MR histology reveals tissue features beneath heterogeneous MRI signal in genetically engineered mouse models of sarcoma.

Purpose: To identify significant relationships between quantitative cytometric tissue features and quantitative MR (qMRI) intratumorally in preclinical undifferentiated pleomorphic sarcomas (UPS). Materials and methods: In a prospective study of genetically engineered mouse models of UPS, we registered imaging libraries consisting of matched multi-contrast in vivo MRI, three-dimensional (3D) multi-contrast high-resolution ex vivo MR histology (MRH), and two-dimensional (2D) tissue slides. From digitized histology we generated quantitative cytometric feature maps from whole-slide automated nuclear segmentation. We automatically segmented intratumoral regions of distinct qMRI values and measured corresponding cytometric features. Linear regression analysis was performed to compare intratumoral qMRI and tissue cytometric features, and results were corrected for multiple comparisons. Linear correlations between qMRI and cytometric features with p values of <0.05 after correction for multiple comparisons were considered significant. Results: Three features correlated with ex vivo apparent diffusion coefficient (ADC), and no features correlated with in vivo ADC. Six features demonstrated significant linear relationships with ex vivo T2*, and fifteen features correlated significantly with in vivo T2*. In both cases, nuclear Haralick texture features were the most prevalent type of feature correlated with T2*. A small group of nuclear topology features also correlated with one or both T2* contrasts, and positive trends were seen between T2* and nuclear size metrics. Conclusion: Registered multi-parametric imaging datasets can identify quantitative tissue features which contribute to UPS MR signal. T2* may provide quantitative information about nuclear morphology and pleomorphism, adding histological insights to radiological interpretation of UPS.

Transcriptome analysis identifies an ASD-Like phenotype in oligodendrocytes and microglia from C58/J amygdala that is dependent on sex and sociability.

BACKGROUND: Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with higher incidence in males and is characterized by atypical verbal/nonverbal communication, restricted interests that can be accompanied by repetitive behavior, and disturbances in social behavior. This study investigated brain mechanisms that contribute to sociability deficits and sex differences in an ASD animal model. METHODS: Sociability was measured in C58/J and C57BL/6J mice using the 3-chamber social choice test. Bulk RNA-Seq and snRNA-Seq identified transcriptional changes in C58/J and C57BL/6J amygdala within which DMRseq was used to measure differentially methylated regions in amygdala. RESULTS: C58/J mice displayed divergent social strata in the 3-chamber test. Transcriptional and pathway signatures revealed immune-related biological processes differ between C58/J and C57BL/6J amygdala. Hypermethylated and hypomethylated genes were identified in C58/J versus C57BL/6J amygdala. snRNA-Seq data in C58/J amygdala identified differential transcriptional signatures within oligodendrocytes and microglia characterized by increased ASD risk gene expression and predicted impaired myelination that was dependent on sex and sociability. RNA velocity, gene regulatory network, and cell communication analysis showed diminished oligodendrocyte/microglia differentiation. Findings were verified using Bulk RNA-Seq and demonstrated oxytocin's beneficial effects on myelin gene expression. LIMITATIONS: Our findings are significant. However, limitations can be noted. The cellular mechanisms linking reduced oligodendrocyte differentiation and reduced myelination to an ASD phenotype in C58/J mice need further investigation. Additional snRNA-Seq and spatial studies would determine if effects in oligodendrocytes/microglia are unique to amygdala or if this occurs in other brain regions. Oxytocin's effects need further examination to understand its' potential as an ASD therapeutic. CONCLUSIONS: Our work demonstrates the C58/J mouse model's utility in evaluating the influence of sex and sociability on the transcriptome in concomitant brain regions involved in ASD. Our single-nucleus transcriptome analysis elucidates potential pathological roles of oligodendrocytes and microglia in ASD. This investigation provides details regarding regulatory features disrupted in these cell types, including transcriptional gene dysregulation, aberrant cell differentiation, altered gene regulatory networks, and changes to key pathways that promote microglia/oligodendrocyte differentiation. Our studies provide insight into interactions between genetic risk and epigenetic processes associated with divergent affiliative behavior and lack of positive sociability.

