Early Regenerative Capacity in the Porcine Heart.

BACKGROUND: The adult mammalian heart has limited ability to repair itself after injury. Zebrafish, newts, and neonatal mice can regenerate cardiac tissue, largely by cardiac myocyte (CM) proliferation. It is unknown whether hearts of young large mammals can regenerate. METHODS: We examined the regenerative capacity of the pig heart in neonatal animals (ages 2, 3, or 14 days postnatal) after myocardial infarction or sham procedure. Myocardial scar and left ventricular function were determined by cardiac magnetic resonance imaging and echocardiography. Bromodeoxyuridine pulse-chase labeling, histology, immunohistochemistry, and Western blotting were performed to study cell proliferation, sarcomere dynamics, and cytokinesis and to quantify myocardial fibrosis. RNA-sequencing was also performed. RESULTS: After myocardial infarction, there was early and sustained recovery of cardiac function and wall thickness in the absence of fibrosis in 2-day-old pigs. In contrast, older animals developed full-thickness myocardial scarring, thinned walls, and did not recover function. Genome-wide analyses of the infarct zone revealed a strong transcriptional signature of fibrosis in 14-day-old animals that was absent in 2-day-old pigs, which instead had enrichment for cytokinesis genes. In regenerating hearts of the younger animals, up to 10% of CMs in the border zone of the myocardial infarction showed evidence of DNA replication that was associated with markers of myocyte division and sarcomere disassembly. CONCLUSIONS: Hearts of large mammals have regenerative capacity, likely driven by cardiac myocyte division, but this potential is lost immediately after birth.

The effect of realistic geometries on the susceptibility-weighted MR signal in white matter.

PURPOSE: To investigate the effect of realistic microstructural geometry on the susceptibility-weighted MR signal in white matter (WM), with application to demyelination. METHODS: Previous work has modeled susceptibility-weighted signals under the assumption that axons are cylindrical. In this study, we explored the implications of this assumption by considering the effect of more realistic geometries. A three-compartment WM model incorporating relevant properties based on the literature was used to predict the MR signal. Myelinated axons were modeled with several cross-sectional geometries of increasing realism: nested circles, warped/elliptical circles, and measured axonal geometries from electron micrographs. Signal simulations from the different microstructural geometries were compared with measured signals from a cuprizone mouse model with varying degrees of demyelination. RESULTS: Simulation results suggest that axonal geometry affects the MR signal. Predictions with realistic models were significantly different compared with circular models under the same microstructural tissue properties, for simulations with and without diffusion. CONCLUSION: The geometry of axons affects the MR signal significantly. Literature estimates of myelin susceptibility, which are based on fitting biophysical models to the MR signal, are likely to be biased by the assumed geometry, as will any derived microstructural properties. Magn Reson Med 79:489-500, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Spatial memory training induces morphological changes detected by manganese-enhanced MRI in the hippocampal CA3 mossy fiber terminal zone.

Hippocampal mossy fibers (MFs) can show plasticity of their axon terminal arbor consequent to learning a spatial memory task. Such plasticity is seen as translaminar sprouting from the stratum lucidum (SL) of CA3 into the stratum pyramidale (SP) and the stratum oriens (SO). However, the functional role of this presynaptic remodeling is still obscure. In vivo imaging that allows longitudinal observation of such remodeling could provide a deeper understanding of this presynaptic growth phenomenon as it occurs over time. Here we used manganese-enhanced magnetic resonance imaging (MEMRI), which shows a high-contrast area that co-localizes with the MFs. This technique was applied in the detection of learning-induced MF plasticity in two strains of rats. Quantitative analysis of a series of sections in the rostral dorsal hippocampus showed increases in the CA3a' area in MEMRI of trained Wistar rats consistent with the increased SO+SP area seen in the Timm's staining. MF plasticity was not seen in the trained Lister-Hooded rats in either MEMRI or in Timm's staining. This indicates the potential of MEMRI for revealing neuro-architectures and plasticity of the hippocampal MF system in vivo in longitudinal studies.

Robust hyperpolarized (13)C metabolic imaging with selective non-excitation of pyruvate (SNEP).

