Multiple-mouse magnetic resonance imaging with cryogenic radiofrequency probes for evaluation of brain development.

Multiple-mouse magnetic resonance imaging (MRI) increases scan throughput by imaging several mice simultaneously in the same magnet bore, enabling multiple images to be obtained in the same time as a single scan. This increase in throughput enables larger studies than otherwise feasible and is particularly advantageous in longitudinal study designs where frequent imaging time points result in high demand for MRI resources. Cryogenically-cooled radiofrequency probes (CryoProbes) have been demonstrated to have significant signal-to-noise ratio benefits over comparable room temperature coils for in vivo mouse imaging. In this work, we demonstrate implementation of a multiple-mouse MRI system using CryoProbes, achieved by mounting four such coils in a 30-cm, 7-Tesla magnet bore. The approach is demonstrated for longitudinal quantification of brain structure from infancy to early adulthood in a mouse model of Sanfilippo syndrome (mucopolysaccharidosis type III), generated by knockout of the Hgsnat gene. We find that Hgsnat-/- mice have regionally increased growth rates compared to Hgsnat+/+ mice in a number of brain regions, notably including the ventricles, amygdala and superior colliculus. A strong sex dependence was also noted, with the lateral ventricle volume growing at an accelerated rate in males, but several structures in the brain parenchyma growing faster in females. This approach is broadly applicable to other mouse models of human disease and the increased throughput may be particularly beneficial in studying mouse models of neurodevelopmental disorders.

Kctd13-deficient mice display short-term memory impairment and sex-dependent genetic interactions.

The 16p11.2 BP4-BP5 deletion and duplication syndromes are associated with a complex spectrum of neurodevelopmental phenotypes that includes developmental delay and autism spectrum disorder, with a reciprocal effect on head circumference, brain structure and body mass index. Mouse models of the 16p11.2 copy number variant have recapitulated some of the patient phenotypes, while studies in flies and zebrafish have uncovered several candidate contributory genes within the region, as well as complex genetic interactions. We evaluated one of these loci, KCTD13, by modeling haploinsufficiency and complete knockout in mice. In contrast to the zebrafish model, and in agreement with recent data, we found normal brain structure in heterozygous and homozygous mutants. However, recapitulating previously observed genetic interactions, we discovered sex-specific brain volumetric alterations in double heterozygous Kctd13xMvp and Kctd13xLat mice. Behavioral testing revealed a significant deficit in novel object recognition, novel location recognition and social transmission of food preference in Kctd13 mutants. These phenotypes were concomitant with a reduction in density of mature spines in the hippocampus, but potentially independent of RhoA abundance, which was unperturbed postnatally in our mutants. Furthermore, transcriptome analyses from cortex and hippocampus highlighted the dysregulation of pathways important in neurodevelopment, the most significant of which was synaptic formation. Together, these data suggest that KCTD13 contributes to the neurocognitive aspects of patients with the BP4-BP5 deletion, likely through genetic interactions with other loci.

Spatial gene expression analysis of neuroanatomical differences in mouse models.

MRI is a powerful modality to detect neuroanatomical differences that result from mutations and treatments. Knowing which genes drive these differences is important in understanding etiology, but candidate genes are often difficult to identify. We tested whether spatial gene expression data from the Allen Brain Institute can be used to inform us about genes that cause neuroanatomical differences. For many single-gene-mutation mouse models, we found that affected neuroanatomy was not strongly associated with the spatial expression of the altered gene and there are specific caveats for each model. However, among models with significant neuroanatomical differences from their wildtype controls, the mutated genes had preferential spatial expression in affected neuroanatomy. In mice exposed to environmental enrichment, candidate genes could be identified by a genome-wide search for genes with preferential spatial expression in the altered neuroanatomical regions. These candidates have functions related to learning and plasticity. We demonstrate that spatial gene expression of single-genes is a poor predictor of altered neuroanatomy, but altered neuroanatomy can identify candidate genes responsible for neuroanatomical phenotypes.

Zebrafish models of idiopathic scoliosis link cerebrospinal fluid flow defects to spine curvature.

