Improved cellular uptake of perfluorocarbon nanoparticles for in vivo murine cardiac 19F MRS/MRI and temporal tracking of progenitor cells.

Herein, we maximize the labeling efficiency of cardiac progenitor cells (CPCs) using perfluorocarbon nanoparticles (PFCE-NP) and 19F MRI detectability, determine the temporal dynamics of single-cell label uptake, quantify the temporal viability/fluorescence persistence of labeled CPCs in vitro, and implement in vivo, murine cardiac CPC MRI/tracking that could be translatable to humans. FuGENEHD-mediated CPC PFCE-NP uptake is confirmed with flow cytometry/confocal microscopy. Epifluorescence imaging assessed temporal viability/fluorescence (up to 7 days [D]). Nonlocalized murine 19F MRS and cardiac MRI studied label localization in terminal/longitudinal tracking studies at 9.4 T (D1-D8). A 4-8 fold 19F concentration increase is evidenced in CPCs for FuGENE vs. directly labeled cells. Cardiac 19F signals post-CPC injections diminished in vivo to ~31% of their values on D1 by D7/D8. Histology confirmed CPC retention, dispersion, and macrophage-induced infiltration. Intra-cardiac injections of PFCE-NP-labeled CPCs with FuGENE can be visualized/tracked in vivo for the first time with 19F MRI.

Temporal accumulation and localization of isoflurane in the C57BL/6 mouse and assessment of its potential contamination in 19 F MRI with perfluoro-crown-ether-labeled cardiac progenitor cells at 9.4 Tesla.

PURPOSE: To assess the uptake, accumulation, temporal stability, and spatial localization of isoflurane (ISO) in the C57BL/6 mouse, and to identify its potential interference with the detection of labeled cardiac progenitor cells using 19 F MRI/MR spectroscopy (MRS). MATERIALS AND METHODS: Objectives are demonstrated using (a) in vitro ISO tests, (b) in vivo temporal accumulation/spatial localization C57BL/6 studies (n = 3), and (c) through injections of perfluoro-crown-ether (PFCE) labeled cardiac progenitor cells into femoral muscle areas of the murine hindlimb post-mortem (n = 1) using 1 H/19 F MRI/MRS at 9.4 Tesla. Data were acquired using double-gated spoiled gradient echo images and pulse-acquire spectra. For the in vivo study, the temporal stability of ISO resonances was quantified using coefficient of variability (CV) (5 min) estimates. RESULTS: Two ISO resonances were observed in vivo that correspond to the -CF3 and -OCHF2 moieties. CV values ranged between 3.2 and 6.4% (-CF3 ) and 6.4 and 11.2% (-OCHF2 ). Reductions of the ISO dose (2.0 to 1.7%) at 80 min postinduction had insignificant effects on ISO signals (P = 0.23; P = 0.71). PFCE-labeled cells exhibited a resonance at -16.25 ppm in vitro that did not overlap with the ISO resonances, a finding that is confirmed with MRS post-mortem using injected, labeled cells. Based on 19 F MRI, similar in vivo/post-mortem ISO compartmentalization was also confirmed in peripheral and thoracic skeletal muscles. CONCLUSION: Significant ISO accumulation was observed by 19 F MRS in vivo with temporally stable signals over 90 min postinduction. ISO effects on PFCE labels are anticipated to be minimal but may be more prominent for perfluoropolyether or perfluorooctyl bromide labels. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 1 J. MAGN. RESON. IMAGING 2017;45:1659-1667.

A high-resolution cardiovascular magnetic resonance diffusion tensor map from ex-vivo C57BL/6 murine hearts.