In vivo metabolic imaging using hyperpolarized [1-(13)C]pyruvate provides localized biochemical information and is particularly useful in detecting early disease changes, as well as monitoring disease progression and treatment response. However, a major limitation of hyperpolarized magnetization is its unrecoverable decay, due not only to T1 relaxation but also to radio-frequency (RF) excitation. RF excitation schemes used in metabolic imaging must therefore be able to utilize available hyperpolarized magnetization efficiently and robustly for the optimal detection of substrate and metabolite activities. In this work, a novel RF excitation scheme called selective non-excitation of pyruvate (SNEP) is presented. This excitation scheme involves the use of a spectral selective RF pulse to specifically exclude the excitation of [1-(13)C]pyruvate, while uniformly exciting the key metabolites of interest (namely [1-(13)C]lactate and [1-(13)C]alanine) and [1-(13)C]pyruvate-hydrate. By eliminating the loss of hyperpolarized [1-(13)C]pyruvate magnetization due to RF excitation, the signal from downstream metabolite pools is increased together with enhanced dynamic range. Simulation results, together with phantom measurements and in vivo experiments, demonstrated the improvement in signal-to-noise ratio (SNR) and the extension of the lifetime of the [1-(13)C]lactate and [1-(13)C]alanine pools when compared with conventional non-spectral selective (NS) excitation. SNEP has also been shown to perform comparably well with multi-band (MB) excitation, yet SNEP possesses distinct advantages, including ease of implementation, less stringent demands on gradient performance, increased robustness to frequency drifts and B0 inhomogeneity as well as easier quantification involving the use of [1-(13)C]pyruvate-hydrate as a proxy for the actual [1-(13)C] pyruvate signal. SNEP is therefore a promising alternative for robust hyperpolarized [1-(13)C]pyruvate metabolic imaging with high fidelity.

Detecting microstructural properties of white matter based on compartmentalization of magnetic susceptibility.

The microscopic structure of neuronal tissue is crucial to brain function, with axon diameter, axonal density and myelination directly influencing signal conduction in the white matter. There is increasing evidence that these microstructural properties alter signal in magnetic resonance imaging (MRI) driven by magnetic susceptibility of different compartments (e.g., myelin sheaths and iron-laden cells). To explain these observations, we have developed a multi-compartmental geometric model of whitematter microstructure. Using a single set of literature parameters, this forward model predicts experimentally observed orientation dependence and temporal evolution of the MRI signal. Where previous models have aimed to explain only the orientation dependence of signal phase, the proposed approach encapsulates the full repertoire of signal behavior. The frequency distribution underlying signal behavior is predicted to be a rich source of microstructural information with relevance to neuronal pathology.

Predictive mouse model reflects distinct stages of human atheroma in a single carotid artery.

Identification of patients with high-risk asymptomatic atherosclerotic plaques remains an elusive but essential step in preventing stroke. However, there is a lack of animal model that provides a reproducible method to predict where, when and what types of plaque formation, which fulfils the American Heart Association (AHA) histological classification of human plaques. We have developed a predictive mouse model that reflects different stages of human plaques in a single carotid artery by means of shear-stress modifying cuff. Validated with over 30000 histological sections, the model generates a specific pattern of plaques with different risk levels along the same artery depending on their position relative to the cuff. The further upstream of the cuff-implanted artery, the lower the magnitude of shear stress, the more unstable the plaques of higher grade according to AHA classification; with characteristics including greater degree of vascular remodeling, plaque size, plaque vulnerability and inflammation, resulting in higher risk plaques. By weeks 20 and 30, this model achieved 80% and near 100% accuracy respectively, in predicting precisely where, when and what stages/AHA types of plaques develop along the same carotid artery. This model can generate clinically-relevant plaques with varying phenotypes fulfilling AHA classification and risk levels, in specific locations of the single artery with near 100% accuracy of prediction. The model offers a promising tool for development of diagnostic tools to target high-risk plaques, increasing accuracy in predicting which individual patients may require surgical intervention to prevent stroke, paving the way for personalized management of carotid atherosclerotic disease.

Translational Molecular Imaging Tool of Vulnerable Carotid Plaque: Evaluate Effects of Statin Therapy on Plaque Inflammation and American Heart Association-Defined Risk Levels in Cuff-Implanted Apolipoprotein E-Deficient Mice.