Idiopathic scoliosis (IS) affects 3% of children worldwide, yet the mechanisms underlying this spinal deformity remain unknown. Here we show that ptk7 mutant zebrafish, a faithful developmental model of IS, exhibit defects in ependymal cell cilia development and cerebrospinal fluid (CSF) flow. Transgenic reintroduction of Ptk7 in motile ciliated lineages prevents scoliosis in ptk7 mutants, and mutation of multiple independent cilia motility genes yields IS phenotypes. We define a finite developmental window for motile cilia in zebrafish spine morphogenesis. Notably, restoration of cilia motility after the onset of scoliosis blocks spinal curve progression. Together, our results indicate a critical role for cilia-driven CSF flow in spine development, implicate irregularities in CSF flow as an underlying biological cause of IS, and suggest that noninvasive therapeutic intervention may prevent severe scoliosis.

Clustering autism: using neuroanatomical differences in 26 mouse models to gain insight into the heterogeneity.

Autism is a heritable disorder, with over 250 associated genes identified to date, yet no single gene accounts for >1-2% of cases. The clinical presentation, behavioural symptoms, imaging and histopathology findings are strikingly heterogeneous. A more complete understanding of autism can be obtained by examining multiple genetic or behavioural mouse models of autism using magnetic resonance imaging (MRI)-based neuroanatomical phenotyping. Twenty-six different mouse models were examined and the consistently found abnormal brain regions across models were parieto-temporal lobe, cerebellar cortex, frontal lobe, hypothalamus and striatum. These models separated into three distinct clusters, two of which can be linked to the under and over-connectivity found in autism. These clusters also identified previously unknown connections between Nrxn1α, En2 and Fmr1; Nlgn3, BTBR and Slc6A4; and also between X monosomy and Mecp2. With no single treatment for autism found, clustering autism using neuroanatomy and identifying these strong connections may prove to be a crucial step in predicting treatment response.

Neuroanatomical phenotypes in a mouse model of the 22q11.2 microdeletion.

Recurrent deletions at the 22q11.2 locus have been established as a strong genetic risk factor for the development of schizophrenia and cognitive dysfunction. Individuals with 22q11.2 deletions have a range of well-defined volumetric abnormalities in a number of critical brain structures. A mouse model of the 22q11.2 deletion (Df(16)A(+/-)) has previously been utilized to characterize disease-associated abnormalities on synaptic, cellular, neurocircuitry, and behavioral levels. We performed a high-resolution MRI analysis of mutant mice compared with wild-type littermates. Our analysis revealed a striking similarity in the specific volumetric changes of Df(16)A(+/-) mice compared with human 22q11.2 deletion carriers, including in cortico-cerebellar, cortico-striatal and cortico-limbic circuits. In addition, higher resolution magnetic resonance imaging compared with neuroimaging in human subjects allowed the detection of previously unknown subtle local differences. The cerebellar findings in Df(16)A(+/-) mice are particularly instructive as they are localized to specific areas within both the deep cerebellar nuclei and the cerebellar cortex. Our study indicates that the Df(16)A(+/-)mouse model recapitulates most of the hallmark neuroanatomical changes observed in 22q11.2 deletion carriers. Our findings will help guide the design and interpretation of additional complementary studies and thereby advance our understanding of the abnormal brain development underlying the emergence of 22q11.2 deletion-associated psychiatric and cognitive symptoms.

Mouse embryonic phenotyping by morphometric analysis of MR images.

A new method is described for automatic detection of subtle morphological phenotypes in mouse embryos. Based on high-resolution magnetic resonance imaging scanning and nonlinear image alignment, this method is demonstrated by comparing the morphology of two inbred strains, C57BL/6J and 129Sv/S1ImJ, at 15.5 days postconception. Mouse embryo morphology was found to be highly amenable to this kind of analysis with very low levels (on average 110 µm) of residual anatomical variation within strains after linear differences in pose and scale are removed. Mapping of local size differences showed that C57BL/6J embryos were larger than 129Sv/S1ImJ embryos, although these differences were not uniformly distributed across the anatomy. Expressed in terms of organ volumes, heart and lung were larger in C57BL/6J embryos, while brain and liver were comparable in volume between strains. The positive relationship between organ size and embryo size was consistent for the two strains but differed by organ, with the brain and liver being the least variable. Together these findings suggest the power of this technique for detecting subtle phenotypic differences arising from mutated genes.

Comparative structural and hemodynamic analysis of vascular trees.