Transcriptome Analysis Identifies An ASD-Like Phenotype In Oligodendrocytes And Microglia From C58/J Amygdala That Is Dependent On Sex and Sociability.

Background: Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with higher incidence in males and is characterized by atypical verbal/nonverbal communication, restricted interests that can be accompanied by repetitive behavior, and disturbances in social behavior. This study investigated brain mechanisms that contribute to sociability deficits and sex differences in an ASD animal model. Methods: Sociability was measured in C58/J and C57BL/6J mice using the 3-chamber social choice test. Bulk RNA-Seq and snRNA-Seq identified transcriptional changes in C58/J and C57BL/6J amygdala within which DMRseq was used to measure differentially methylated regions in amygdala. Results: C58/J mice displayed divergent social strata in the 3-chamber test. Transcriptional and pathway signatures revealed immune-related biological processes differ between C58/J and C57BL/6J amygdala. Hypermethylated and hypomethylated genes were identified in C58/J versus C57BL/6J amygdala. snRNA-Seq data in C58/J amygdala identified differential transcriptional signatures within oligodendrocytes and microglia characterized by increased ASD risk gene expression and predicted impaired myelination that was dependent on sex and sociability. RNA velocity, gene regulatory network, and cell communication analysis showed diminished oligodendrocyte/microglia differentiation. Findings were verified using bulk RNA-Seq and demonstrated oxytocin's beneficial effects on myelin gene expression. Limitations: Our findings are significant. However, limitations can be noted. The cellular mechanisms linking reduced oligodendrocyte differentiation and reduced myelination to an ASD phenotype in C58/J mice need further investigation. Additional snRNA-Seq and spatial studies would determine if effects in oligodendrocytes/microglia are unique to amygdala or if this occurs in other brain regions. Oxytocin's effects need further examination to understand its potential as an ASD therapeutic. Conclusions: Our work demonstrates the C58/J mouse model's utility in evaluating the influence of sex and sociability on the transcriptome in concomitant brain regions involved in ASD. Our single-nucleus transcriptome analysis elucidates potential pathological roles of oligodendrocytes and microglia in ASD. This investigation provides details regarding regulatory features disrupted in these cell types, including transcriptional gene dysregulation, aberrant cell differentiation, altered gene regulatory networks, and changes to key pathways that promote microglia/oligodendrocyte differentiation. Our studies provide insight into interactions between genetic risk and epigenetic processes associated with divergent affiliative behavior and lack of positive sociability.

Merged magnetic resonance and light sheet microscopy of the whole mouse brain.

We have developed workflows to align 3D magnetic resonance histology (MRH) of the mouse brain with light sheet microscopy (LSM) and 3D delineations of the same specimen. We start with MRH of the brain in the skull with gradient echo and diffusion tensor imaging (DTI) at 15 µm isotropic resolution which is ~ 1,000 times higher than that of most preclinical MRI. Connectomes are generated with superresolution tract density images of ~5 µm. Brains are cleared, stained for selected proteins, and imaged by LSM at 1.8 µm/pixel. LSM data are registered into the reference MRH space with labels derived from the ABA common coordinate framework. The result is a high-dimensional integrated volume with registration (HiDiver) with alignment precision better than 50 µm. Throughput is sufficiently high that HiDiver is being used in quantitative studies of the impact of gene variants and aging on mouse brain cytoarchitecture and connectomics.

Prenatal heroin exposure alters brain morphology and connectivity in adolescent mice.

