Jon O. Cleary

Magnetic resonance virtual histology for embryos: 3D atlases for automated high-throughput phenotyping

Ambitious international efforts are underway to produce gene-knockout mice for each of the 25,000 mouse genes, providing a new platform to study mammalian development and disease. Robust, large-scale methods for morphological assessment of prenatal mice will be essential to this work. Embryo phenotyping currently relies on histological techniques but these are not well suited to large volume screening. The qualitative nature of these approaches also limits the potential for detailed group analysis. Advances in non-invasive imaging techniques such as magnetic resonance imaging (MRI) may surmount these barriers. We present a high-throughput approach to generate detailed virtual histology of the whole embryo, combined with the novel use of a whole-embryo atlas for automated phenotypic assessment. Using individual 3D embryo MRI histology, we identified new pituitary phenotypes in Hesx1 mutant mice. Subsequently, we used advanced computational techniques to produce a whole-body embryo atlas from 6 CD-1 embryos, creating an average image with greatly enhanced anatomical detail, particularly in CNS structures. This methodology enabled unsupervised assessment of morphological differences between CD-1 embryos and Chd7 knockout mice (n=5 Chd7(+/+) and n=8 Chd7(+/-), C57BL/6 background). Using a new atlas generated from these three groups, quantitative organ volumes were automatically measured. We demonstrated a difference in mean brain volumes between Chd7(+/+) and Chd7(+/-) mice (42.0 vs. 39.1mm(3), p<0.05). Differences in whole-body, olfactory and normalised pituitary gland volumes were also found between CD-1 and Chd7(+/+) mice (C57BL/6 background). Our work demonstrates the feasibility of combining high-throughput embryo MRI with automated analysis techniques to distinguish novel mouse phenotypes.

In vivo photoacoustic imaging of mouse embryos

The ability to noninvasively image embryonic vascular anatomy in mouse models is an important requirement for characterizing the development of the normal cardiovascular system and malformations in the heart and vascular supply. Photoacoustic imaging, which can provide high resolution non invasive images of the vasculature based upon optical absorption by endogenous hemoglobin, is well suited to this application. In this study, photoacoustic images of mouse embryos were obtained ex vivo and in vivo. The images show intricate details of the embryonic vascular system to depths of up to 10 mm, which allowed whole embryos to be imaged in situ. To achieve this, an all-optical photoacoustic scanner and a novel time reversal image reconstruction algorithm, which provide deep tissue imaging capability while maintaining high spatial resolution and contrast were employed. This technology may find application as an imaging tool for preclinical embryo studies in developmental biology as well as more generally in preclinical and clinical medicine for studying pathologies characterized by changes in the vasculature.

Cardiovascular magnetic resonance imaging in experimental models.

Cardiovascular magnetic resonance (CMR) imaging is the modality of choice for clinical studies of the heart and vasculature, offering detailed images of both structure and function with high temporal resolution. Small animals are increasingly used for genetic and translational research, in conjunction with models of common pathologies such as myocardial infarction. In all cases, effective methods for characterising a wide range of functional and anatomical parameters are crucial for robust studies. CMR is the gold-standard for the non-invasive examination of these models, although physiological differences, such as rapid heart rate, make this a greater challenge than conventional clinical imaging. However, with the help of specialised magnetic resonance (MR) systems, novel gating strategies and optimised pulse sequences, high-quality images can be obtained in these animals despite their small size. In this review, we provide an overview of the principal CMR techniques for small animals for example cine, angiography and perfusion imaging, which can provide measures such as ejection fraction, vessel anatomy and local blood flow, respectively. In combination with MR contrast agents, regional dysfunction in the heart can also be identified and assessed. We also discuss optimal methods for analysing CMR data, particularly the use of semi-automated tools for parameter measurement to reduce analysis time. Finally, we describe current and emerging methods for imaging the developing heart, aiding characterisation of congenital cardiovascular defects. Advanced small animal CMR now offers an unparalleled range of cardiovascular assessments. Employing these methods should allow new insights into the structural, functional and molecular basis of the cardiovascular system.

Comparison of segmentation methods for MRI measurement of cardiac function in rats

To establish the accuracy, intra‐ and inter‐observer variabilities of four different segmentation methods for measuring cardiac functional parameters in healthy and infarcted rat hearts.

Cardiac phenotyping in ex vivo murine embryos using microMRI.

Microscopic MRI (µMRI) is an emerging technique for high‐throughput phenotyping of transgenic mouse embryos, and is capable of visualising abnormalities in cardiac development. To identify cardiac defects in embryos, we have optimised embryo preparation and MR acquisition parameters to maximise image quality and assess the phenotypic changes in chromodomain helicase DNA‐binding protein 7 (Chd7) transgenic mice. µMRI methods rely on tissue penetration with a gadolinium chelate contrast agent to reduce tissue T1, thus improving signal‐to‐noise ratio (SNR) in rapid gradient echo sequences. We investigated 15.5 days post coitum (dpc) wild‐type CD‐1 embryos fixed in gadolinium‐diethylene triamine pentaacetic acid (Gd‐DTPA) solutions for either 3 days (2 and 4 mM) or 2 weeks (2, 4, 8 and 16 mM). To assess penetration of the contrast agent into heart tissue and enable image contrast simulations, T1 and T  *2 were measured in heart and background agarose. Compared to 3‐day, 2‐week fixation showed reduced mean T1 in the heart at both 2 and 4 mM concentrations (p < 0.0001), resulting in calculated signal gains of 23% (2 mM) and 29% (4 mM). Using T1 and T  *2 values from 2‐week concentrations, computer simulation of heart and background signal, and ex vivo 3D gradient echo imaging, we demonstrated that 2‐week fixed embryos in 8 mM Gd‐DTPA in combination with optimised parameters (TE/TR/α/number of averages: 9 ms/20 ms/60°/7) produced the largest SNR in the heart (23.2 ± 1.0) and heart chamber contrast‐to‐noise ratio (CNR) (27.1 ± 1.6). These optimised parameters were then applied to an MRI screen of embryos heterozygous for the gene Chd7, implicated in coloboma of the eye, heart defects, atresia of the choanae, retardation of growth, genital/urinary abnormalities, ear abnormalities and deafness (CHARGE) syndrome (a condition partly characterised by cardiovascular birth defects in humans). A ventricular septal defect was readily identified in the screen, consistent with the human phenotype. Copyright

