efforts with construction of human brain atlases [Young 2009], Frangi et al. [Frangi 2002], Mitchell et al. [Mitchell 2002], and Lotjonen et al. [Lotjonen 2004] were the first to develop human ventricular statistical shape models. More recently Ordas et al. ] developed a computational atlas of the entire heart using registration-based techniques. Perperidis et al. [Perperidis 1995, Perperidis 2005] and Hoogendoorn et al. [Hoogendoorn 2007] proposed 4D spatio-temporal human cardiac probability atlases from (MRI), while Beg et al. [Beg 2004] developed a large deformation diffeomorphic mapping (LDDM) for construction of cardiac statistical atlases. As an extension to Beg's work, Helm et al. [Helm 2006] employed LDMM to achieve inter-subject registration to a reference anatomical template to compare cardiac geometric variability using Principle Component Analysis (PCA) from diffusion tensor MRI in normal and failing human hearts.

Manual and semi-automated segmentation and registration approaches
Critical to successful constructions of atlases and to the transformation of constructed surface models to the finite element models (for subsequent computational work of mechanical function), are efficient and accurate segmentation and registration techniques (Figures 13, 14). Using recently developed techniques in our group [Perperidis 2011], a segmented (template) reference MRI sequence is selected from the mouse database consisting of imaged anatomic structures (left and right ventricular myocardium, left and right ventricular blood pools, and papillary muscles) as representative sets of typical anatomic objects of interest. This template is subsequently used to register population images via global and local non-rigid transformations. Although such high-dimensional transformation differs from established one-to-one (invertible) diffeomorphic transformations [Helm 2006], its performance is comparable in accuracy, precision, simplicity, and computational intensity, as recently reported by a multi-center quantitative ranking of 14 different non-linear deformation algorithms [Klein 2009]. Figure 13 show examples of manual, user-based spline segmentation and subsequent correction for left and right ventricular feature extraction, as discussed previously [Perperidis 2011]. Implementation of a global and local registration scheme is also diagrammatically summarized in Figure 14   Manual, image-based techniques seem to provide the best avenue for segmentation (despite their inherent intra-and inter-observer variability inaccuracies).
Certainly, atlas-based approaches are envisaged to be of tremendous benefit and value for cardiac phenotyping characterization in the upcoming years, aiding spatial mapping of gene expression using novel cellular and sub-cellular probes and markers, understanding embryogenesis and development, accurately mapping strain-, age-, and sex-based morphological dependencies, quantifying patterns of motional variability, and efficiently screening cardiac functional changes based on semi-automatic template segmentation techniques for efficient estimation of hemodynamic indices of function, and high throughput phenotypic screening.  Despite its potential usefulness, the major limitations of such a technique is associated with the significant image post-processing, the need for accurate registration methods (often a challenging task for cardiac datasets), the inherent assumptions associated with the normality of distribution of independent datasets, and confounding factors on attempts to analyze motion and variability (often as a direct modulation of anesthesia effects).

Interstrain comparisons of global cardiac function
Apart from early attempts ], little work has been published on interstrain comparisons of global cardiac functional differences in various mouse strains [Constantinides_SBI 2010, Bucholz 2010]. Instead, studies of cardiac dysfunction have often included quantitative comparisons with normal control or sham mice ].
Interstrain variability on cardiac function exists in different mouse strains, which may be dependent or independent of strain, but there are certainly developmental factors and other factors that ought to be investigated. Reported results must thus be carefully considered for age, weight, sex, and genetic background. In recent findings from our group ( Figure 15) no significant variations in cardiac function were observed from age-, sex-matched (male), normal C57BL/6J and DBA/2J mice. Image-derived hemodynamic indices of function reported in Figure 16 exemplify similar cardiac functional responses from both the right and left ventricular cavities, in agreement with prior reports ].