The United States is experiencing a dramatic increase in maternal opioid misuse and, consequently, the number of individuals exposed to opioids in utero. Prenatal opioid exposure has both acute and long-lasting effects on health and wellbeing. Effects on the brain, often identified at school age, manifest as cognitive impairment, attention deficit, and reduced scholastic achievement. The neurobiological basis for these effects is poorly understood. Here, we examine how in utero exposure to heroin affects brain development into early adolescence in a mouse model. Pregnant C57BL/6J mice received escalating doses of heroin twice daily on gestational days 4-18. The brains of offspring were assessed on postnatal day 28 using 9.4 T diffusion MRI of postmortem specimens at 36 µm resolution. Whole-brain volumes and the volumes of 166 bilateral regions were compared between heroin-exposed and control offspring. We identified a reduction in whole-brain volume in heroin-exposed offspring and heroin-associated volume changes in 29 regions after standardizing for whole-brain volume. Regions with bilaterally reduced standardized volumes in heroin-exposed offspring relative to controls include the ectorhinal and insular cortices. Regions with bilaterally increased standardized volumes in heroin-exposed offspring relative to controls include the periaqueductal gray, septal region, striatum, and hypothalamus. Leveraging microscopic resolution diffusion tensor imaging and precise regional parcellation, we generated whole-brain structural MRI diffusion connectomes. Using a dimension reduction approach with multivariate analysis of variance to assess group differences in the connectome, we found that in utero heroin exposure altered structure-based connectivity of the left septal region and the region that acts as a hub for limbic regulatory actions. Consistent with clinical evidence, our findings suggest that prenatal opioid exposure may have effects on brain morphology, connectivity, and, consequently, function that persist into adolescence. This work expands our understanding of the risks associated with opioid misuse during pregnancy and identifies biomarkers that may facilitate diagnosis and treatment.

Volumetric analysis of the aging auditory pathway using high resolution magnetic resonance histology.

Numerous shown consequences of age-related hearing loss have been unveiled; however, the relationship of the cortical and subcortical structures of the auditory pathway with aging is not well known. Investigations into neural structure analysis remain sparse due to difficulties of doing so in animal models; however, recent technological advances have been able to achieve a resolution adequate to perform such studies even in the small mouse. We utilize 12 members of the BXD family of recombinant inbred mice and aged separate cohorts. Utilizing novel magnetic resonance histology imaging techniques, we imaged these mice and generated high spatial resolution three dimensional images which were then comprehensively labeled. We completed volumetric analysis of 12 separate regions of interest specific to the auditory pathway brainstem nuclei and cortical areas with focus on the effect of aging upon said structures. Our results showed significant interstrain variation in the age-related effect on structure volume supporting a genetic influence in this interaction. Through multivariable modeling, we observed heterogenous effects of aging between different structures. Six of the 12 regions of interests demonstrated a significant age-related effect. The auditory cortex and ventral cochlear nucleus were found to decrease in volume with age, while the medial division of the medial geniculate nucleus, lateral lemniscus and its nucleus, and the inferior colliculus increased in size with age. Additionally, no sex-based differences were noted, and we observed a negative relationship between auditory cortex volume and mouse weight. This study is one of the first to perform comprehensive magnetic resonance imaging and quantitative analysis in the mouse brain auditory pathway cytoarchitecture, offering both novel insights into the neuroanatomical basis of age-related changes in hearing as well as evidence toward a genetic influence in this interaction. High resonance magnetic resonance imaging provides a promising efficacious avenue in future mouse model hearing loss investigations.

Tractography of Porcine Meniscus Microstructure Using High-Resolution Diffusion Magnetic Resonance Imaging.

To noninvasively evaluate the three-dimensional collagen fiber architecture of porcine meniscus using diffusion MRI, meniscal specimens were scanned using a 3D diffusion-weighted spin-echo pulse sequence at 7.0 T. The collagen fiber alignment was revealed in each voxel and the complex 3D collagen network was visualized for the entire meniscus using tractography. The proposed automatic segmentation methods divided the whole meniscus to different zones (Red-Red, Red-White, and White-White) and different parts (anterior, body, and posterior). The diffusion tensor imaging (DTI) metrics were quantified based on the segmentation results. The heatmap was generated to investigate the connections among different regions of meniscus. Strong zonal-dependent diffusion properties were demonstrated by DTI metrics. The fractional anisotropy (FA) value increased from 0.13 (White-White zone) to 0.26 (Red-Red zone) and the radial diffusivity (RD) value changed from 1.0 × 10-3 mm2/s (White-White zone) to 0.7 × 10-3 mm2/s (Red-Red zone). Coexistence of both radial and circumferential collagen fibers in the meniscus was evident by diffusion tractography. Weak connections were found between White-White zone and Red-Red zone in each part of the meniscus. The anterior part and posterior part were less connected, while the body part showed high connections to both anterior part and posterior part. The tractography based on diffusion MRI may provide a complementary method to study the integrity of meniscus and nondestructively visualize the 3D collagen fiber architecture.