Identification of high-risk carotid plaques in asymptomatic patients remains a challenging but crucial step in stroke prevention. The challenge is to accurately monitor the development of high-risk carotid plaques and promptly identify patients, who are unresponsive to best medical therapy, and hence targeted for carotid surgical interventions to prevent stroke. Inflammation is a key operator in destabilisation of plaques prior to clinical sequelae. Currently, there is a lack of imaging tool in routine clinical practice, which allows assessment of inflammatory activity within the atherosclerotic plaque. Herein, we have used a periarterial cuff to generate a progressive carotid atherosclerosis model in apolipoprotein E-deficient mice. This model produced clinically relevant plaques with different levels of risk, fulfilling American Heart Association (AHA) classification, at specific timepoints and locations, along the same carotid artery. Exploiting this platform, we have developed smart molecular magnetic resonance imaging (MRI) probes consisting of dual-targeted microparticles of iron oxide (DT-MPIO) against VCAM-1 and P-selectin, to evaluate the anti-inflammatory effect of statin therapy on progressive carotid atherosclerosis. We demonstrated that in vivo DT-MPIO-enhanced MRI can (i) quantitatively track plaque inflammation from early to advanced stage; (ii) identify and characterise high-risk inflamed, vulnerable plaques; and (iii) monitor the response to statin therapy longitudinally. Moreover, this molecular imaging-defined therapeutic response was validated using AHA classification of human plaques, a clinically relevant parameter, approximating the clinical translation of this tool. Further development and translation of this molecular imaging tool into the clinical arena may potentially facilitate more accurate risk stratification, permitting timely identification of the high-risk patients for prophylactic carotid intervention, affording early opportunities for stroke prevention in the future.

Paraspinal myopathy-induced intervertebral disc degeneration and thoracolumbar kyphosis in TSC1mKO mice model-a preliminary study.

BACKGROUND: Increasing kyphosis of the spine in a human is a well-recognized clinical phenomenon that has been associated with back pain, poor physical performance and disability. The pathophysiology of age-related kyphosis is complex and has been associated with physiological changes in vertebrae, intervertebral disc (IVD) and paraspinal musculature, which current cross-sectional studies are unable to demonstrate. Creating an in vivo, paraspinal myopathic animal model for longitudinal study of these changes under controlled conditions is thus warranted. PURPOSE: To confirm the TSC1 gene knockout effect on paraspinal muscle musculature; to analyze the development of spinal kyphosis, IVD degeneration and vertebra structural changes in a longitudinal manner to gain insights into the relationship between these processes. STUDY DESIGN: A prospective cohort study of 28 female mice, divided into 4 groups-9-month-old TSC1mKO (n=7), 9-month-old control (n=4), 12-month-old TSC1mKO (n=8), and 12-month-old controls (n=9). METHODS: High resolution micro-computed tomography was used to measure sagittal spinal alignment (Cobb's angle), vertebral height, vertebral body wedging, disc height index (DHI), disc wedge index (DWI), histomorphometry of trabecular bone and erector spinae muscle cross-sectional area. Paraspinal muscle specimens were harvested to assess for myopathic features with H&E stain, muscle fiber size, density of triangular fiber and central nucleus with WGA/DAPI stain, and percentage of fibers with PGC-1α stain. Intervertebral discs were evaluated for disc score using FAST stain. RESULTS: Compared to controls, paraspinal muscle sections revealed features of myopathy in TSC1mKO mice similar to human sarcopenic paraspinal muscle. While there was significantly greater presence of small triangular fiber and density of central nucleus in 9-and 12-month-old TSC1mKO mice, significantly larger muscle fibers and decreased erector spinae muscle cross-sectional area were only found in 12-month-old TSC1mKO mice compared to controls. TSC1mKO mice developed accelerated thoracolumbar kyphosis, with significantly larger Cobb angles found only at 12 months old. Structural changes to the trabecular bone in terms of higher bone volume fraction and quality, as well as vertebral body wedging were observed only in 12-month-old TSC1mKO mice when compared to controls. Disc degeneration was observed as early as 9 months in TSC1mKO mice and corresponded with disc wedging. However, significant disc height loss was only observed when comparing 12-month-old TSC1mKO mice with controls. CONCLUSIONS: This study successfully shows the TSC1 gene knockout effect on the development of paraspinal muscle myopathy in a mouse which is characteristic of sarcopenia. The TSC1mKO mice is by far the best model available to study the pathological consequence of sarcopenia on mice spine. With paraspinal muscle myopathy established as early as 9 months, TSC1mKO mice developed disc degeneration and disc wedging. This is followed by kyphosis of the spine at 12 months with concomitant disc height loss and vertebral body wedging due to bone remodeling. Age-related bone loss was not found in our study, suggesting osteoporosis and myopathy-induced vertebral body wedging are likely two independent processes. CLINICAL SIGNIFICANCE: This is the first study to provide key insights on the early and late consequences of paraspinal myopathy on intervertebral disc degeneration, spinal kyphosis, and vertebral body changes. With this new understanding, future studies evaluating therapies for spinal degeneration may be performed to develop time-sensitive interventions.