The availability of detailed three-dimensional images of vascular trees from mammalian organs provides a wealth of essential data for understanding the processes and mechanisms of vascular patterning. Using this detailed geometric data requires the ability to compare individual representations of vascular trees in statistically meaningful ways. This article provides some comparisons of geometry and also of simulated hemodynamics, enabling the identification of similarities and differences among 10 individual specimens (5 placenta specimens and 5 lung specimens). Similar comparisons made with a series of models (starting with the simplest and increasing in complexity) enable the identification of essential features that are needed to account for the patterns and function of vascular arborization.

The ubiquitin ligase Nedd4-1 is required for heart development and is a suppressor of thrombospondin-1.

Nedd4 (Nedd4-1) is a Hect domain E3 ubiquitin ligase that also contains a C2 domain and three WW domains. Despite numerous in vitro studies, its biological function in vivo is not well understood. Here we show that disruption of Nedd4-1 in mice (leaving Nedd4-2 intact) caused embryonic lethality at mid gestation, with pronounced heart defects (double-outlet right ventricle and atrioventricular cushion defects) and vasculature abnormalities. Quantitative mass spectrometry and immunoblot analyses of lysates from the wild type and knock-out mouse embryonic fibroblasts to identify Nedd4-1 in vivo targets revealed dramatically increased amounts of thrombospondin-1 (Tsp-1) in the knock-out mouse embryonic fibroblasts and embryos. Tsp-1 is an inhibitor of angiogenesis, and its elevated level was mediated primarily by enhanced transcription. Interestingly, the administration of aspirin (an inhibitor of Tsp-1) to the pregnant heterozygote mothers led to a reduction in Tsp-1 levels and a substantial rescue of the embryonic lethality. These results suggest that Nedd4-1 is a suppressor of Tsp1 and that increased levels of Tsp-1 in the Nedd4-1 knock-out mice may have contributed to the developmental defect observed in the embryos.

Age-dependent endothelial nitric oxide synthase uncoupling in pulmonary arteries of endoglin heterozygous mice.

Endoglin is a TGF-beta superfamily receptor critical for endothelial cell function. Mutations in this gene are associated with hereditary hemorrhagic telangiectasia type I (HHT1), and clinical signs of disease are generally more evident later in life. We previously showed that systemic vessels of adult Eng heterozygous (Eng(+/-)) mice exhibit increased vasorelaxation due to uncoupling of endothelial nitric oxide synthase (eNOS). We postulated that these changes may develop with age and evaluated pulmonary arteries from newborn and adult Eng(+/-) mice for eNOS-dependent, acetylcholine (ACh-induced) vasorelaxation, compared with that of age-matched littermate controls. While ACh-induced vasorelaxation was similar in all newborn mice, it was significantly increased in the adult Eng(+/-) vs. control vessels. The vasodilatory responses were inhibited by l-NAME suggesting eNOS dependence. eNOS uncoupling was observed in lung tissues of adult, but not newborn, heterozygous mice and was associated with increased production of reactive O(2) species (ROS) in adult Eng(+/-) vs. control lungs. Interestingly, ROS generation was higher in adult than newborn mice and so were the levels of NADPH oxidase 4 and SOD 1, 2, 3 isoforms. However, enzyme protein levels and NADPH activity were normal in adult Eng(+/-) lungs indicating that the developmental maturation of ROS generation and scavenging cannot account for the increased vasodilatation observed in adult Eng(+/-) mice. Our data suggest that eNOS-dependent H(2)O(2) generation in Eng(+/-) lungs accounts for the heightened pulmonary vasorelaxation. To the extent that these mice mimic human HHT1, age-associated pulmonary vascular eNOS uncoupling may explain the late childhood and adult onset of clinical lung manifestations.

Information content of SNR/resolution trade-offs in three-dimensional magnetic resonance imaging.

In MRI, a trade-off exists between resolution and signal-to-noise ratio, since different fractions of the available scan time can be used to acquire data at higher spatial frequencies and to perform signal averaging. By comparing a wide variety of 3D isotropic MR scans with different combinations of SNR, resolution, and scan duration, the impact of this trade-off on the image information content was assessed. The information content of mouse brain, mouse whole-body, and human brain images was evaluated using a simple numerical approach, which sums the information contribution of each individual k-space data point. Results show that, with a fixed receiver bandwidth and field of view, the information content of trade-off images is always maximized when the SNR is equal to about 16. The optimal imaging resolution is dependent on the scan duration, as well as certain MR system properties, such as field strength and coil sensitivity. These properties are, however, easily accounted for with the acquisition of a single scout MR image, and the optimal imaging resolution can then be calculated using a simple mathematical relationship. If the imaging task is approached with a predetermined resolution requirement, the same scout scan can be used to calculate the scan duration that will provide the maximum possible information. Using these relationships to maximize the image information content is an excellent technique for guiding the initial selection of imaging parameters.