Resolution and b value dependent structural connectome in ex vivo mouse brain.

Diffusion magnetic resonance imaging has been widely used in both clinical and preclinical studies to characterize tissue microstructure and structural connectivity. The diffusion MRI protocol for the Human Connectome Project (HCP) has been developed and optimized to obtain high-quality, high-resolution diffusion MRI (dMRI) datasets. However, such efforts have not been fully explored in preclinical studies, especially for rodents. In this study, high quality dMRI datasets of mouse brains were acquired at 9.4T system from two vendors. In particular, we acquired a high-spatial resolution dMRI dataset (25 µm isotropic with 126 diffusion encoding directions), which we believe to be the highest spatial resolution yet obtained; and a high-angular resolution dMRI dataset (50 µm isotropic with 384 diffusion encoding directions), which we believe to be the highest angular resolution compared to the dMRI datasets at the microscopic resolution. We systematically investigated the effects of three important parameters that affect the final outcome of the connectome: b value (1000s/mm2 to 8000 s/mm2), angular resolution (10 to 126), and spatial resolution (25 µm to 200 µm). The stability of tractography and connectome increase with the angular resolution, where more than 50 angles is necessary to achieve consistent results. The connectome and quantitative parameters derived from graph theory exhibit a linear relationship to the b value (R2 > 0.99); a single-shell acquisition with b value of 3000 s/mm2 shows comparable results to the multi-shell high angular resolution dataset. The dice coefficient decreases and both false positive rate and false negative rate gradually increase with coarser spatial resolution. Our study provides guidelines and foundations for exploration of tradeoffs among acquisition parameters for the structural connectome in ex vivo mouse brain.

A time-course study of actively stained mouse brains: Diffusion tensor imaging parameters and connectomic stability over 1 year.

While the application of diffusion tensor imaging (DTI), tractography, and connectomics to fixed tissue is a common practice today, there have been limited studies examining the effects of fixation on brain microstructure over extended periods. This mouse model time-course study reports the changes of regional brain volumes and diffusion scalar parameters, such as fractional anisotropy, across 12 representative brain regions as measures of brain structural stability. The scalar DTI parameters and regional volumes were highly variable over the first 2 weeks after fixation. The same parameters were consistent over a 2-8-week window after fixation, which means confounds from tissue stability over that scanning window were minimal. Quantitative connectomes were analyzed over the same time with extension out to 1 year. While there was some change in the scalar metrics at 1 year after fixation, these changes were sufficiently small, particularly in white matter, to support reproducible connectomes over a period ranging from 2-weeks to 1-year post-fixation. These findings delineate a scanning period, during which brain volumes, diffusion scalar metrics, and connectomes are remarkably consistent.

A multicontrast MR atlas of the Wistar rat brain.

We describe a multi-contrast, multi-dimensional atlas of the Wistar rat acquired at microscopic spatial resolution using magnetic resonance histology (MRH). Diffusion weighted images, and associated scalar images were acquired of a single specimen with a fully sampled Fourier reconstruction, 61 angles and b=3000 s/mm2 yielding 50 um isotropic spatial resolution. The higher angular sampling allows use of the GQI algorithm improving the angular invariance of the scalar images and yielding an orientation distribution function to assist in delineating subtle boundaries where there are crossing fibers  and track density images providing insight into local fiber architecture.  A multigradient echo image of the same specimen was acquired at 25 um isotropic spatial resolution. A quantitative susceptibility map enhances fiber architecture relative to the magnitude images.  An accompanying multi-specimen atlas (n=6) was acquired with compressed sensing with the same diffusion protocol as used for the single specimen atlas.  An average was created using diffeomorphic mapping. Scalar volumes from the diffusion data, a T2* weighted volume, a quantitative susceptibility map, and a track density volume, all registered to the same space provide multiple contrasts to assist in anatomic delineation. The new template  provides significantly increased contrast in the scalar DTI images when compared to previous atlases. A compact interactive viewer based on 3D Slicer is provided to facilitate comparison among the contrasts in the multiple volumes. The single volume and average atlas with multiple 3D volumes provide an improved template for anatomic interrogation of the Wistar rat brain. The improved contrast to noise in the scalar DTI images and the addition of other volumes (eg. QA,QSM,TDI ) will facilitate automated label registration for MR histology and preclinical imaging.

