Fully self-gated whole-heart 4D flow imaging from a 5-minute scan.

PURPOSE: To develop and validate an acquisition and processing technique that enables fully self-gated 4D flow imaging with whole-heart coverage in a fixed 5-minute scan. THEORY AND METHODS: The data are acquired continuously using Cartesian sampling and sorted into respiratory and cardiac bins using the self-gating signal. The reconstruction is performed using a recently proposed Bayesian method called ReVEAL4D. ReVEAL4D is validated using data from 8 healthy volunteers and 2 patients and compared with compressed sensing technique, L1-SENSE. RESULTS: Healthy subjects-Compared with 2D phase-contrast MRI (2D-PC), flow quantification from ReVEAL4D shows no significant bias. In contrast, the peak velocity and peak flow rate for L1-SENSE are significantly underestimated. Compared with traditional parallel MRI-based 4D flow imaging, ReVEAL4D demonstrates small but significant biases in net flow and peak flow rate, with no significant bias in peak velocity. All 3 indices are significantly and more markedly underestimated by L1-SENSE. Patients-Flow quantification from ReVEAL4D agrees well with the 2D-PC reference. In contrast, L1-SENSE markedly underestimated peak velocity. CONCLUSIONS: The combination of highly accelerated 5-minute Cartesian acquisition, self-gating, and ReVEAL4D enables whole-heart 4D flow imaging with accurate flow quantification.

CArtesian sampling with Variable density and Adjustable temporal resolution (CAVA).

PURPOSE: To develop a variable density Cartesian sampling method that allows retrospective adjustment of temporal resolution for dynamic MRI applications and to validate it in real-time phase contrast MRI (PC-MRI). THEORY AND METHODS: The proposed method, called CArtesian sampling with Variable density and Adjustable temporal resolution (CAVA), begins by producing a sequence of phase encoding indices based on the golden ratio increment. Then, variable density is introduced by nonlinear stretching of the indices. Finally, the elements of the resulting sequence are rounded up to the nearest integer. The performance of CAVA is evaluated using PC-MRI data from a pulsatile flow phantom and real-time, free-breathing data from ten healthy volunteers. RESULTS: CAVA enabled image recovery at various temporal resolutions that were selected retrospectively. For the pulsatile flow phantom, image quality and flow quantification accuracy from CAVA were comparable to that from another pseudo-random sampling pattern with fixed temporal resolution. In addition, flow quantification results based on CAVA were in good agreement with a breath-held segmented acquisition. CONCLUSIONS: By allowing retrospective binning of the MRI data, CAVA provides an avenue to retrospectively adjust the temporal resolution of PC-MRI.

A Bayesian approach for 4D flow imaging of aortic valve in a single breath-hold.

PURPOSE: To develop and validate a data processing technique that allows phase-contrast MRI-based 4D flow imaging of the aortic valve in a single breath-hold. THEORY AND METHODS: To regularize the ill-posed inverse problem, we extend a recently proposed 2D phase-contrast MRI method to 4D flow imaging. Adopting an empirical Bayes approach, spatial and temporal redundancies are exploited via sparsity in the wavelet domain, and the voxel-wise magnitude and phase structure across encodings is captured in a conditional mixture prior that applies regularizing constraints based on the presence of flow. We validate the proposed technique using data from a mechanical flow phantom and five healthy volunteers. RESULTS: The flow parameters derived from the proposed technique are in good agreement with those derived from reference datasets for both in vivo and mechanical flow experiments at accelerations rates as high as R = 27. Additionally, the proposed technique outperforms kt SPARSE-SENSE and a method that exploits spatio-temporal sparsity but does not utilize signal structure across encodings. CONCLUSIONS: Using the proposed technique, it is feasible to highly accelerate 4D flow acquisition and thus enable aortic valve imaging within a single breath-hold.

Quantification of aortic stenosis diagnostic parameters: comparison of fast 3 direction and 1 direction phase contrast CMR and transthoracic echocardiography.