BACKGROUND: The complex cardiac fiber structural organization and spatial arrangement of cardiomyocytes in laminar sheetlets contributes greatly to cardiac functional and contractile ejection patterns. This study presents the first comprehensive, ultra-high resolution, fully quantitative statistical tensor map of the fixed murine heart at isotropic resolution of 43 µm using diffusion tensor (DT) cardiovascular magnetic resonance (CMR). METHODS: Imaging was completed in approximately 12 hours using a six-directional encoding scheme, in five ex vivo healthy C57BL/6 mouse hearts. The tensor map constructed from this data provides an average description of the murine fiber architecture visualized with fiber tractography, and its population variability, using the latest advances in image tensor analysis and statistics. RESULTS: Results show that non-normalized cardiac tensor maps are associated with mean fractional anisotropy of 0.25 ± 0.07 and mean diffusivity of 8.9 ± 1.6 × 10⁻4mm²/s. Moreover, average mid-ventricular helical angle distributions ranged between -41 ± 3° and +52 ± 5° and were highly correlated with transmural depth, in agreement with prior published results in humans and canines. Calculated variabilities of local myocyte orientations were 2.0° and 1.4°. Laminar sheet orientation variability was found to be less stable at 2.6°. Despite such variations, the murine heart seems to be highly structured, particularly when compared to canines and humans. CONCLUSIONS: This tensor map has the potential to yield an accurate mean representation and identification of common or unique features of the cardiac myocyte architecture, to establish a baseline standard reference of DTI indices, and to improve detection of biomarkers, especially in pathological states or post-transgenetic modifications.

Is There Preclinical and Clinical Value for 19F MRI in Stem Cell Cardiac Regeneration?

Cardiovascular regeneration aims to renew damaged or necrotic tissue and to enhance cardiac functional performance. Despite the hope arisen from the introduction and use of stem cells (SCs) as a novel cardiac regenerative approach, to-this-date, clinical trial findings are still ambivalent despite preclinical successes. Concurrently, noninvasive magnetic resonance imaging (MRI) advances have been based on nanotechnological breakthroughs that have (a) allowed fluorinated nanoparticles and ultrasmall iron oxide single-cell labeling, (b) explored imaging detection sensitivity limits (for preclinical/low-field clinical settings), and (c) accomplished cellular tracking in vivo. Nevertheless, outcomes have been far from ideal. Herein, the recently developed preclinical and clinical 1H and 19F MRI approaches for direct cardiac SC labeling techniques intended for cellular implantation and their potential for tracking these cells in health and infarcted states are summarized. To this extent, the potential preclinical and clinical values of 19F MRI and tracking of SCs for cardiac regeneration in myocardial infarction are questioned and challenged.

Correction: Fast, quantitative, murine cardiac 19F MRI/MRS of PFCE-labeled progenitor stem cells and macrophages at 9.4T.

[This corrects the article DOI: 10.1371/journal.pone.0190558.].

Fast, quantitative, murine cardiac 19F MRI/MRS of PFCE-labeled progenitor stem cells and macrophages at 9.4T.

PURPOSE: To a) achieve cardiac 19F-Magnetic Resonance Imaging (MRI) of perfluoro-crown-ether (PFCE) labeled cardiac progenitor stem cells (CPCs) and bone-derived bone marrow macrophages, b) determine label concentration and cellular load limits, and c) achieve spectroscopic and image-based quantification. METHODS: Theoretical simulations and experimental comparisons of spoiled-gradient echo (SPGR), rapid acquisition with relaxation enhancement (RARE), and steady state at free precession (SSFP) pulse sequences, and phantom validations, were conducted using 19F MRI/Magnetic Resonance Spectroscopy (MRS) at 9.4 T. Successful cell labeling was confirmed using flow cytometry and confocal microscopy. For CPC and macrophage concentration quantification, in vitro and post-mortem cardiac validations were pursued with the use of the transfection agent FuGENE. Feasibility of fast imaging is demonstrated in murine cardiac acquisitions in vivo, and in post-mortem murine skeletal and cardiac applications. RESULTS: SPGR/SSFP proved favorable imaging sequences yielding good signal-to-noise ratio values. Confocal microscopy confirmed heterogeneity of cellular label uptake in CPCs. 19F MRI indicated lack of additional benefits upon label concentrations above 7.5-10 mg/ml/million cells. The minimum detectable CPC load was ~500k (~10k/voxel) in two-dimensional (2D) acquisitions (3-5 min) using the butterfly coil. Additionally, absolute 19F based concentration and intensity estimates (trifluoroacetic-acid solutions, macrophages, and labeled CPCs in vitro and post-CPC injections in the post-mortem state) scaled linearly with fluorine concentrations. Fast, quantitative cardiac 19F-MRI was demonstrated with SPGR/SSFP and MRS acquisitions spanning 3-5 min, using a butterfly coil. CONCLUSION: The developed methodologies achieved in vivo cardiac 19F of exogenously injected labeled CPCs for the first time, accelerating imaging to a total acquisition of a few minutes, providing evidence for their potential for possible translational work.