A high-resolution interactive atlas of the human brainstem using magnetic resonance imaging.

Conventional atlases of the human brainstem are limited by the inflexible, sparsely-sampled, two-dimensional nature of histology, or the low spatial resolution of conventional magnetic resonance imaging (MRI). Postmortem high-resolution MRI circumvents the challenges associated with both modalities. A single human brainstem specimen extending from the rostral diencephalon through the caudal medulla was prepared for imaging after the brain was removed from a 65-year-old male within 24 h of death. The specimen was formalin-fixed for two weeks, then rehydrated and placed in a custom-made MRI compatible tube and immersed in liquid fluorocarbon. MRI was performed in a 7-Tesla scanner with 120 unique diffusion directions. Acquisition time for anatomic and diffusion images were 14 h and 208 h, respectively. Segmentation was performed manually. Deterministic fiber tractography was done using strategically chosen regions of interest and avoidance, with manual editing using expert knowledge of human neuroanatomy. Anatomic and diffusion images were rendered with isotropic resolutions of 50 µm and 200 µm, respectively. Ninety different structures were segmented and labeled, and 11 different fiber bundles were rendered with tractography. The complete atlas is available online for interactive use at https://www.civmvoxport.vm.duke.edu/voxbase/login.php?return_url=%2Fvoxbase%2F. This atlas presents multiple contrasting datasets and selected tract reconstruction with unprecedented resolution for MR imaging of the human brainstem. There are immediate applications in neuroanatomical education, with the potential to serve future applications for neuroanatomical research and enhanced neurosurgical planning through "safe" zones of entry into the human brainstem.

Mapping the peripheral nervous system in the whole mouse via compressed sensing tractography.

Objective.The peripheral nervous system (PNS) connects the central nervous system with the rest of the body to regulate many physiological functions and is therapeutically targeted to treat diseases such as epilepsy, depression, intestinal dysmotility, chronic pain, and more. However, we still lack understanding of PNS innervation in most organs because the large span, diffuse nature, and small terminal nerve bundle fibers have precluded whole-organism, high resolution mapping of the PNS. We sought to produce a comprehensive peripheral nerve atlas for use in future interrogation of neural circuitry and selection of targets for neuromodulation.Approach.We used diffusion tensor magnetic resonance imaging (DT-MRI) with high-speed compressed sensing to generate a tractogram of the whole mouse PNS. The tractography generated from the DT-MRI data is validated using lightsheet microscopy on optically cleared, antibody stained tissue.Main results.Herein we demonstrate the first comprehensive PNS tractography in a whole mouse. Using this technique, we scanned the whole mouse in 28 h and mapped PNS innervation and fiber network in multiple organs including heart, lung, liver, kidneys, stomach, intestines, and bladder at 70µm resolution. This whole-body PNS tractography map has provided unparalleled information; for example, it delineates the innervation along the gastrointestinal tract by multiple sacral levels and by the vagal nerves. The map enabled a quantitative tractogram that revealed relative innervation of the major organs by each vertebral foramen as well as the vagus nerve.Significance.This novel high-resolution nerve atlas provides a potential roadmap for future neuromodulation therapies and other investigations into the neural circuits which drive homeostasis and disease throughout the body.

Ex Vivo MR Histology and Cytometric Feature Mapping Connect Three-dimensional in Vivo MR Images to Two-dimensional Histopathologic Images of Murine Sarcomas.