BACKGROUND: Aortic stenosis (AS) is a common valvular disorder, and disease severity is currently assessed by transthoracic echocardiography (TTE). However, TTE results can be inconsistent in some patients, thus other diagnostic modalities such as cardiovascular magnetic resonance (CMR) are demanded. While traditional unidirectional phase-contrast CMR (1Dir PC-CMR) underestimates velocity if the imaging plane is misaligned to the flow direction, multi-directional acquisitions are expected to improve velocity measurement accuracy. Nonetheless, clinical use of multidirectional techniques has been hindered by long acquisition times. Our goal was to quantify flow parameters in patients using 1Dir PC-CMR and a faster multi-directional technique (3Dir PC-CMR), and compare to TTE. METHODS: Twenty-three patients were prospectively assessed with TTE and CMR. Slices above the aortic valve were acquired for both PC-CMR techniques and cine SSFP images were acquired to quantify left ventricular stroke volume. 3Dir PC-CMR implementation included a variable density sampling pattern with acceleration rate of 8 and a reconstruction method called ReVEAL, to significantly accelerate acquisition. 3Dir PC-CMR reconstruction was performed offline and ReVEAL-based image recovery was performed on the three (x, y, z) encoding pairs. 1Dir PC-CMR was acquired with GRAPPA acceleration rate of 2 and reconstructed online. CMR derived flow parameters and aortic valve area estimates were compared to TTE. RESULTS: ReVEAL based 3Dir PC-CMR derived parameters correlated better with TTE than 1Dir PC-CMR. Correlations ranged from 0.61 to 0.81 between TTE and 1Dir PC-CMR and from 0.61 to 0.87 between TTE and 3Dir-PC-CMR. The correlation coefficients between TTE, 1Dir and 3Dir PC-CMR Vpeakwere 0.81 and 0.87, respectively. In comparison to ReVEAL, TTE slightly underestimates peak velocities, which is not surprising as TTE is only sensitive to flow that is parallel to the acoustic beam. CONCLUSIONS: By exploiting structure unique to PC-CMR, ReVEAL enables multi-directional flow imaging in clinically feasible acquisition times. Results support the hypothesis that ReVEAL-based 3Dir PC-CMR provides better estimation of hemodynamic parameters in AS patients in comparison to 1Dir PC-CMR. While TTE can accurately measure velocity parallel to the acoustic beam, it is not sensitive to the other directions of flow. Therefore, multi-directional flow imaging, which encodes all three components of the velocity vector, can potentially outperform TTE in patients with eccentric or multiple jets.

A Bayesian model for highly accelerated phase-contrast MRI.

PURPOSE: Phase-contrast magnetic resonance imaging is a noninvasive tool to assess cardiovascular disease by quantifying blood flow; however, low data acquisition efficiency limits the spatial and temporal resolutions, real-time application, and extensions to four-dimensional flow imaging in clinical settings. We propose a new data processing approach called Reconstructing Velocity Encoded MRI with Approximate message passing aLgorithms (ReVEAL) that accelerates the acquisition by exploiting data structure unique to phase-contrast magnetic resonance imaging. THEORY AND METHODS: The proposed approach models physical correlations across space, time, and velocity encodings. The proposed Bayesian approach exploits the relationships in both magnitude and phase among velocity encodings. A fast iterative recovery algorithm is introduced based on message passing. For validation, prospectively undersampled data are processed from a pulsatile flow phantom and five healthy volunteers. RESULTS: The proposed approach is in good agreement, quantified by peak velocity and stroke volume (SV), with reference data for acceleration rates R≤10. For SV, Pearson r≥0.99 for phantom imaging (n = 24) and r≥0.96 for prospectively accelerated in vivo imaging (n = 10) for R≤10. CONCLUSION: The proposed approach enables accurate quantification of blood flow from highly undersampled data. The technique is extensible to four-dimensional flow imaging, where higher acceleration may be possible due to additional redundancy. Magn Reson Med 76:689-701, 2016. © 2015 Wiley Periodicals, Inc.

Ultra-structural identification of interstitial cells of Cajal in the zebrafish Danio rerio.