Quantification of regional cardiac function -Comparison of mouse and human
Despite the usefulness of global cardiac index comparisons, the value and importance of regional cardiac functional analyses in disease is paramount. Further to the use of tagging [Liu 2006] and DENSE , Zhong 2010] as non-invasive techniques for assessment of regional cardiac displacement and strain in mice, similar patterns of motional responses were observed in mice and humans with noted finite, but distinct, differences. Particularly, Gilson ] reports basal displacement but almost no apical displacements in the mouse during the cardiac cycle, in comparison to both apical and basal motion in humans [Moore 2000]. Furthermore, circumferential and radial displacements seem to scale proportionally with values previously reported in humans [Constantinides_Phantom 2012]. Finite differences may exist but careful consideration of other factors (such as age, sex, or anesthesia effects) must also be considered in such analyses. Apart from tissue displacement encoding techniques other motion tracking [Osman 1999], velocity [Streif 2003], and acceleration techniques [Staehle 2011] have been reported.
Despite the importance of all such techniques they nevertheless necessitate access to dedicated high-field MRI scanners and invariably require development, or use, of complex algorithms and reconstruction software. An easier methodology to assess regional cardiac function for comparison with humans was recently reported by Constantinides Based on such analyses, bullseye-plots of regional cardiac function were generated in 17sector representations of the murine and human hearts (from independent studies in two separate Institutions) showing similar patterns in transmural variations in wall motion and thickness, and regional ejection fraction ( Figure 18) in mouse and man. Such spatial patterns observed for mouse and human (in agreement with prior tagging work [Moore 2000]) are supported by two-tailed paired t-tests indicating absence of statistical significant differences in the mean values of wall thickness (p=0.07), wall motion (p=0.051), or regional EF (p=0.065) at the 1% significance. Repeated measures ANOVA indicated significant differences in regional mouse and human for wall thickness (p=0.002) and regional EF (p<0.0001) and insignificant differences for wall motion (p=0.016) at the 1% significance. However, despite the similarity in such patterns, quantification of global and regional functional indices (Table 2) shows distinct, finite differences, in agreement with prior reports. Unknown at this stage is whether such differences can be attributed to species variability or endogenous or exogenous parameter dependencies, as they relate to the conduct of such studies and data analyses, or the modus operandi of the human and murine hearts. Figure 17. (Left to right) Epicardial and endocardial LV contour definition in mouse long and shortaxis MRI, and 3D LV blood cavity segmentation; 3D ventriculogram rendition using Vitrea from murine MRI; short axis human MRI, and Vitrea reconstruction of wall motion, wall thickening, and regional ejection fraction from a typical mouse dataset.
Therefore, numerous practical benefits are associated with dedicated, state-of-the-art mouse cardiac MR imaging, including the non-invasive nature of the techniques, the inherent capability to map cardiac morphology and function, for both LV and RV chambers, and their motional patterns. High spatial and temporal resolution imaging can thus be achieved, through execution of high-throughput protocols, yielding direct, accurate estimates of global and regional indices of cardiac function, avoiding any assumptions whatsoever or model-based derivation approaches endorsed by other imaging techniques such as ultrasound.
Study of the Murine Cardiac Mechanical Function Using Magnetic Resonance Imaging: The Current Status, Challenges, and Future Perspectives 371 Figure 18. (Left, middle) Regional parameter quantification and comparisons in the mouse and the human using VITREA. Mean bullseye plots for wall motion, wall thickening, and regional ejection fraction for C57BL/6J mice (n=9) and human (n=8) datasets over the entire cardiac cycle. Schematic diagram of sectoral representation of the heart according to AHA guidelines; (right) Regional cardiac performance inter-comparison of mouse and human, including wall thickness, wall motion, and rEF variation in the various sectors of the murine and human hearts (sector 17 is excluded from presented results).
As an extension of the development and use of such techniques (DENSE, tagging, HARP) has been the tremendous value for regional cardiac functional quantification and direct applicability to transgenic mice and in pathological states (myocardial infarction, heart failure).
Such approaches are, however, associated with a number of limitations and drawbacks, including the necessity for use of complex algorithms and laborious data post-processing (tagging, HARP, and DENSE), the inherently low spatial resolution for strain quantification (tagging vs. DENSE), the T1-tag dependence, the low SNR performance of DENSE (often with a temporal decreasing dependence following ECG-triggering), and the necessity to eliminate the anti-echo and free-induction decay signals in DENSE through proper acquisition adjustments and/or image subtractions.  Table 2. Summary of mean global and regional cardiac mechanical functional indices (±sd) of murine and human myocardium.

Future perspectives
In the new era of molecular imaging, MRI faces major challenges in accomplishing detection of molecular probes with increased sensitivity and specificity, comparable to other diagnostic techniques (such as PET/SPECT). Currently, 10-100 μmolar sensitivity is attained by MRI in contrast to the established nano-and picomolar sensitivity of PET and SPECT. While a new generation of contrast agents is anticipated to extend current limits shedding new light into cellular and molecular mechanisms, current efforts focus primarily on stem cell technology, cellular tracking, and construction of hybrid PET/MRI systems, as discussed below.

Regenerative techniques -Stem cell technology and cellular tracking
Advances in the biology of stem cells have evoked great interest in cell replacement therapies for the regeneration of heart tissue after myocardial infarction. Despite the initial controversial results from human trials [Rosenzweig 2006] due to the uncertain and unclear long-term fate, target-destination of the injected cells, their engraftment and viability, scientific interest and excitement remains high.
Employing high-resolution MRI in association with metabolism, iron-oxide labeled cells [Stuckey 2006] can be tracked and visualized thereby monitoring their migration patterns and ultimate engraftment fate in tissues of interest.