Purpose To establish a platform for quantitative tissue-based interpretation of cytoarchitecture features from tumor MRI measurements. Materials and Methods In a pilot preclinical study, multicontrast in vivo MRI of murine soft-tissue sarcomas in 10 mice, followed by ex vivo MRI of fixed tissues (termed MR histology), was performed. Paraffin-embedded limb cross-sections were stained with hematoxylin-eosin, digitized, and registered with MRI. Registration was assessed by using binarized tumor maps and Dice similarity coefficients (DSCs). Quantitative cytometric feature maps from histologic slides were derived by using nuclear segmentation and compared with registered MRI, including apparent diffusion coefficients and transverse relaxation times as affected by magnetic field heterogeneity (T2* maps). Cytometric features were compared with each MR image individually by using simple linear regression analysis to identify the features of interest, and the goodness of fit was assessed on the basis of R2 values. Results Registration of MR images to histopathologic slide images resulted in mean DSCs of 0.912 for ex vivo MR histology and 0.881 for in vivo MRI. Triplicate repeats showed high registration repeatability (mean DSC, >0.9). Whole-slide nuclear segmentations were automated to detect nuclei on histopathologic slides (DSC = 0.8), and feature maps were generated for correlative analysis with MR images. Notable trends were observed between cell density and in vivo apparent diffusion coefficients (best line fit: R2 = 0.96, P < .001). Multiple cytoarchitectural features exhibited linear relationships with in vivo T2* maps, including nuclear circularity (best line fit: R2 = 0.99, P < .001) and variance in nuclear circularity (best line fit: R2 = 0.98, P < .001). Conclusion An infrastructure for registering and quantitatively comparing in vivo tumor MRI with traditional histologic analysis was successfully implemented in a preclinical pilot study of soft-tissue sarcomas. Keywords: MRI, Pathology, Animal Studies, Tissue Characterization Supplemental material is available for this article. © RSNA, 2021.

Microcephaly with altered cortical layering in GIT1 deficiency revealed by quantitative neuroimaging.

G Protein-Coupled Receptor Kinase-Interacting Protein-1 (GIT1) regulates neuronal functions, including cell and axon migration and synapse formation and maintenance, and GIT1 knockout (KO) mice exhibit learning and memory deficits. We noted that male and female GIT1-KO mice exhibit neuroimaging phenotypes including microcephaly, and altered cortical layering, with a decrease in neuron density in cortical layer V. Micro-CT and magnetic resonance microscopy (MRM) were used to identify morphometric phenotypes for the skulls and throughout the GIT1-KO brains. High field MRM of actively-stained mouse brains from GIT1-KO and wild type (WT) controls (n = 6 per group) allowed segmenting 37 regions, based on co-registration to the Waxholm Space atlas. Overall brain size in GIT1-KO mice was ~32% smaller compared to WT controls. After correcting for brain size, several regions were significantly different in GIT1-KO mice relative to WT, including the gray matter of the ventral thalamic nuclei and the rest of the thalamus, the inferior colliculus, and pontine nuclei. GIT1-KO mice had reduced volume of white matter tracts, most notably in the anterior commissure (~26% smaller), but also in the cerebral peduncle, fornix, and spinal trigeminal tract. On the other hand, the basal ganglia appeared enlarged in GIT1-KO mice, including the globus pallidus, caudate putamen, and particularly the accumbens - supporting a possible vulnerability to addiction. Volume based morphometry based on high-resolution MRM (21.5 µm isotropic voxels) was effective in detecting overall, and local differences in brain volumes in GIT1-KO mice, including in white matter tracts. The reduced relative volume of specific brain regions suggests a critical, but not uniform, role for GIT1 in brain development, conducive to brain microcephaly, and aberrant connectivity.

Cytoarchitecture of the mouse brain by high resolution diffusion magnetic resonance imaging.

MRI has been widely used to probe the neuroanatomy of the mouse brain, directly correlating MRI findings to histology is still challenging due to the limited spatial resolution and various image contrasts derived from water relaxation or diffusion properties. Magnetic resonance histology has the potential to become an indispensable research tool to mitigate such challenges. In the present study, we acquired high spatial resolution MRI datasets, including diffusion MRI (dMRI) at 25 â€‹µm isotropic resolution and quantitative susceptibility mapping (QSM) at 21.5 â€‹µm isotropic resolution to validate with conventional mouse brain histology. Diffusion weighted images (DWIs) show better delineation of cortical layers and glomeruli in the olfactory bulb than fractional anisotropy (FA) maps. However, among all the image contrasts, including quantitative susceptibility mapping (QSM), T1/T2∗ images and DTI metrics, FA maps highlight unique laminar architecture in sub-regions of the hippocampus, including the strata of the dentate gyrus and CA fields of the hippocampus. The mean diffusivity (MD) and axial diffusivity (AD) yield higher correlation with DAPI (0.62 and 0.71) and NeuN (0.78 and 0.74) than with NF-160 (-0.34 and -0.49). The correlations between FA and DAPI, NeuN, and NF-160 are 0.31, -0.01, and -0.49, respectively. Our findings demonstrate that MRI at microscopic resolution deliver a three-dimensional, non-invasive and non-destructive platform for characterization of fine structural detail in both gray matter and white matter of the mouse brain.