The interstitial cells of Cajal (ICCs) are important mediators of gastrointestinal (GI) motility because of their role as pacemakers in the GI tract. In addition to their function, ICCs are also structurally distinct cells most easily identified by their ultra-structural features and expression of the tyrosine kinase receptor c-KIT. ICCs have been described in mammals, rodents, birds, reptiles, and amphibians, but there are no reports at the ultra-structural level of ICCs within the GI tract of an organism from the teleost lineage. We describe the presence of cells in the muscularis of the zebrafish intestine; these cells have similar features to ICCs in other vertebrates. The ICC-like cells are associated with the muscularis, are more electron-dense than surrounding smooth muscle cells, possess long cytoplasmic processes and mitochondria, and are situated opposing enteric nervous structures. In addition, immunofluorescent and immunoelectron-microscopic studies with antibodies targeting the zebrafish ortholog of a putative ICC marker, c-KIT (kita), showed c-kit immunoreactivity in zebrafish ICCs. Taken together, these data represent the first ultra-structural characterization of cells in the muscularis of the zebrafish Danio rerio and suggest that ICC differentiation in vertebrate evolution dates back to the teleost lineage.

Increasing Robustness of Brain-Computer Interfaces Through Automatic Detection and Removal of Corrupted Input Signals.

For brain-computer interfaces (BCIs) to be viable for long-term daily usage, they must be able to quickly identify and adapt to signal disruptions. Furthermore, the detection and mitigation steps need to occur automatically and without the need for user intervention while also being computationally tractable for the low-power hardware that will be used in a deployed BCI system. Here, we focus on disruptions that are likely to occur during chronic use that cause some recording channels to fail but leave the remaining channels unaffected. In these cases, the algorithm that translates recorded neural activity into actions, the neural decoder, should seamlessly identify and adjust to the altered neural signals with minimal inconvenience to the user. First, we introduce an adapted statistical process control (SPC) method that automatically identifies disrupted channels so that both decoding algorithms can be adjusted, and technicians can be alerted. Next, after identifying corrupted channels, we demonstrate the automated and rapid removal of channels from a neural network decoder using a masking approach that does not change the decoding architecture, making it amenable for transfer learning. Finally, using transfer and unsupervised learning techniques, we update the model weights to adjust for the corrupted channels without requiring the user to collect additional calibration data. We demonstrate with both real and simulated neural data that our approach can maintain high-performance while simultaneously minimizing computation time and data storage requirements. This framework is invisible to the user but can dramatically increase BCI robustness and usability.

Dynamic detection and reversal of myocardial ischemia using an artificially intelligent bioelectronic medicine.

Myocardial ischemia is spontaneous, frequently asymptomatic, and contributes to fatal cardiovascular consequences. Importantly, myocardial sensory networks cannot reliably detect and correct myocardial ischemia on their own. Here, we demonstrate an artificially intelligent and responsive bioelectronic medicine, where an artificial neural network (ANN) supplements myocardial sensory networks, enabling reliable detection and correction of myocardial ischemia. ANNs were first trained to decode spontaneous cardiovascular stress and myocardial ischemia with an overall accuracy of ~92%. ANN-controlled vagus nerve stimulation (VNS) significantly mitigated major physiological features of myocardial ischemia, including ST depression and arrhythmias. In contrast, open-loop VNS or ANN-controlled VNS following a caudal vagotomy essentially failed to reverse cardiovascular pathophysiology. Last, variants of ANNs were used to meet clinically relevant needs, including interpretable visualizations and unsupervised detection of emerging cardiovascular stress. Overall, these preclinical results suggest that ANNs can potentially supplement deficient myocardial sensory networks via an artificially intelligent bioelectronic medicine system.

The alpha1H Ca2+ channel subunit is expressed in mouse jejunal interstitial cells of Cajal and myocytes.