Dynamic Contrast-Enhanced Magnetic Resonance Imaging as Imaging Biomarker for Vascular Normalization Effect of Infigratinib in High-FGFR-Expressing Hepatocellular Carcinoma Xenografts.

PURPOSE: Overexpression of fibroblast growth factor receptor (FGFR) contributes to tumorigenesis, metastasis, and poor prognosis of hepatocellular carcinoma (HCC). Infigratinib-a pan-FGFR inhibitor-potently suppresses the growth of high-FGFR-expressing HCCs in part via alteration of the tumor microenvironment and vessel normalization. In this study, we aim to assess the utility of dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) as a non-invasive imaging technique to detect microenvironment changes associated with infigratinib and sorafenib treatment in high-FGFR-expressing HCC xenografts. PROCEDURES: Serial DCE-MRIs were performed on 12 nude mice bearing high-FGFR-expressing patient-derived HCC xenografts to quantify tumor microenvironment pre- (day 0) and post-treatment (days 3, 6, 9, and 15) of vehicle, sorafenib, and infigratinib. DCE-MRI data were analyzed using extended generalized kinetic model and two-compartment distributed parameter model. After treatment, immunohistochemistry stains were performed on the harvested tumors to confirm DCE-MRI findings. RESULTS: By treatment day 15, infigratinib induced tumor regression (70 % volume reduction from baseline) while sorafenib induced relative growth arrest (185 % volume increase from baseline versus 694 % volume increase from baseline of control). DCE-MRI analysis revealed different changes in microcirculatory parameters upon exposure to sorafenib versus infigratinib. While sorafenib induced microenvironment changes similar to those of rapidly growing tumors, such as a decrease in blood flow (F), fractional intravascular volume (vp), and permeability surface area product (PS), infigratinib induced the exact opposite changes as early as day 3 after treatment: increase in F, vp, and PS. CONCLUSIONS: Our study demonstrated that DCE-MRI is a reliable non-invasive imaging technique to monitor tumor microcirculatory response to FGFR inhibition and VEGF inhibition in high-FGFR-expressing HCC xenografts. Furthermore, the microcirculatory changes from FGFR inhibition manifested early upon treatment initiation and were reliably detected by DCE-MRI, creating possibilities of combinatorial therapy for synergistic effect.

Downregulating serine hydroxymethyltransferase 2 (SHMT2) suppresses tumorigenesis in human hepatocellular carcinoma.

Serine-glycine biosynthetic pathway diverts the glycolytic intermediate 3-phosphoglycerate to synthesize serine and glycine, of which the latter was found to correlate with cancer cell proliferation. Increased de novo biosynthesis of glycine by serine hydroxymethyltransferase 2 (SHMT2) is the central mechanism to fuel one-carbon pools supporting tumorigenesis. However, the therapeutic potential in targeting SHMT2 in hepatocellular carcinoma (HCC) is unknown. In this study we showed that SHMT2 inhibition significantly suppressed liver tumorigenesis. In vitro, SHMT2-knockdown was found to reduce cell growth and tumorigenicity in Huh-7 and HepG2 liver cancer cells. Moreover SHMT2-knockdown Huh-7 cells failed to form tumor xenograft after subcutaneous inoculation into nude mice. Similarly, inducible SHMT2 inhibition, via doxycycline-added drinking water, was found to reduce tumor incidence and tumor growth in a human tumor xenograft mouse model. SHMT2-knockdown increased the susceptibility of Huh-7 cells to doxorubicin suggesting its potential in combination chemotherapy. Through isotopomer tracing of [2-13C] glycine metabolism, we demonstrated that SHMT2 activity is associated with cancer phenotype. However, overexpression of SHMT2 was insufficient to transform immortalized hepatic cells to malignancy, suggesting that SHMT2 is one of the building blocks in liver cancer metabolism but does not initiate malignant transformation. Moreover, our results suggest that glycine, but not 5,10-methylenetetrahydrofolate, from the SHMT2-mediated enzymatic reaction is instrumental in tumorigenesis. Indeed, we found that SHMT2-knockdown cells exhibited increased glycine uptake. Taken together, our data suggest that SHMT2 may be a potential target in the treatment of human HCC.