A Novel Concept of a Phased-Array HIFU Transducer Optimized for MR-Guided Hepatic Ablation: Embodiment and First In-Vivo Studies.

Purpose: High-intensity focused ultrasound (HIFU) is challenging in the liver due to the respiratory motion and risks of near-/far-field burns, particularly on the ribs. We implemented a novel design of a HIFU phased-array transducer, dedicated to transcostal hepatic thermo-ablation. Due to its large acoustic window and strong focusing, the transducer should perform safely for this application. Material and Methods: The new HIFU transducer is composed of 256 elements distributed on 5 concentric segments of a specific radius (either 100, 111, or 125 mm). It has been optimally shaped to fit the abdominal wall. The shape and size of the acoustic elements were optimized for the largest emitting surface and the lowest symmetry. Calibration tests have been conducted on tissue-mimicking gels under 3-T magnetic resonance (MR) guidance. In-vivo MR-guided HIFU treatment was conducted in two pigs, aiming to create thermal ablation deep in the liver without significant side effects. Imaging follow-up was performed at D0 and D7. Sacrifice and post-mortem macroscopic examination occurred at D7, with the ablated tissue being fixed for pathology. Results: The device showed -3-dB focusing capacities in a volume of 27 × 46 × 50 mm3 as compared with the numerical simulation volume of 18 × 48 × 60 mm3. The shape of the focal area was in millimeter-range agreement with the numerical simulations. No interference was detected between the HIFU sonication and the MR acquisition. In vivo, the temperature elevation in perivascular liver parenchyma reached 28°C above physiological temperature, within one breath-hold. The lesion was visible on Gd contrast-enhanced MRI sequences and post-mortem examination. The non-perfused volume was found in pig #1 and pig #2 of 8/11, 6/8, and 7/7 mm along the LR, AP, and HF directions, respectively. No rib burns or other near-field side effects were visually observed on post-mortem gross examination. High-resolution contrast-enhanced 3D MRI indicated a minor lesion on the sternum. Conclusion: The performance of this new HIFU transducer has been demonstrated in vitro and in vivo. The transducer meets the requirement to perform thermal lesions in deep tissues, without the need for rib-sparing means.

Diffusion-magnetic resonance imaging predicts decline of kidney function in chronic kidney disease and in patients with a kidney allograft.

Kidney cortical interstitial fibrosis is highly predictive of kidney prognosis and is currently assessed by evaluation of a biopsy. Diffusion-weighted magnetic resonance imaging is a promising non-invasive tool to evaluate kidney fibrosis. We recently adapted diffusion-weighted imaging sequence for discrimination between the kidney cortex and medulla and found that the cortico-medullary difference in apparent diffusion coefficient (ΔADC) correlated with histological interstitial fibrosis. Here, we assessed whether ΔADC as measured with diffusion-weighted magnetic resonance imaging is predictive of kidney function decline and dialysis initiation in chronic kidney disease (CKD) and patients with a kidney allograft in a prospective study encompassing 197 patients. We measured ΔADC in 43 patients with CKD (estimated GFR (eGFR) 55ml/min/1.73m2) and 154 patients with a kidney allograft (eGFR 53ml/min/1.73m2). Patients underwent a kidney biopsy and diffusion-weighted magnetic resonance imaging within one week of biopsy; median follow-up of 2.2 years with measured laboratory parameters. The primary outcome was a rapid decline of kidney function (eGFR decline over 30% or dialysis initiation) during follow up. Significantly, patients with a negative ΔADC had 5.4 times more risk of rapid decline of kidney function or dialysis (95% confidence interval: 2.29-12.58). After correction for kidney function at baseline and proteinuria, low ADC still predicted significant kidney function loss with a hazard ratio of 4.62 (95% confidence interval 1.56-13.67) independent of baseline age, sex, eGFR and proteinuria. Thus, low ΔADC can be a predictor of kidney function decline and dialysis initiation in patients with native kidney disease or kidney allograft, independent of baseline kidney function and proteinuria.

Vascular-targeted micelles as a specific MRI contrast agent for molecular imaging of fibrin clots and cancer cells.

Molecular medical imaging is intended to increase the accuracy of diagnosis, particularly in cardiovascular and cancer-related diseases, where early detection could significantly increase the treatment success rate. In this study, we present mixed micelles formed from four building blocks as a magnetic resonance imaging targeted contrast agent for the detection of atheroma and cancer cells. The building blocks are a gadolinium-loaded DOTA ring responsible for contrast enhancement, a fibrin-specific CREKA pentapeptide responsible for targeting, a fluorescent dye and DSPE-PEG2000. The micelles were fully characterized in terms of their size, zeta potential, stability, relaxivity and toxicity. Target binding assays performed on fibrin clots were quantified by fluorescence and image signal intensities and proved the binding power. An additional internalization assay showed that the micelles were also designed to specifically enter into cancer cells. Overall, these multimodal mixed micelles represent a potential formulation for MRI molecular imaging of atheroma and cancer cells.

