Multimodal Correlative Preclinical Whole Body Imaging and Segmentation.

Segmentation of anatomical structures and particularly abdominal organs is a fundamental problem for quantitative image analysis in preclinical research. This paper presents a novel approach for whole body segmentation of small animals in a multimodal setting of MR, CT and optical imaging. The algorithm integrates multiple imaging sequences into a machine learning framework, which generates supervoxels by an efficient hierarchical agglomerative strategy and utilizes multiple SVM-kNN classifiers each constrained by a heatmap prior region to compose the segmentation. We demonstrate results showing segmentation of mice images into several structures including the heart, lungs, liver, kidneys, stomach, vena cava, bladder, tumor, and skeleton structures. Experimental validation on a large set of mice and organs, indicated that our system outperforms alternative state of the art approaches. The system proposed can be generalized to various tissues and imaging modalities to produce automatic atlas-free segmentation, thereby enabling a wide range of applications in preclinical studies of small animal imaging.

MR Imaging-derived Oxygen-Hemoglobin Dissociation Curves and Fetal-Placental Oxygen-Hemoglobin Affinities.

Purpose To generate magnetic resonance (MR) imaging-derived, oxygen-hemoglobin dissociation curves and to map fetal-placental oxygen-hemoglobin affinity in pregnant mice noninvasively by combining blood oxygen level-dependent (BOLD) T2* and oxygen-weighted T1 contrast mechanisms under different respiration challenges. Materials and Methods All procedures were approved by the Weizmann Institutional Animal Care and Use Committee. Pregnant mice were analyzed with MR imaging at 9.4 T on embryonic days 14.5 (eight dams and 58 fetuses; imprinting control region ICR strain) and 17.5 (21 dams and 158 fetuses) under respiration challenges ranging from hyperoxia to hypoxia (10 levels of oxygenation, 100%-10%; total imaging time, 100 minutes). A shorter protocol with normoxia to hyperoxia was also performed (five levels of oxygenation, 20%-100%; total imaging time, 60 minutes). Fast spin-echo anatomic images were obtained, followed by sequential acquisition of three-dimensional gradient-echo T2*- and T1-weighted images. Automated registration was applied to align regions of interest of the entire placenta, fetal liver, and maternal liver. Results were compared by using a two-tailed unpaired Student t test. R1 and R2* values were derived for each tissue. MR imaging-based oxygen-hemoglobin dissociation curves were constructed by nonlinear least square fitting of 1 minus the change in R2*divided by R2*at baseline as a function of R1 to a sigmoid-shaped curve. The apparent P50 (oxygen tension at which hemoglobin is 50% saturated) value was derived from the curves, calculated as the R1 scaled value (x) at which the change in R2* divided by R2*at baseline scaled (y) equals 0.5. Results The apparent P50 values were significantly lower in fetal liver than in maternal liver for both gestation stages (day 14.5: 21% ± 5 [P = .04] and day 17.5: 41% ± 7 [P < .0001]). The placenta showed a reduction of 18% ± 4 in mean apparent P50 values from day 14.5 to day 17.5 (P = .003). Reproduction of the MR imaging-based oxygen-hemoglobin dissociation curves with a shorter protocol that excluded the hypoxic periods was demonstrated. Conclusion MR imaging-based oxygen-hemoglobin dissociation curves and oxygen-hemoglobin affinity information were derived for pregnant mice by using 9.4-T MR imaging, which suggests a potential to overcome the need for direct sampling of fetal or maternal blood. Online supplemental material is available for this article.

Genetic and Pharmacological Modulation of Akt1 for Improving Ovarian Graft Revascularization in a Mouse Model.