T-type Ca(2+) currents have been detected in cells from the external muscular layers of gastrointestinal smooth muscles and appear to contribute to the generation of pacemaker potentials in interstitial cells of Cajal from those tissues. However, the Ca(2+) channel alpha subunit responsible for these currents has not been determined. We established that the alpha subunit of the alpha(1H) Ca(2+) channel is expressed in single myocytes and interstitial cells of Cajal using reverse transcription and polymerase chain reaction from whole tissue, laser capture microdissected tissue and single cells isolated from the mouse jejunum. Whole-cell voltage clamp recordings demonstrated that a nifedipine and Cd(2+) resistant, mibefradil-sensitive current is present in myocytes dissociated from the jejunum. Electrical recordings from the circular muscle layer demonstrated that mibefradil reduced the frequency and initial rate of rise of the electrical slow wave. Gene targeted knockout of both alleles of the cacna1h gene, which encodes the alpha(1H) Ca(2+) channel subunit, resulted in embryonic lethality because of death of the homozygous knockouts prior to E13.5 days in utero. We conclude that a channel with the pharmacological and molecular characteristics of the alpha(1H) Ca(2+) channel subunit is expressed in interstitial cells of Cajal and myocytes from the mouse jejunum, and that ionic conductances through the alpha(1H) Ca(2+) channel contribute to the upstroke of the pacemaker potential. Furthermore, the survival of mice that do not express the alpha(1H) Ca(2+) channel protein is dependent on the genetic background and targeting approach used to generate the knockout mice.

Expression and comparative genomics of two serum response factor genes in zebrafish.

Serum response factor (SRF) is a single copy, highly conserved transcription factor that governs the expression of hundreds of genes involved with actin cytoskeletal organization, cellular growth and signaling, neuronal circuitry and muscle differentiation. Zebrafish have emerged as a facile and inexpensive vertebrate model to delineate gene expression, regulation, and function, and yet the study of SRF in this animal has been virtually unexplored. Here, we report the existence of two srf genes in zebrafish, with partially overlapping patterns of expression in 3 and 7 day old developing animals. The mammalian ortholog (srf1) encodes for a 520 amino acid protein expressed in adult vascular and visceral smooth muscle cells, cardiac and skeletal muscle, as well as neuronal cells. The second zebrafish srf gene (srf2), encoding for a presumptive protein of only 314 amino acids, is transcribed at lower levels and appears to be less widely expressed across adult tissues. Both srf genes are induced by the SRF coactivator myocardin and attenuated with a short hairpin RNA to mammalian SRF. Promoter studies with srf1 reveal conserved CArG boxes that are the targets of SRF-myocardin in embryonic zebrafish cells. These results reveal that SRF was duplicated in the zebrafish genome and that its protein expression in all three muscle cell types is highly conserved across vertebrate animals suggesting an ancient code for transcriptional regulation of genes unique to muscle cell lineages.

Design and SAR of selective T-type calcium channel antagonists containing a biaryl sulfonamide core.

T-type calcium channel antagonists were designed using a protocol involving the program SPROUT and constrained by a ComFA-based pharmacophore model. Scaffolds generated by SPROUT were evaluated based on their ability to be translated into structures that were synthetically tractable. From this exercise, a novel series of potent and selective T-type channel antagonists containing a biaryl sulfonamide core were discovered.

Improved Imaging of Zebrafish Motility.

Zebrafish larvae are transparent and the entire gastrointestinal (GI) tract is easily visualized. Application of a new image analysis technique is reported in this issue of Neurogastroenterology and Motility (Neurogastroenterol Motil., 2018, volume 30, e13351). The technique quantifies movement in images collected in a timed sequence, and characterizes smooth muscle contractions based on contraction distance and frequency. The technique also reports the contraction amplitude, or the distance moved. This technique, and current spatiotemporal mapping techniques, are essential tools enabling characterization of GI motility patterns in intact physiological settings. Advances and development of transgenic zebrafish that lack pigmentation, with calcium reporters expressed in specific cell types, or with inactivation of specific genes contribute to our understanding of the generation, and regulation of GI motility at the molecular, cellular, and systemic level. Finally, development of chambers that immobilize zebrafish larvae for long-duration imaging will contribute to our technique toolbox, and will provide an increased experimental throughput.

Intestinal Transit Time and Cortisol-Mediated Stress in Zebrafish.