Characterization complex collagen fiber architecture in knee joint using high-resolution diffusion imaging.

PURPOSE: To evaluate the complex fiber orientations and 3D collagen fiber network of knee joint connective tissues, including ligaments, muscle, articular cartilage, and meniscus using high spatial and angular resolution diffusion imaging. METHODS: Two rat knee joints were scanned using a modified 3D diffusion-weighted spin echo pulse sequence with the isotropic spatial resolution of 45 µm at 9.4T. The b values varied from 250 to 1250 s/mm2 with 31 diffusion encoding directions for 1 rat knee. The b value was fixed to 1000 s/mm2 with 147 diffusion encoding directions for the second knee. Both the diffusion tensor imaging (DTI) model and generalized Q-sampling imaging (GQI) method were used to investigate the fiber orientation distributions and tractography with the validation of polarized light microscopy. RESULTS: To better resolve the crossing fibers, the b value should be great than or equal to 1000 s/mm2 . The tractography results were comparable between the DTI model and GQI method in ligament and muscle. However, the tractography exhibited apparent difference between DTI and GQI in connective tissues with more complex collagen fibers network, such as cartilage and meniscus. In articular cartilage, there were numerous crossing fibers found in superficial zone and transitional zone. Tractography generated with GQI also resulted in more intact tracts in articular cartilage than DTI. CONCLUSION: High-resolution diffusion imaging with GQI method can trace the complex collagen fiber orientations and architectures of the knee joint at microscopic resolution.

Optimizing Diffusion Imaging Protocols for Structural Connectomics in Mouse Models of Neurological Conditions.

Network approaches provide sensitive biomarkers for neurological conditions, such as Alzheimer's disease (AD). Mouse models can help advance our understanding of underlying pathologies, by dissecting vulnerable circuits. While the mouse brain contains less white matter compared to the human brain, axonal diameters compare relatively well (e.g., ~0.6 µm in the mouse and ~0.65-1.05 µm in the human corpus callosum). This makes the mouse an attractive test bed for novel diffusion models and imaging protocols. Remaining questions on the accuracy and uncertainty of connectomes have prompted us to evaluate diffusion imaging protocols with various spatial and angular resolutions. We have derived structural connectomes by extracting gradient subsets from a high-spatial, high-angular resolution diffusion acquisition (120 directions, 43-µm-size voxels). We have simulated protocols with 12, 15, 20, 30, 45, 60, 80, 100, and 120 angles and at 43, 86, or 172-µm voxel sizes. The rotational stability of these schemes increased with angular resolution. The minimum condition number was achieved for 120 directions, followed by 60 and 45 directions. The percentage of voxels containing one dyad was exceeded by those with two dyads after 45 directions, and for the highest spatial resolution protocols. For the 86- or 172-µm resolutions, these ratios converged toward 55% for one and 39% for two dyads, respectively, with <7% from voxels with three dyads. Tractography errors, estimated through dyad dispersion, decreased most with angular resolution. Spatial resolution effects became noticeable at 172 µm. Smaller tracts, e.g., the fornix, were affected more than larger ones, e.g., the fimbria. We observed an inflection point for 45 directions, and an asymptotic behavior after 60 directions, corresponding to similar projection density maps. Spatially downsampling to 86 µm, while maintaining the angular resolution, achieved a subgraph similarity of 96% relative to the reference. Using 60 directions with 86- or 172-µm voxels resulted in 94% similarity. Node similarity metrics indicated that major white matter tracts were more robust to downsampling relative to cortical regions. Our study provides guidelines for new protocols in mouse models of neurological conditions, so as to achieve similar connectomes, while increasing efficiency.