Ovarian tissue cryopreservation and transplantation is one of a few available treatments for fertility preservation in women diagnosed with cancer. Rapid revascularization is essential for reducing hypoxic damage after grafting and protecting the primordial follicles reserve. Using a mouse model of heterotopic ovarian graft transplantation, we have delineated the role of endothelial Akt1 expression using longitudinal magnetic resonance imaging follow-up to quantify angiogenic response. Endothelial Akt1 activation in ovarian grafts promoted angiogenesis to support the graft during posttransplantation hypoxic period. Similarly, simvastatin therapy activated Akt1 at the transplantation site and improved the revascularization and vascular support of ovarian grafts. These results serve as an important first step toward pharmacological intervention to improve revascularization of ovarian grafts and restoration of fertility in cancer survivors. The pro-angiogenic effects reported here may extend beyond improving ovarian graft reception in fertility preservation and could potentially be used for different organ or tissue transplantation.

Major mouse placental compartments revealed by diffusion-weighted MRI, contrast-enhanced MRI, and fluorescence imaging.

Mammalian models, and mouse studies in particular, play a central role in our understanding of placental development. Magnetic resonance imaging (MRI) could be a valuable tool to further these studies, providing both structural and functional information. As fluid dynamics throughout the placenta are driven by a variety of flow and diffusion processes, diffusion-weighted MRI could enhance our understanding of the exchange properties of maternal and fetal blood pools--and thereby of placental function. These studies, however, have so far been hindered by the small sizes, the unavoidable motions, and the challenging air/water/fat heterogeneities, associated with mouse placental environments. The present study demonstrates that emerging methods based on the spatiotemporal encoding (SPEN) of the MRI information can robustly overcome these obstacles. Using SPEN MRI in combination with albumin-based contrast agents, we analyzed the diffusion behavior of developing placentas in a cohort of mice. These studies successfully discriminated the maternal from the fetal blood flows; the two orders of magnitude differences measured in these fluids' apparent diffusion coefficients suggest a nearly free diffusion behavior for the former and a strong flow-based component for the latter. An intermediate behavior was observed by these methods for a third compartment that, based on maternal albumin endocytosis, was associated with trophoblastic cells in the interphase labyrinth. Structural features associated with these dynamic measurements were consistent with independent intravital and ex vivo fluorescence microscopy studies and are discussed within the context of the anatomy of developing mouse placentas.

In search of signaling pathways critical for ovarian graft reception: Akt1 is essential for long-term survival of ovarian grafts.

OBJECTIVE: To explore the role of Akt1, a principle modulator of angiogenesis, in ovarian graft reception and to investigate whether Akt1 deficiency can alter ovarian graft reception. DESIGN: Experimental mouse model. SETTING: Research institute. ANIMAL(S): Donors: Akt1 knockout (Akt1(-/-)) and wild types (Akt1(+/+)) mice. Recipients: CD-1 nude immune deficient female mice. INTERVENTION(S): Ovaries from Akt1(-/-) and Akt1(+/+) mice transplanted in the biceps femoris muscle of immunocompromised CD-1 mice, and ovarian graft viability, perfusion, and revascularization explored in vivo by magnetic resonance imaging (MRI). MAIN OUTCOME MEASURE(S): Vascular density and permeability of newly formed graft blood vessels quantified by dynamic contrast-enhanced MRI 7, 14, 30, and 60 days after grafting as indicators for angiogenesis and reestablishment of blood perfusion. RESULT(S): The Akt1(-/-) ovarian grafts showed a gradual decrease in angiogenic response with time after transplantation, ultimately leading to complete or near-complete graft destruction coinciding with massive follicular loss. Sixty days after transplantation, the mean blood volume fraction (fBV) and vessel permeability (PS) were statistically significantly lower in Akt1(-/-) transplants compared with Akt1(+/+). CONCLUSION(S): Akt1 is essential for ovarian graft reception. However, surprisingly the impact of Akt1 deficiency was most profound not in the early stages of angiogenesis but rather in long-term survival of the graft.

Unique in utero identification of fetuses in multifetal mouse pregnancies by placental bidirectional arterial spin labeling MRI.