Intestinal motility, the spontaneous and rhythmic smooth muscle contraction, is a complex process that is regulated by overlapping and redundant regulatory mechanisms. Primary regulators intrinsic to the gastrointestinal tract include interstitial cells of Cajal, enteric neurons, and smooth muscle cells. Extrinsic primary regulators include the autonomic nervous system, immune system, and the endocrine system. Due to this complexity, a reductionist approach may be inappropriate if the ultimate goal is to understand motility regulation in vivo. Motility can be directly visualized in intact zebrafish, with intact regulatory systems, because larvae are transparent. Intestinal motility can therefore be measured in a complete system. However, the intestinal tract may respond to external influences, such as handling, which may invoke a stress response and influence intestinal transit. We used SR4G transgenic zebrafish, which express green fluorescent protein following activation of glucocorticoid receptors, and showed that handling required for the intestinal motility assay induces stress. Separate experiments showed that exogenous application of hydrocortisone did not influence intestinal transit, suggesting that handling may not interfere with transit measurements in intact zebrafish larvae. These experiments contribute to further development of the zebrafish model for intestinal motility research.

Discovery of a potent and novel motilin agonist.

A novel series of dihydro- and tetrahydrotriazolopyridazine-1,3-dione-based amino acid derivatives were identified as very potent motilin receptor agonists. Incorporating one additional phenylethyl glycinamide subunit to 1 (EC(50) = 660 nM) was found to improve in vitro potency approximately 3000-fold, resulting in compound 10 (EC(50) = 0.22 nM). The more potent enantiomer 11A has an EC(50) of 0.047 nM in the motilin receptor functional assay and a K(i) of 0.7 nM in the binding assay. In addition, compound 11A was shown to have a significantly reduced tendency to cause receptor desensitization as compared with the motilin receptor agonist ABT-229.

Kit signaling is required for development of coordinated motility patterns in zebrafish gastrointestinal tract.

Interstitial cells of Cajal (ICC) provide a pacemaker signal for coordinated motility patterns in the mammalian gastrointestinal (GI) tract. Kit signaling is required for development and maintenance of ICC, and these cells can be identified by Kit-like immunoreactivity. The zebrafish GI tract has two distinct ICC networks similar to mammals, suggesting a similar role in the generation of GI motility; however, a functional role for Kit-positive cells in zebrafish has not been determined. Analysis of GI motility in intact zebrafish larvae was performed during development and after disruption of Kit signaling. Development of coordinated motility patterns occurred after 5 days post-fertilization (dpf) and correlated with appearance of Kit-positive cells. Disruptions of Kit signaling using the Kit antagonist imatinib mesylate, and in Sparse, a null kita mutant, also disrupted development of coordinated motility patterns. These data suggest that Kit signaling is necessary for development of coordinated motility patterns and that Kit-positive cells in zebrafish are necessary for coordinated motility patterns.

Characterization of agonist-induced motilin receptor trafficking and its implications for tachyphylaxis.

The motilin receptor (MR) is a member of the seven-transmembrane receptor family and is expressed throughout the gastrointestinal tract of humans and other species. Motilin, the natural MR peptide ligand, has profound stimulatory effects on gastrointestinal contractility, indicating a therapeutic potential for MR modulators. However, long-term clinical use of certain MR agonists is limited by tachyphylaxis, a reduced responsiveness to repeated compound exposure. This study was meant to characterize the ligand-induced endocytosis of MR and to test whether receptor trafficking contributes to tachyphylaxis. A cell-based assay was developed by fusing a green fluorescent protein (GFP) moiety to the motilin receptor, and high-content biology instrumentation was used to quantify time and dose dependence of MR-GFP endocytosis. Maximal internalization of MR-GFP was induced after 45 min of constant exposure to 80 nM motilin. This process was disrupted by nocodazole, suggesting an essential role for microtubules. Internalized MR-GFP vesicles disappeared within 15 to 45 min of motilin withdrawal but did not overlap with the lysosomal compartment, indicating that MR-GFP escaped degradation and was recycled back to the plasma membrane. It is noteworthy that the kinetics of MR-GFP redistribution varied substantially when stimulated with motilin, erythromycin, 6,9-hemiacetal 8,9-anhydro-4''-deoxy-3'-N-desmethyl-3'-N-ethylerythromycin B (ABT-229), or N-[(1S)-1-[[[(1S)-1-(aminocarbonyl)-3-phenylpropyl]amino]carbonyl]-3-phenylpropyl]-2'-(1,3-benzodioxol-5-ylmethyl)tetrahydro-1',3'-dioxo-spiro[piperidine-4,5'(6'H)-[1H][1,2,4]triazolo[1,2-a]pyridazine]-8'-carboxamide (BMS-591348) at equipotent doses for Ca(2+)-mobilization. Retardation of the intracellular MR-GFP sorting cycle seemed to correlate with the tachyphylaxis-inducing properties of each compound, but not its EC(50). These results indicate that MR internalization, desensitization, and resensitization are ligand-dependent and that appropriate screening strategies may enable the development of small molecule agonists with ideal combinations of these distinct properties.