Hybrid system imaging
The superb ability of PET to detect ligand-receptor binding at the nano-to picomolar concentrations and the excellent spatial resolution of MR imaging, have stimulated efforts for the construction of hybrid PET/MRI systems. While the first prototype systems have already been completed [Herzog 2010, Pichler 2008], it will be of interest to see if corporate interest will aid establish such hybrid imaging techniques as tools of the arsenal of other diagnostic tools for clinical practice and for basic science research.

Conclusion
In the short-lived period of mouse cardiac MRI of the past 15 years, tremendous strides have been made for image-based phenotyping of the cardiovascular system. Such were realized in terms of the scalability of equipment, ease of handling and maintaining animals under proper physiological homeostatic conditions, adaptation of conventional imaging techniques, and inception of new, fast imaging acquisition schemes that have revolutionized cardiac, image-based phenotyping with efficient, high-throughput imaging protocols.
In conclusion, despite the usefulness, practicality, and low costs associated with the study of the mouse, important genetic, developmental, morphological, and cardiovascular system differences exist between mouse and man especially as such are unmasked in pathological conditions or under stress. Physiological results indicate that the optimal ISO anesthetic regimen for mouse studies under anesthesia is approximately 1.5% v/v, yielding stable MAP and HR values comparable to those observed in the animal's conscious state, with a minute-tominute variability in MAP and HR of ≤11%. Based on such recordings, the optimal FiO2 appears to be 50%. The additional use of N2O was associated with higher and more stable values of MAP and HR (at a mixture of 25-50% O2 and 75-50% N2O). Arterial pH values are within the physiological range and varied between 7.20 and 7.43. ISO anesthesia at 1.5% v/v is also associated with mild hyperglycemia (+47%) whereas insulin levels remain unaltered. The protocol described for physiological studies of mice under anesthesia has the potential for high reproducibility in diagnostic modalities, including MRI, microCT, ultrasound, and microPET. This regimen can be useful in phenotypic screening and pharmacological studies of cardiac function in mice and can facilitate the transfer of such work to noninvasive imaging platforms, with tremendous potential for both basic science and translational research.
Basic MRI studies of murine global cardiac structure and function under optimal physiological conditions, in combination with PCA and other image processing techniques, can identify modal components of shape variability and disseminate components of global mechanical motion. Such atlases can be population-based instead of single-subject based and can serve as a powerful reference tool for morphological and functional inter-strain mouse studies, complementary to current ongoing efforts for image-based phenotyping that target the cardiovascular system. Based on constructed morphometric maps and atlases using principal component analysis in C57BL/6J, it is found that in probabilistic atlases, a gradient of probability exists for both strains in longitudinal locations from base to apex. Based on the statistical atlases, differences in size (49.8%), apical direction (15.6%), basal ventricular blood pool size (13.2%), and papillary muscle shape and position (17.2%) account for the most significant modes of shape variability for the left ventricle of the C57BL/6J mice. Correspondingly, for DBA mice, differences in left ventricular size and direction (67.4%), basal size (15.7%), and position of papillary muscles (16.8%) account for significant variability. These data reason in favor of existing variability in the apical location in both strains, a direct consequence of the heart's effort to re-establish the position of the apex in a consistent manner at end-diastole. Additionally, higher variability exists in DBA mice in the location of the papillary muscles, as well as in the epicardial areas of the left ventricle.
On the forefront of direct, high-field acquisitions using RF technologies with mouse cardiac MRI, the commercially available birdcage outperforms cylindrical spiral multi-turn surface coils in relative signal-to-ratio (SNR) by a factor of 3-5 times as assessed by experimental measurements, simulations, and experiments in free space, and under phantom and animal loading conditions. Nevertheless, quantitative comparison of the performance of the two spiral coil geometries in anterior, lateral, inferior, and septal regions of the murine heart yield maximum mean percentage rSNR increases to the order of 27-167% post-mortem (cylindrical compared to flat coil), values that are by far higher than previous designs of surface coils and comparable to receiver phased array performance.
Such hardware improvements, in association with fast radial pulse sequence acquisitions, may also lead to quantification of global and regional functional parameters in various mouse strains. While morphological differences appear only to relate to increased papillary muscle variability in the DBA/2J mice, nevertheless interstrain cardiac hemodynamics, based on dynamic cardiac MRI acquisitions, do not exhibit significant differences for neither the LV nor RV in C57BL/6J or DBA/2J mice. Comparative to previous reports of global functional indices [Bucholz 2010], similar mouse and human results were also observed. The work supports the validity of the hypothesis of functional scaling in mice and humans.