Performance evaluation of GPU parallelization, space-time adaptive algorithms, and their combination for simulating cardiac electrophysiology.

The use of computer models as a tool for the study and understanding of the complex phenomena of cardiac electrophysiology has attained increased importance nowadays. At the same time, the increased complexity of the biophysical processes translates into complex computational and mathematical models. To speed up cardiac simulations and to allow more precise and realistic uses, 2 different techniques have been traditionally exploited: parallel computing and sophisticated numerical methods. In this work, we combine a modern parallel computing technique based on multicore and graphics processing units (GPUs) and a sophisticated numerical method based on a new space-time adaptive algorithm. We evaluate each technique alone and in different combinations: multicore and GPU, multicore and GPU and space adaptivity, multicore and GPU and space adaptivity and time adaptivity. All the techniques and combinations were evaluated under different scenarios: 3D simulations on slabs, 3D simulations on a ventricular mouse mesh, ie, complex geometry, sinus-rhythm, and arrhythmic conditions. Our results suggest that multicore and GPU accelerate the simulations by an approximate factor of 33×, whereas the speedups attained by the space-time adaptive algorithms were approximately 48. Nevertheless, by combining all the techniques, we obtained speedups that ranged between 165 and 498. The tested methods were able to reduce the execution time of a simulation by more than 498× for a complex cellular model in a slab geometry and by 165× in a realistic heart geometry simulating spiral waves. The proposed methods will allow faster and more realistic simulations in a feasible time with no significant loss of accuracy.

In Vivo Tracking and 1H/19F Magnetic Resonance Imaging of Biodegradable Polyhydroxyalkanoate/Polycaprolactone Blend Scaffolds Seeded with Labeled Cardiac Stem Cells.

Medium-chain length polyhydroxyalkanoates (MCL-PHAs) have demonstrated exceptional properties for cardiac tissue engineering (CTE) applications. Despite prior work on MCL-PHA/polycaprolactone (PCL) blends, optimal scaffold production and use as an alternative delivery route for controlled release of seeded cardiac progenitor cells (CPCs) in CTE applications in vivo has been lacking. We present herein applicability of MCL-PHA/PCL (95/5 wt %) blends fabricated as thin films with an improved performance compared to the neat MCL-PHA. Polymer characterization confirmed the chemical structure and composition of the synthesized scaffolds, while thermal, wettability, and mechanical properties were also investigated and compared in neat and porous counterparts. In vitro cytocompatibility studies were performed using perfluorocrown-ether-nanoparticle-labeled murine CPCs and studied using confocal microscopy and 19F magnetic resonance spectroscopy and magnetic resonance imaging (MRI). Seeded scaffolds were implanted and studied in the postmortem murine heart in situ and in two additional C57BL/6 mice in vivo (using single-layered and double-layered scaffolds) and imaged immediately after and at 7 days postimplantation. Superior MCL-PHA/PCL scaffold performance has been demonstrated compared to MCL-PHA through experimental comparisons of (a) morphological data using scanning electron microscopy and (b) contact angle measurements attesting to improved CPC adhesion, (c) in vitro confocal microscopy showing increased SC proliferative capacity, and (d) mechanical testing that elicited good overall responses. In vitro MRI results justify the increased seeding density, increased in vitro MRI signal, and improved MRI visibility in vivo, in the double-layered compared to the single-layered scaffolds. Histological evaluations [bright-field, cytoplasmic (Atto647) and nuclear (4',6-diamidino-2-phenylindole) stains] performed in conjunction with confocal microscopy imaging attest to CPC binding within the scaffold, subsequent release and migration to the neighboring myocardium, and increased retention in the murine myocardium in the case of the double-layered scaffold. Thus, MCL-PHA/PCL blends possess tremendous potential for controlled delivery of CPCs and for maximizing possible regeneration in myocardial infarction.