Enhancement of HIFU thermal therapy in perfused tissue models using micron-sized FTAC-stabilized PFOB-core endovascular sonosensitizers.

BACKGROUND: High intensity focused ultrasound (HIFU) is clinically accepted for the treatment of solid tumors but remains challenging in highly perfused tissue due to the heat sink effect. Endovascular liquid-core sonosensitizers have been previously suggested to enhance the thermal energy deposition at the focal area and to lower the near-/far-field heating. We are investigating the therapeutic potential of PFOB-FTAC micro-droplets in a perfused tissue-mimicking model and postmortem excised organs. METHOD: A custom-made in vitro perfused tissue-mimicking model, freshly excised pig kidneys (n = 3) and liver (n = 1) were perfused and subjected to focused ultrasound generated by an MR-compatible HIFU transducer. PFOB-FTAC sonosensitizers were injected in the perfusion fluid up to 0.235% v/v ratio. Targeting and on-line PRFS thermometry were performed on a 3 T MR scanner. Assessment of the fluid perfusion was performed with pulsed color Doppler in vitro and with dynamic contrast-enhanced (DCE)-MRI in excised organs. RESULTS: Our in vitro model of perfused tissue demonstrated re-usability. Sonosensitizer concentration and perfusion rate were tunable in situ. Differential heating under equivalent HIFU sonications demonstrated a dramatic improvement in the thermal deposition due to the sonosensitizers activity. Typically, the energy deposition was multiplied by a factor between 2.5 and 3 in perfused organs after the administration of micro-droplets, while DCE-MRI indicated an effective perfusion. CONCLUSION: The current PFOB-FTAC micro-droplet sonosensitizers provided a large and sustained enhancement of the HIFU thermal deposition at the focal area, suggesting solutions for less technological constraints, lower risk for the near-/far- field heating. We also report a suitable experimental model for other MRgHIFU studies.

Validation of the corticomedullary difference in magnetic resonance imaging-derived apparent diffusion coefficient for kidney fibrosis detection: a cross-sectional study.

BACKGROUND: Kidney cortical interstitial fibrosis (IF) is highly predictive of renal prognosis and is currently assessed by the evaluation of a biopsy. Diffusion magnetic resonance imaging (MRI) is a promising tool to evaluate kidney fibrosis via the apparent diffusion coefficient (ADC), but suffers from inter-individual variability. We recently applied a novel MRI protocol to allow calculation of the corticomedullary ADC difference (ΔADC). We here present the validation of ΔADC for fibrosis assessment in a cohort of 164 patients undergoing biopsy and compare it with estimated glomerular filtration rate (eGFR) and other plasmatic parameters for the detection of fibrosis. METHODS: This monocentric cross-sectional study included 164 patients undergoing renal biopsy at the Nephrology Department of the University Hospital of Geneva between October 2014 and May 2018. Patients underwent diffusion-weighted imaging, and T1 and T2 mappings, within 1 week after biopsy. MRI results were compared with gold standard histology for fibrosis assessment. RESULTS: Absolute cortical ADC or cortical T1 values correlated poorly to IF assessed by the biopsy, whereas ΔADC was highly correlated to IF (r=-0.52, P < 0.001) and eGFR (r = 0.37, P < 0.01), in both native and allograft patients. ΔT1 displayed a lower, but significant, correlation to IF and eGFR, whereas T2 did not correlate to IF nor to eGFR. ΔADC, ΔT1 and eGFR were independently associated with kidney fibrosis, and their combination allowed detection of extensive fibrosis with good specificity. CONCLUSION: ΔADC is better correlated to IF than absolute cortical or medullary ADC values. ΔADC, ΔT1 and eGFR are independently associated to IF and allow the identification of patients with extensive IF.

Cardiomyocyte-Specific JunD Overexpression Increases Infarct Size following Ischemia/Reperfusion Cardiac Injury by Downregulating Sirt3.