Structural correlates of active-staining following magnetic resonance microscopy in the mouse brain

Extensive worldwide efforts are underway to produce knockout mice for each of the ~ 25,000 mouse genes, which may give new insights into the underlying pathophysiology of neurological disease. Microscopic magnetic resonance imaging (μMRI) is a key method for non-invasive morphological phenotyping, capable of producing high-resolution 3D images of ex-vivo brains, after fixation with an MR contrast agent. These agents have been suggested to act as active-stains, enhancing structures not normally visible on MRI. In this study, we investigated the structural correlates of the MRI agent Gd-DTPA, together with the optimal preparation and scan parameters for contrast-enhanced gradient-echo imaging of the mouse brain. We observed that in-situ preparation was preferential to ex-situ due to the degree of extraction damage. In-situ brains scanned with optimised parameters, enabled images with a high signal-to-noise-ratio (SNR ~ 30) and comprehensive anatomical delineation. Direct correlation of the MR brain structures to histology, detailed fine histoarchitecture in the cortex, cerebellum, olfactory bulb and hippocampus. Neurofilament staining demonstrated that regions of negative MR contrast strongly correlated to myelinated white-matter structures, whilst structures of more positive MR contrast corresponded to areas with high grey matter content. We were able to identify many sub-regions, particularly within the hippocampus, such as the unmyelinated mossy fibres (stratum lucidum) and their region of synapse in the stratum pyramidale, together with the granular layer of the dentate gyrus, an area of densely packed cell bodies, which was clearly visible as a region of hyperintensity. This suggests that cellular structure influences the site-specific distribution of the MR contrast agent, resulting in local variations in T2*, which leads to enhanced tissue discrimination. Our findings provide insights not only into the cellular distribution and mechanism of MR active-staining, but also allow for three dimensional analysis, which enables interpretation of magnetic resonance microscopy brain data and highlights cellular structure for investigation of disease processes in development and disease.

Segmentation propagation using a 3D embryo atlas for high-throughput MRI phenotyping: comparison and validation with manual segmentation.

Effective methods for high‐throughput screening and morphometric analysis are crucial for phenotyping the increasing number of mouse mutants that are being generated. Automated segmentation propagation for embryo phenotyping is an emerging application that enables noninvasive and rapid quantification of substructure volumetric data for morphometric analysis. We present a study to assess and validate the accuracy of brain and kidney volumes generated via segmentation propagation in an ex vivo mouse embryo MRI atlas comprising three different groups against the current “gold standard”—manual segmentation. Morphometric assessment showed good agreement between automatically and manually segmented volumes, demonstrating that it is possible to assess volumes for phenotyping a population of embryos using segmentation propagation with the same variation as manual segmentation. As part of this study, we have made our average atlas and segmented volumes freely available to the community for use in mouse embryo phenotyping studies. These MRI datasets and automated methods of analyses will be essential for meeting the challenge of high‐throughput, automated embryo phenotyping. Magn Reson Med, 2013.

Photoacoustic imaging of vascular networks in transgenic mice

The preferential absorption of near infrared light by blood makes photoacoustic imaging well suited to visualising vascular structures in soft tissue. In addition, the spectroscopic specificity of tissue chromophores can be exploited by acquiring images at multiple excitation wavelengths. This allows the quantification of endogenous chromophores, such as oxy- and deoxyhaemoglobin, and hence blood oxygenation, and the detection of exogenous chromophores, such as functionalised contrast agents. More importantly, this approach has the potential to visualise the spatial distribution of low concentrations of functionalised contrast agents against the strong background absorption of the endogenous chromophores. This has a large number of applications in the life sciences. One example is the structural and functional phenotyping of transgenic mice for the study of the genetic origins of vascular malformations, such as heart defects. In this study, photoacoustic images of mouse embryos have been acquired to study the development of the vasculature following specific genetic knockouts.

Enhanced tissue differentiation in the developing mouse brain using magnetic resonance micro-histology

Purpose: Worldwide efforts to understand developmental processes demand new high‐resolution 3D imaging methods to detect the consequences of gene function in embryo development and diseases. Encouragingly, recent studies have shown that MRI contrast agents can highlight specific tissue structures in ex vivo adult mouse brains. MR imaging of mouse embryos is currently limited by a lack of tissue staining capabilities that would provide the flexibility and specificity offered by histological stains conventionally used for mouse embryo phenotyping.

3D‐multi‐echo radial imaging of 23Na (3D‐MERINA) for time‐efficient multi‐parameter tissue compartment mapping

This work demonstrates a 3D radial multi‐echo acquisition scheme for time‐efficient sodium (23Na) MR‐signal acquisition and analysis. Echo reconstructions were used to produce signal‐to‐noise ratio (SNR)‐enhanced 23Na‐images and parameter maps of the biexponential observed transverse relaxation time ( T2* ) decay.