EphB2 and EphA4 receptors regulate formation of the principal inter-hemispheric tracts of the mammalian forebrain.

Previously, we have demonstrated that EphB2 activity is required for proper development of the posterior branch of the anterior commissure (ACpp) within the mammalian forebrain. In the present study, using magnetic resonance imaging (MRI), immunohistochemistry, and in vivo stereotactic fluorescence tracing of EphB2, B3, A4 and combinatorial Eph receptor mutants, we have developed a detailed three-dimensional model of how EphB-class receptors interact to regulate commissural formation within the forebrain. The results demonstrate that EphB2 and EphA4 each regulate distinct aspects of axon guidance within the ACpp. Specifically, while EphB2 is required to retard ACpp axons from projecting aberrantly into the ventral forebrain, EphA4 is required to restrict axons from entering the anterior branch of the anterior commissure (ACpa). Together, EphB2 and EphA4 act synergistically to prevent a subpopulation of axons within the anterior branch of the AC from mis-projecting caudally. Analysis of EphA4 null mice using high resolution MRI reveals for the first time that, in addition to errors in midline guidance, loss of EphA4 results in aberrant lateral and ventral displacement of the ACpa tract. In addition, tracing studies in alpha-chimerin null mice reveal that EphA4-mediated effects are not regulated through this pathway. Taken together, the results demonstrate that each of the principal guidance decisions within both anterior and posterior tracts of the anterior commissure can be accounted for by the individual and combinatorial actions of EphB2/A4 receptors.

Volume ordering for analysis and modeling of vascular systems.

Morphological characteristics of vascular systems are commonly presented in terms of Strahler order because the logarithms of quantities such as vessel diameter and length are often linearly related to Strahler order. However, the ability to interpret Strahler order geometrically or physiologically is compromised because the precision of the order number is limited to integer values. This limitation is overcome by the volume ordering scheme, in which volume order number is defined as the logarithm of the estimated perfused tissue volume for each vascular segment. While Strahler and volume order numbers are equivalent for completely symmetrical branching trees, they deviate in the presence of asymmetries. The physiology-based definition of volume ordering offers benefits in the analysis of vascular design, fractal characterization of vascular systems, and blood flow modeling. These benefits are illustrated based on arterial kidney data that show a linear relationship of logarithmic vessel diameter and conductance as a function of both Strahler order and volume order with differing proportionality constants, which are expected to depend on the branching characteristics of the particular organ investigated.

High resolution three-dimensional brain atlas using an average magnetic resonance image of 40 adult C57Bl/6J mice.

Detailed anatomical atlases can provide considerable interpretive power in studies of both human and rodent neuroanatomy. Here we describe a three-dimensional atlas of the mouse brain, manually segmented into 62 structures, based on an average of 32 mum isotropic resolution T(2)-weighted, within skull images of forty 12 week old C57Bl/6J mice, scanned on a 7 T scanner. Individual scans were normalized, registered, and averaged into one volume. Structures within the cerebrum, cerebellum, and brainstem were painted on each slice of the average MR image while using simultaneous viewing of the coronal, sagittal and horizontal orientations. The final product, which will be freely available to the research community, provides the most detailed MR-based, three-dimensional neuroanatomical atlas of the whole brain yet created. The atlas is furthermore accompanied by ancillary detailed descriptions of boundaries for each structure and provides high quality neuroanatomical details pertinent to MR studies using mouse models in research.

Oxygen-enhanced MR imaging of mice lungs.