Noninvasive imaging is a critical part of the study of developing embryos/fetuses, particularly in the context of alterations of gene expression in genetically modified animals. However, in litter-bearing animals, such as mice, the inability to accurately identify individual embryo/fetus in utero is a major obstacle to longitudinal, noninvasive in vivo studies. Arterial spin labeling MRI was adopted here to determine the fetal order along the uterine horns in vivo, based on the specific pattern of dual arterial blood supply within the mouse uterine horns. Blood enters the mouse uterus cranially through the ovarian artery and caudally through the uterine artery. Saturation slices were alternately placed on the maternal heart or on the bifurcation point of the common iliac artery, thereby saturating either downward inflow via the ovarian arteries or upward inflow via the uterine arteries, respectively. Saturation maps provided a unique signature with highly significant correlation between the direction-dependent magnetization transfer and the position of the fetuses/placentas along the uterine horns. The bidirectional arterial spin labeling-MRI method reported here opens possibilities to determine and pursue phenotypic alterations in fetuses and placentas in longitudinal studies of transgenic and knockout mice models, and for studying defects in placental vascular architecture.

The hemodynamic basis for positional- and inter-fetal dependent effects in dual arterial supply of mouse pregnancies.

In mammalian pregnancy, maternal cardiovascular adaptations must match the requirements of the growing fetus(es), and respond to physiologic and pathologic conditions. Such adaptations are particularly demanding for mammals bearing large-litter pregnancies, with their inherent conflict between the interests of each individual fetus and the welfare of the entire progeny. The mouse is the most common animal model used to study development and genetics, as well as pregnancy-related diseases. Previous studies suggested that in mice, maternal blood flow to the placentas occurs via a single arterial uterine loop generated by arterial-arterial anastomosis of the uterine artery to the uterine branch of the ovarian artery, resulting in counter bi-directional blood flow. However, we provide here experimental evidence that each placenta is actually supplied by two distinct arterial inputs stemming from the uterine artery and from the uterine branch of the ovarian artery, with position-dependent contribution of flow from each source. Moreover, we report significant positional- and inter-fetal dependent alteration of placental perfusion, which were detected by in vivo MRI and fluorescence imaging. Maternal blood flow to the placentas was dependent on litter size and was attenuated for placentas located centrally along the uterine horn. Distinctive apposing, inter-fetal hemodynamic effects of either reduced or elevated maternal blood flow, were measured for placenta of normal fetuses that are positioned adjacent to either pathological, or to hypovascular Akt1-deficient placentas, respectively. The results reported here underscore the critical importance of confounding local and systemic in utero effects on phenotype presentation, in general and in the setting of genetically modified mice. The unique robustness and plasticity of the uterine vasculature architecture, as reported in this study, can explain the ability to accommodate varying litter sizes, sustain large-litter pregnancies and overcome pathologic challenges. Remarkably, the dual arterial supply is evolutionary conserved in mammals bearing a single offspring, including primates.

Hypoxic stress and cancer: imaging the axis of evil in tumor metastasis.

Tumors emerge as a result of the sequential acquisition of genetic, epigenetic and somatic alterations promoting cell proliferation and survival. The maintenance and expansion of tumor cells rely on their ability to adapt to changes in their microenvironment, together with the acquisition of the ability to remodel their surroundings. Tumor cells interact with two types of interconnected microenvironments: the metabolic cell autonomous microenvironment and the nonautonomous cellular-molecular microenvironment comprising interactions between tumor cells and the surrounding stroma. Hypoxia is a central player in cancer progression, affecting not only tumor cell autonomous functions, such as cell division and invasion, resistance to therapy and genetic instability, but also nonautonomous processes, such as angiogenesis, lymphangiogenesis and inflammation, all contributing to metastasis. Closely related microenvironmental stressors affecting cancer progression include, in addition to hypoxia, elevated interstitial pressure and oxidative stress. Noninvasive imaging offers multiple means to monitor the tumor microenvironment and its consequences, and can thus assist in the understanding of the biological basis of hypoxia and microenvironmental stress in cancer progression, and in the development of strategies to monitor therapies targeted at stress-induced tumor progression.

Novel MRI and fluorescent probes responsive to the Factor XIII transglutaminase activity.