Molecular and Integrative Physiological Effects of Isoflurane Anesthesia: The Paradigm of Cardiovascular Studies in Rodents using Magnetic Resonance Imaging.

To-this-date, the exact molecular, cellular, and integrative physiological mechanisms of anesthesia remain largely unknown. Published evidence indicates that anesthetic effects are multifocal and occur in a time-dependent and coordinated manner, mediated via central, local, and peripheral pathways. Their effects can be modulated by a range of variables, and their elicited end-effect on the integrative physiological response is highly variable. This review summarizes the major cellular and molecular sites of anesthetic action with a focus on the paradigm of isoflurane (ISO) - the most commonly used anesthetic nowadays - and its use in prolonged in vivo rodent studies using imaging modalities, such as magnetic resonance imaging (MRI). It also presents established evidence for normal ranges of global and regional physiological cardiac function under ISO, proposes optimal, practical methodologies relevant to the use of anesthetic protocols for MRI and outlines the beneficial effects of nitrous oxide supplementation.

Elimination of mutual inductance in NMR phased arrays: the paddle design revisited.

This study proposes a method to empirically minimize mutual inductance, using passive end-ring circular paddles, with neighboring coil loops placed in a non-overlapped configuration. The proposed concepts are validated through B(1)-field simulations for resonant coils at f(o)=300.5 MHz, having various sizes (3-10 cm), and for paddles with sizes ranging from 16 to 30 mm, and bench tests on constructed 4×4cm(2) two- (1×2) and four-coil loop (2×2) planar arrays. Simulation results yield total mean percentage B(1)-field differences of only 7.03% between the two non-overlapping coil array configurations (paddles vs. no-paddles). Pair-wise comparisons of elicited mean B(1)-field differences from the use of different circular and rectangular paddle sizes, yield values <5.3%. Theoretical calculation of the normalized mutual coupling coefficient in the non-overlapped coil configuration reduces to almost zero with optimally sized-paddles having a radius of approximately 28% the coil's largest dimension. In the absence of paddles, differences in the split of resonance peaks of 9.9 MHz were observed for the two coils in the 1×2 array, which vanished with paddle placement. Single coil responses (unloaded/loaded) without paddles, and responses from array coils with use of optimally-sized paddles yielded quality factor ratios that ranged between 1.1-1.86 and 1.0-1.5, respectively. Phantom and mouse loaded reflection coefficients S(11)/S(22) were -16.7/-16.2dB and -28.2/-16.1 dB, for the two array loops, respectively. Under unloaded conditions and in the absence of paddles, split resonances were observed for the 1×2 array, yielding transmission coefficients of -5.5 to -8.1 dB, reversing to single resonance responses upon paddle placements, with transmission coefficients of -14.4 to -15.6 dB.

Morphological studies of the murine heart based on probabilistic and statistical atlases.

This study directly compares morphological features of the mouse heart in its end-relaxed state based on constructed morphometric maps and atlases using principal component analysis in C57BL/6J (n=8) and DBA (n=5) mice. In probabilistic atlases, a gradient probability exists for both strains in longitudinal locations from base to apex. Based on the statistical atlases, differences in size (49.8%), apical direction (15.6%), basal ventricular blood pool size (13.2%), and papillary muscle shape and position (17.2%) account for the most significant modes of shape variability for the left ventricle of the C57BL/6J mice. For DBA mice, differences in left ventricular size and direction (67.4%), basal size (15.7%), and position of papillary muscles (16.8%) account for significant variability.

Effects of isoflurane anesthesia on the cardiovascular function of the C57BL/6 mouse.