Ischemia/reperfusion (I/R) injury in acute myocardial infarction activates several deleterious molecular mechanisms. The transcription factor JunD regulates pathways involved in oxidative stress as well as in cellular proliferation, differentiation, and death. The present study investigated the potential role of JunD as a modulator of myocardial injury pathways in a mouse model of cardiac I/R injury. Infarct size, systemic and local inflammation, and production of reactive oxygen species, as well as cytosolic and mitochondrial apoptotic pathways were investigated in adult males after myocardial I/R. In wild-type (WT) mice, 30 minutes after ischemia and up to 24 hours following reperfusion, cardiac JunD messenger ribonucleic acid expression was reduced while JunB increased. Cardiac-specific JunD overexpressing mice (JunDTg/0 ) displayed larger infarcts compared with WT. However, postischemic inflammatory or oxidative responses did not differ. JunD overexpression reduced Sirt3 transcription by binding to its promoter, thus leading to mitochondrial dysfunction, myocardial cell death, and increased infarct size. On the other hand, JunD silencing reduced, while Sirt3 silencing increased infarct size. In human myocardial autopsy specimens, JunD-positive areas within the infarcted left ventricle staining corresponded to undetectable Sirt3 areas in consecutive sections of the same heart. Cardiac-specific JunD overexpression increases myocardial infarct size following I/R. These effects are mediated via Sirt3 transcriptional repression, mitochondrial swelling, and increased apoptosis, suggesting that JunD is a key regulator of myocardial I/R injury. The present data set the stage for further investigation of the potential role of Sirt3 activation as a novel target for the treatment of acute myocardial infarction.

Mild hyperthermia by MR-guided focused ultrasound in an ex vivo model of osteolytic bone tumour: optimization of the spatio-temporal control of the delivered temperature.

BACKGROUND: Magnetic resonance guided focused ultrasound was suggested for the induction of deep localized hyperthermia adjuvant to radiation- or chemotherapy. In this study we are aiming to validate an experimental model for the induction of uniform temperature elevation in osteolytic bone tumours, using the natural acoustic window provided by the cortical breakthrough. MATERIALS AND METHODS: Experiments were conducted on ex vivo lamb shank by mimicking osteolytic bone tumours. The cortical breakthrough was exploited to induce hyperthermia inside the medullar cavity by delivering acoustic energy from a phased array HIFU transducer. MR thermometry data was acquired intra-operatory using the proton resonance frequency shift (PRFS) method. Active temperature control was achieved via a closed-loop predictive controller set at 6 °C above the baseline. Several beam geometries with respect to the cortical breakthrough were investigated. Numerical simulations were used to further explain the observed phenomena. Thermal safety of bone heating was assessed by cross-correlating MR thermometry data with the measurements from a fluoroptic temperature sensor inserted in the cortical bone. RESULTS: Numerical simulations and MR thermometry confirmed the feasibility of spatio-temporal uniform hyperthermia (± 0.5 °C) inside the medullar cavity using a fixed focal point sonication. This result was obtained by the combination of several factors: an optimal positioning of the focal spot in the plane of the cortical breakthrough, the direct absorption of the HIFU beam at the focal spot, the "acoustic oven effect" yielded by the beam interaction with the bone, and a predictive temperature controller. The fluoroptical sensor data revealed no heating risks for the bone and adjacent tissues and were in good agreement with the PRFS thermometry from measurable voxels adjacent to the periosteum. CONCLUSION: To our knowledge, this is the first study demonstrating the feasibility of MR-guided focused ultrasound hyperthermia inside the medullar cavity of bones affected by osteolytic tumours. Our results are considered a promising step for combining adjuvant mild hyperthermia to external beam radiation therapy for sustained pain relief in patients with symptomatic bone metastases.

Micron-sized PFOB liquid core droplets stabilized with tailored-made perfluorinated surfactants as a new class of endovascular sono-sensitizers for focused ultrasound thermotherapy.

The purpose of this study was to develop micron-sized droplet emulsions able to increase the heat deposition of high intensity focused ultrasound (HIFU), aiming to accelerate the tumour ablation in highly perfused organs with reduced side effects. The investigated droplets consisted of a perfluorooctyl bromide (PFOB) core coated with a biocompatible fluorinated surfactant called F-TAC. The novelty of this work relies on the use, for this application, of a high boiling point perfluorocarbon core (142 °C), combined with an in-house fluorinated surfactant to formulate the emulsion, yielding quasi-reversible strong interactions between the HIFU beam and the droplets. In order to fine-tune the emulsion size, surfactants with different hydrophobic/hydrophilic ratios were screened. Different concentrations of PFOB droplets were homogeneously embedded in two different MRI compatible materials, exhibiting either ultrasound (US) absorbing or non-absorbing properties. For the US absorbing TMM, the speed of sound at each droplet concentration was also assessed. These TMM were sonicated by 1 MHz HIFU with acoustical power of 94 W at two different duty cycles. The temperature elevation was monitored accurately by MRI proton shift resonance frequency in near real-time. The presence of sono-sensitive droplets induced a significant increase of the HIFU thermal effect that persisted under repeated sonication of the same locus. Optimal enhancement was observed at the lowest concentration tested (0.1%) with an additional temperature rise at the focal point of approximately 4 °C per applied kJ of acoustic energy corresponding to one order of magnitude augmentation of the thermal dose. Furthermore, no deformation of the heating pattern pre- or post-focal was observed.