Inhaled molecular oxygen has been widely used in humans to evaluate pulmonary ventilation using MRI. MR imaging has recently played a greater role in examining the morphologic and physiologic characteristics of mouse models of lung disease where structural changes are highly correlated to abnormalities in respiratory function. The motivation of this work is to develop oxygen-enhanced MR imaging for mice. Conventional human MR techniques cannot be directly applied to mouse imaging due to smaller dimensions and faster cardiac and respiratory physiology. This study examines the development of oxygen-enhanced MR as a noninvasive tool to assess regional ventilation in spontaneously breathing mice. An optimized cardiac-triggered, respiratory-gated fast spin-echo imaging sequence was developed to address demands of attaining adequate signal from the parenchyma, maintaining practical acquisition times, and compensating for rapid physiological motion. On average, a 20% T1-shortening effect was observed in mice breathing 100% oxygen as compared to air. The effect of ventilation was shown as a significant signal intensity increase of 11% to 16% in the mouse parenchyma with 100% oxygen inhalation. This work demonstrates that adequate contrast and resolution can be achieved using oxygen-enhanced MR to visualize ventilation, providing an effective technique to study ventilation defects in mice.

Development of a high resolution three-dimensional surgical atlas of the murine head for strains 129S1/SvImJ and C57Bl/6J using magnetic resonance imaging and micro-computed tomography.

The mouse has emerged as a major experimental model system for examining the functional properties of the mammalian CNS; both during development and following CNS injury. Histologic procedures currently used to determine the relative position of structures within the CNS are presently limited in their ability to take full advantage of this system for surgical and morphometric procedures. We present here the first three-dimensional interactive digital atlas of the murine brain and skull for two genetically important strains of mice; 129S1/SvImJ and C57Bl/6J. The final resolution of these digital atlases is 54 micro m(3). These representations of the murine brain and skull, in conjunction with our development of a new, more dynamic master coordinate system, provide improved accuracy with respect to targeting CNS structures during surgery compared with previous systems. The interactive three-dimensional nature of these atlases also provide users with stereotactic information necessary to perform accurate "off-axis" surgical procedures, as is commonly required for experiments such as in vivo micro-electroporation. In addition, three-dimensional analysis of the brain and skull shape in C57Bl, 129Sv, CD1, and additional murine strains, suggests that a stereotactic coordinate system based upon the lambda and rostral confluence of the sinuses at the sagittal midline, provides improved accuracy compared with the traditional lambda-bregma landmark system. These findings demonstrate the utility of developing highly accurate and robust three-dimensional representations of the murine brain and skull, in which experimental outputs can be directly compared using a unified coordinate system. The aim of these studies is to enhance comparative morphometric analyses and stereotactic surgical procedures in mice.

Comparing microsphere deposition and flow modeling in 3D vascular trees.

Blood perfusion in organs has been shown to be heterogeneous in a number of cases. At the same time, a number of models of vascular structure and flow have been proposed that also generate heterogeneous perfusion. Although a relationship between local perfusion and vascular structure has to exist, no model has yet been validated as an accurate description of this relationship. A study of perfusion and three-dimensional (3D) arterial structure in individual rat kidneys is presented, which allows comparison between local measurements of perfusion and model-based predictions. High-resolution computed tomography is used to obtain images of both deposited microspheres and of an arterial cast in the same organ. Microsphere deposition is used as an estimate of local perfusion. A 3D cylindrical pipe model of the arterial tree is generated based on an image of the arterial cast. Results of a flow model are compared with local microsphere deposition. High correlation (r(2) > 0.94) was observed between measured and modeled flows through the vascular tree segments. However, the relative dispersion of the microsphere perfusion measurement was two- to threefold higher than perfusion heterogeneity calculated in the flow model. Also, there was no correlation in the residual deviations between the methods. This study illustrates the importance of comparing models of local perfusion with in vivo measurements of perfusion in the same biologically realistic vascular tree.

Estimating perfusion using microCT to locate microspheres.

The injection of microspheres into the blood stream has been a common method to measure the spatial distribution of blood flow (perfusion). A technique to conduct this kind of measurement in small animal organs is presented using silver-coated microspheres with a diameter of 16 microm and high-resolution computed tomography (microCT) to detect individual microspheres. Phantom experiments demonstrate the detectability of individual spheres. The distribution of microspheres within a rat heart is given as an example. Using non-destructive, three-dimensional imaging for microsphere detection avoids the cumbersome dissection of the organ into samples or slices and their subsequent registration. The detection of individual spheres allows high-resolution measurements of perfusion and arbitrary definition of regions of interest. These, in turn, allow for accurate statistical analysis of perfusion such as relative dispersion curves.