Isoflurane (ISO) is the most commonly used inhalational anesthetic for experimental interventions in mice and is preferred for imaging technologies that require the mouse to remain anesthetized for relatively long time periods. This study compares the stability of mean arterial pressure (MAP), heart rate (HR), and body temperature under ISO concentrations of 1%, 1.5%, and 2% (volume-to-volume, v/v) for up to 90 minutes postinduction. At all three levels of anesthesia, we examined evoked physiological responses to fractional inspiratory ratio variations of oxygen (FiO2) and nitrous oxide (N2O). In addition, we determined the hemodynamic effects of anesthesia on pH, glucose, insulin, glucocorticoids, and partial pressure of oxygen and of carbon dioxide in the blood (paO2, paCO2). The results indicate that the most appropriate ISO dose level was 1.5% v/v, yielding stable MAP and HR values comparable to those observed in the animal's conscious state, with a minute-to-minute variability in MAP and HR of .11%. Based on such recordings, the optimal FiO2 appeared to be 50%. The additional use of N2O was associated with higher and more stable values of MAP and HR. Arterial pH values were within the physiological range and varied between 7.20 and 7.43. ISO anesthesia at 1.5% v/v was also associated with mild hyperglycemia (+47%), whereas insulin levels and corticosteroids remained unaltered. We conclude that the application of isoflurane as an inhalational anesthetic in the mouse can be optimized to attain stable hemodynamics by administering it at 1.5% v/v and by supplementing it with N2O.

Murine cardiac catheterizations and hemodynamics: on the issue of parallel conductance.

Catheter-based measurements are extensively used nowadays in animal models to quantify global left ventricular (LV) cardiac function and hemodynamics. Conductance catheter measurements yield estimates of LV volumes. Such estimates, however, are confounded by the catheter's nonhomogeneous emission field and the contribution to the total conductance of surrounding tissue or blood conductance values (other than LV blood), a term often known as parallel conductance. In practice, in most studies, volume estimates are based on the assumptions that the catheter's electric field is homogeneous and that parallel conductance is constant, despite prior results showing that these assumptions are incorrect. This study challenges the assumption for spatial homogeneity of electric field excitation of miniature catheters and investigated the electric field distribution of miniature catheters in the murine heart, based on cardiac model-driven (geometric, lump component) simulations and noninvasive imaging, at both systolic and diastolic cardiac phases. Results confirm the nonuniform catheter emission field, confined spatially within the LV cavity and myocardium, falling to 10% of its peak value at the ring electrode surface, within 1.1-2.0 mm, given a relative tissue permittivity of 33,615. Additionally, <1% of power leaks were observed into surrounding cavities or organs at end-diastole. Temporally varying parallel conductance effects are also confirmed, becoming more prominent at end-systole.

INTERCOMPARISON OF PERFORMANCE OF RF COIL GEOMETRIES FOR HIGH FIELD MOUSE CARDIAC MRI.

Multi-turn spiral surface coils are constructed in flat and cylindrical arrangements and used for high field (7.1 T) mouse cardiac MRI. Their electrical and imaging performances, based on experimental measurements, simulations, and MRI experiments in free space, and under phantom, and animal loading conditions, are compared with a commercially available birdcage coil. Results show that the four-turn cylindrical spiral coil exhibits improved relative SNR (rSNR) performance to the flat coil counterpart, and compares fairly well with a commercially available birdcage coil. Phantom experiments indicate a 50% improvement in the SNR for penetration depths ≤ 6.1 mm from the coil surface compared to the birdcage coil, and an increased penetration depth at the half-maximum field response of 8 mm in the 4-spiral cylindrical coil case, in contrast to 2.9 mm in the flat 4-turn spiral case. Quantitative comparison of the performance of the two spiral coil geometries in anterior, lateral, inferior, and septal regions of the murine heart yield maximum mean percentage rSNR increases of the order of 27-167% in vivo post-mortem (cylindrical compared to flat coil). The commercially available birdcage outperforms the cylindrical spiral coil in rSNR by a factor of 3-5 times. The comprehensive approach and methodology adopted to accurately design, simulate, implement, and test radiofrequency coils of any geometry and type, under any loading conditions, can be generalized for any application of high field mouse cardiac MRI.