Neurite orientation dispersion and density imaging of mouse brain microstructure.

Advanced biophysical models like neurite orientation dispersion and density imaging (NODDI) have been developed to estimate the microstructural complexity of voxels enriched in dendrites and axons for both in vivo and ex vivo studies. NODDI metrics derived from high spatial and angular resolution diffusion MRI using the fixed mouse brain as a reference template have not yet been reported due in part to the extremely long scan time required. In this study, we modified the three-dimensional diffusion-weighted spin-echo pulse sequence for multi-shell and undersampling acquisition to reduce the scan time. This allowed us to acquire several exhaustive datasets that would otherwise not be attainable. NODDI metrics were derived from a complex 8-shell diffusion (1000-8000 s/mm2) dataset with 384 diffusion gradient-encoding directions at 50 µm isotropic resolution. These provided a foundation for exploration of tradeoffs among acquisition parameters. A three-shell acquisition strategy covering low, medium, and high b values with at least angular resolution of 64 is essential for ex vivo NODDI experiments. The good agreement between neurite density index (NDI) and the orientation dispersion index (ODI) with the subsequent histochemical analysis of myelin and neuronal density highlights that NODDI could provide new insight into the microstructure of the brain. Furthermore, we found that NDI is sensitive to microstructural variations in the corpus callosum using a well-established demyelination cuprizone model. The study lays the ground work for developing protocols for routine use of high-resolution NODDI method in characterizing brain microstructure in mouse models.

Diffusion tractography of the rat knee at microscopic resolution.

PURPOSE: To evaluate whole knee joint tractography, including articular cartilage, ligaments, meniscus, and growth plate using diffusion tensor imaging (DTI) at microscopic resolution. METHODS: Three rat knee joints were scanned using a modified 3D diffusion-weighted spin echo pulse sequence with 90- and 45-µm isotropic spatial resolution at 9.4T. The b values varied from 250 to 1250 s/mm2 with 4 times undersampling in phase directions. Fractional anisotropy (FA) and mean diffusivity (MD) were compared at different spatial resolution and b values. Tractography was evaluated at multiple b values and angular resolutions in different connective tissues, and compared with conventional histology. The mean tract length and tract volume in various types of tissues were also quantified. RESULTS: DTI metrics (FA and MD) showed consistent quantitative results at 90- and 45-µm isotropic spatial resolutions. Tractography of various connective tissues was found to be sensitive to the spatial resolution, angular resolution, and diffusion weightings. Higher spatial resolution (45 µm) supported tracking the cartilage collagen fiber tracts from the superficial zone to the deep zone, in a continuous and smooth progression in the transitional zone. Fiber length and fiber volume in the growth plate were strongly dependent on angular resolution and b values, whereas tractography in ligaments was found to be less dependent on spatial resolution. CONCLUSION: High spatial and angular resolution DTI and diffusion tractography can be valuable for knee joint research because of its visualization capacity for collagen fiber orientations and quantitative evaluation of tissue's microscopic properties.

Whole mouse brain connectomics.

Methods have been developed to allow quantitative connectivity of the whole fixed mouse brain by means of magnetic resonance imaging (MRI). We have translated what we have learned in clinical imaging to the very special domain of the mouse brain. Diffusion tensor imaging (DTI) of perfusion fixed specimens can now be performed with spatial resolution of 45 µm3 , that is, voxels that are 21,000 times smaller than the human connectome protocol. Specimen preparation has been optimized through an active staining protocol using a Gd chelate. Compressed sensing has been integrated into high performance reconstruction and post processing pipelines allowing acquisition of a whole mouse brain connectome in <12 hr. The methods have been validated against retroviral tracer studies. False positive tracts, which are especially problematic in clinical studies, have been reduced substantially to ~28%. The methods have been streamlined to provide high-fidelity, whole mouse brain connectomes as a routine study. The data package provides holistic insight into the mouse brain with anatomic definition at the meso-scale, quantitative volumes of subfields, scalar DTI metrics, and quantitative tractography.