Transglutaminases, including factor XIII and tissue transglutaminase, participate in multiple extracellular processes associated with remodeling of the extracellular matrix during wound repair, blood clotting, tumor progression and fibrosis of ischemic injuries. The aim of this work was to evaluate a novel substrate analog for transglutaminase optimized by molecular modeling calculations (DCCP16), which can serve for molecular imaging of transglutaminase activity by magnetic resonance imaging and by near-infrared imaging. Experimental data showed covalent binding of Gd-DCCP16 and DCCP16-IRIS Blue to human clots, to basement membrane components and to casein in purified systems as well as in three-dimensional multicellular spheroids. In vivo, DCCP16 showed enhancement with a prolonged retention in clots and tumors, demonstrating the ability to detect both factor XIII and tissue transglutaminase mediated covalent binding of the contrast material.

Mice with vestibular deficiency display hyperactivity, disorientation, and signs of anxiety.

Previous studies revealed that vestibular cues are crucial for exploration in the absence of visual cues. The working hypothesis of this study was, accordingly, that mice with vestibular dysfunction would become disoriented or unable to globally explore an unfamiliar environment. In 2- and 3-month-old mutant headbanger (Hdb) mice, stereocilia of hair cells are abnormally elongated, yet maintain partial staircase arrangement, suggesting some spared vestibular function at these ages. Here we tested a group of 3-month-old mutant Hdb and a group of non-mutant mice obtained from the same litters (Wt mice). Each individual mouse was introduced into a dark 120 cm x 120 cm arena and its behavior was followed for 10 min. Hdb mice were hyperactive and appeared to engage in local exploration, traveling in a restricted zone for a while and then shifting to travel in another zone. In contrast, Wt mice traveled across zones incessantly with fewer visits to recently entered zones. Thus, Hdb seemed to display local compared with the global exploration of Wt mice, indicating that they were less oriented in the global environment. In addition, Hdb exhibited numerous stretch-attends, which is suggested as a sign of elevated anxiety. Altogether, the three comorbidities of hyperactivity, anxiety, and disorientation can be presented as a syndrome associated with vestibular deficiency in this animal model, and serve in studying vestibular deficiency in humans.

Magnetization transfer magic-angle-spinning z-spectroscopy of excised tissues.

NMR experiments devised to aid in analyses of tissues include magnetization transfer (MT), which can highlight the signals of biological macromolecules through cross-relaxation and/or chemical exchange processes with the bulk (1)H water resonance, and high-resolution magic-angle-spinning (HRMAS) methods, akin to those used in solid-state NMR to introduce additional spectral resolution via the averaging of spin anisotropies. This paper explores the result of combining these methodologies, and reports on MT "z-spectroscopy" between water and cell components in excised tissues under a variety of HRMAS conditions. Main features arising from the resulting (1)H "MTMAS" experiments include strong spinning sideband manifolds centered at the liquid water shift, high-resolution isotropic features coinciding with aliphatic and amide proton resonances, and a second sideband manifold arising as spinning speeds are increased. Interpretations are given for the origin of these various features, including simulations shedding further light onto the nature of MT NMR signals observed for tissue samples. Concurrently, histological examinations are reported validating the limits of HRMAS NMR procedures to the analysis of tissue samples preserved in a number of different ways.

Exploration and navigation in the blind mole rat (Spalax ehrenbergi): global calibration as a primer of spatial representation.

The aim of this study was to uncover the process of initial spatial mapping of the environment. For this, blind mole rats (Spalax ehrenbergi), were tested in an unfamiliar square arena, in order to reveal how they construct a spatial representation. The mole rats first displayed a build-up phase, in which they gradually formed a path along the perimeter while travelling slowly, frequently pausing and repeating previously travelled segments of the path. This behaviour was followed by a free-travel phase, in which the mole rats appeared to locomote smoothly along the perimeter and through the centre of the arena while travelling faster with fewer stops or repetitions of path segments. Familiarity with the environment was reflected in local shortcuts at the arena corners and global shortcuts (crosscuts) through the arena centre. We suggest that scanning the perimeter throughout the build-up phase constitute a process of calibration, i.e. forming an initial representation of the size and perhaps the shape of the environment--a sort of basic global map. We further suggest that this calibration is later used for navigation, as indicated by the emergence of global crosscuts in the subsequent phase. Further investigation of the build-up phase, e.g. by manipulating environment size, might provide additional insight into the course of establishment of global environment representation (mapping).