Ultrastructure of zebrafish dorsal aortic cells.

Expression of vascular smooth muscle cell (VSMC) markers such as serum response factor (SRF) is complicated in zebrafish because of the ill-defined histology of the dorsal aorta and the presence of perivascular pigment. We report the ultrastructure of aortic cells in 7-day, 1-month, and 3-month-old zebrafish and provide clear evidence for the presence of perivascular melanocytes harboring an abundance of melanin. In 7-day-old larvae, endothelial cells (EC) and synthetic mural cells that display little evidence of VSMC differentiation comprise the dorsal aorta. The latter mural cells appear to fully differentiate into VSMC by 1 month of age. In 3-month-old adult zebrafish, EC exhibit greater differentiation as evidenced by the accumulation of electron-dense bodies having a diameter of approximately 200 nm. Adult zebrafish aortae also exhibit at least one clear layer of VSMC with the characteristic array of membrane-associated dense plaques, myofilament bundles, and a basement membrane. Subjacent to VSMC are collagen-producing adventitial fibroblasts and melanocytes. These studies indicate that fully differentiated VSMC occur only after day 7 in zebrafish and that such cells are arranged in at least one lamellar unit circumscribing the endothelium. These findings provide new data about the timing and accumulation of VSMC around the zebrafish aorta, which will be useful in phenotyping mutant zebrafish that exhibit defects in blood circulation.

Local presentation of Steel factor increases expression of c-kit immunoreactive interstitial cells of Cajal in culture.

The binding of Steel factor (SF) to c-kit initiates a signaling pathway essential for development and maintenance of interstitial cells of Cajal (ICC). Soluble and membrane-bound isoforms of SF are expressed in the gastrointestinal tract, but the role for either isoform in supporting ICC development is unknown. The aim of this study was to determine the role of SF in supporting ICC in culture. ICC were cultured from dissociated mouse jejunum and grown with fibroblast cell lines that produced either soluble, membrane-bound or membrane-restricted SF. ICC were identified and counted by c-kit immunoreactivity. The number of c-kit immunoreactive cells was greater in the coculture system compared with cultures grown without SF-producing fibroblasts. All forms of SF-producing fibroblasts increased ICC number in culture but physical separation of the fibroblasts from the c-kit immunoreactive cells, the addition of exogenous SF to the culture medium, or fibroblast-conditioned media did not. These results are consistent with the hypothesis that the membrane-bound form of SF preferentially contributes to expression of c-kit-positive ICC under cell culture conditions.

Cytoskeletal modulation of sodium current in human jejunal circular smooth muscle cells.

A Na(+) current is present in human jejunal circular smooth muscle cells. The aim of the present study was to determine the role of the cytoskeleton in the regulation of the Na(+) current. Whole cell currents were recorded by using standard patch-clamp techniques with Cs(+) in the pipette to block K(+) currents. Cytochalasin D and gelsolin were used to disrupt the actin cytoskeleton and phalloidin to stabilize it. Colchicine was used to disassemble the microtubule cytoskeleton (and intermediate filaments) and paclitaxel to stabilize it. Acrylamide was used to disrupt the intermediate filament cytoskeleton. Perfusion of the recording chamber at 10 ml/min increased peak Na(+) current recorded from jejunal smooth muscle cells by 27 +/- 3%. Cytochalasin D and gelsolin abolished the perfusion-induced increase in Na(+) current, whereas incubation with phalloidin, colchicine, paclitaxel, or acrylamide had no effect. In conclusion, the Na(+) current expressed in human jejunal circular smooth muscle cells appears to be regulated by the cytoskeleton. An intact actin cytoskeleton is required for perfusion-induced activation of the Na(+) current.