On the border: perimeter patrolling as a transitional exploratory phase in a diurnal rodent, the fat sand rat (Psammomys obesus).

Exploration is an initial phase of constructing spatial representation. In an illuminated environment, exploration by nocturnal rodents takes the form of home-base behavior, with the rodents organizing their activity in relation to the base, repeatedly orienting and returning to it. In the dark, home base behavior in gerbils is preceded by looping exploration, in which travel paths tangle into loops that close at various locations so that the gerbils pilot from one loop to the next. In the present study we tested a diurnal gerbil, the fat sand rat, Psammomys obesus, in both a lit and a dark open field in order to compare its exploratory behavior with that of nocturnal rodents. We found that under lit conditions, fat sand rats used perimeter patrolling, traveling mainly along the walls of the open field. In perimeter patrolling the animal probably monitors its location in relation to the perimeter (arena walls), and not to a specific location as in home base. In the dark, fat sand rats first used looping, and gradually shifted to perimeter patrolling exploration. We suggest that perimeter patrolling is a transient phase in which the animal evaluates possible locations for a home base. Thus, perimeter patrolling is an intermediate phase between looping, which is based on piloting from one landmark to the next, and home base exploration, in which the animal continuously orients to a specific location. This spatial behavior of perimeter patrolling may shed light on phases of information processing and spatial representation during exploration and navigation.

Exploration in a dark open field: a shift from directional to positional progression and a proposed model of acquiring spatial information.

Exploration in a dark open field undergoes three progressive changes: (i) an initial phase of spending equal amounts of time in various zones of the arena changes to staying in the corners, and ultimately spending most of the time in one corner; (ii) travel paths are first circular and scattered all over the arena, but gradually become anchored to one corner at which they start and end; (iii) traveled distance gradually decreases to that of the initial level seen in a lit open field. Altogether, rodents shift from a 'looping' exploration mechanism with feeble coupling with the environment, to 'home base' exploration which is firmly anchored to the environment. This shift also involves switching from momentary and sporadic to repeated returns to a specific, presumably familiar place, to which the animal navigates back from various other places. We suggest that this switching illustrates navigation first by directional and then by positional environmental cues, as hypothesized in the 'parallel map theory'. We also suggest that the transition from looping to home base behavior is part of a hierarchal construction of space representation via three modes of spatial information processing: (i) piloting--sequential processing, based on moving from one landmark to the next; (ii) orienting--parallel processing, based on moving from one point to the next, with the same starting and ending point; (iii) navigating--continuous processing, based on continuously updating the position in relation to several locations in the environment (map navigation).

'Looping'-an exploration mechanism in a dark open field.

The behavior of Tristram's jird (a species of gerbil) in an illuminated open field resembled that of other rodents, comprising round trips to a home base and alternating between periods of progression (locomoting) and of stopping. In this study, we compared the characteristics of exploration in a dark arena with exploration by the same individuals in a lit arena. In the dark arena, stopping episodes were brief and fewer, suggesting almost continuous locomotion by the rodents. The clear distinction between progression and stopping that had characterized locomotion in an illuminated arena, thus diminished in the dark. There was also no apparent home base in the dark and traveling consisted in moving in a circular path, closing a loop to a recently traveled place that varied from one loop to the next. Locomotion in the dark may thus be regarded as a set of loops (round trips) to a continuously shifting home base, whereas with lights on the round trips converge to a home base using visible environmental landmarks. We suggest that a similar looping mechanism may be applicable to the behavior of hippocampal rats displaying hyperactivity and diversified locomotion, reminiscent of that seen in jirds in a dark arena.