Molecular oxygen loading in candidate theranostic droplets stabilized with biocompatible fluorinated surfactants: Particle size effect and application to in situ19F MRI mapping of oxygen partial pressure.

OBJECTIVE: Perfluorocarbon nano- and micron-sized emulsions are a new field of investigation in cancer treatment due to their ability to be used as imaging contrast agents, or as delivery vectors for pharmaceuticals. They also demonstrated capability to enhance the efficiency of high intensity focused ultrasound thermo-therapy. In the context of new biomedical applications we investigated perfluorooctyl bromide (PFOB) theranostic droplets using 19F NMR. Each droplet contains biocompatible fluorinated surfactants composed of a polar Tris(hydroxymethyl)aminomethane head unit and hydrophobic perfluorinated tail (abbreviated as F-TAC). The influence of the droplet size on the oxygen loading capacity was determined from longitudinal relaxation (T1) data of 19F NMR signal. MATERIAL AND METHODS: Liquid PFOB and five samples of PFOB droplets of average diameter 0.177, 0.259, 1.43, 3.12 and 4.53 µm were tested with different oxygen levels. A dedicated gas exchange system was validated to maintain steady state oxygen concentrations, including a spatial gradient of oxygen concentration. A prototyped transmit-receive switchable 19F/1H quadrature coil was integrated on a 3 T clinical scanner. The coil is compatible with focused ultrasound sonication for future application. A spectroscopy FID inversion-recovery (IR) sequence was used to measure the T1 value per sample and per value of equilibrium oxygen pressure. Pixel wise, spatial T1 mapping was performed with magnetization prepared 2D gradient echo sequences in tissue mimicking gels doped with theranostic droplets. RESULTS: Experimental data indicated that the longitudinal relaxation rate of 19F signal of the investigated theranostic droplets depended approximately linearly on the oxygen level and its slope decreased with the particle size according to a second order polynomial over the investigated range. This semi-empirical model was derived from general thermodynamics and weak electrostatic forces theory and fitted the experimental data within 0.75% precision. The capacity of oxygen transportation for the described theranostic droplets tended to that of pure PFOB, while micron-sized droplets lost up to 50% of this capacity. In a specific setup producing a steady state gradient of oxygen concentration, we demonstrated spatial mapping of oxygen pressure gradient of 6 kPa/mm with 1 mm in-plane resolution. CONCLUSION: The size-tunable PFOB theranostic droplets stabilized with F-TAC surfactants could be characterized by 19F MRI in a clinical setup readily compatible with interventional in vivo studies under MR guidance. Current precision and spatial resolution of T1 mapping are promising. A potential challenge for further in vivo studies is the reduction of the imaging time.

Dynamic Volume Assessment of Hepatocellular Carcinoma in Rat Livers Using a Clinical 3T MRI and Novel Segmentation.

PURPOSE: In vivo liver cancer research commonly uses rodent models. One of the limitations of such models is the lack of accurate and reproducible endpoints for a dynamic assessment of growing tumor nodules. The aim of this study was to validate a noninvasive, true volume segmentation method using two rat hepatocellular carcinoma (HCC) models, correlating magnetic resonance imaging (MRI) with histological volume measurement, and with blood levels of α-fetoprotein. MATERIALS AND METHODS: We used 3T clinical MRI to quantify tumor volume with follow-up over time. Using two distinct rat HCC models, calculated MRI tumor volumes were correlated with volumes from histological sections, or with blood levels of α-fetoprotein. Eleven rats, comprising six Buffalo rats (n = 9 scans) and five Fischer rats (n = 14 tumors), were injected in the portal vein with 2.5 × 105 and 2.0 × 106 syngeneic HCC cells, respectively. Longitudinal (T1) relaxation time- and transverse (T2) relaxation time-weighted MR images were acquired. RESULTS: The three-dimensional (3D) T1-weighted gradient echo had 0.35-mm isotropic resolution allowing accurate semi-automatic volume segmentation. 2D T2-weighted imaging provided high tumor contrast. Segmentation of combined 3D gradient echo T1-weighted images and 2D turbo spin echo T2-weighted images provided excellent correlation with histology (y = 0.866x + 0.034, R² = 0.997 p < .0001) and with α-fetoprotein (y = 0.736x + 1.077, R² = 0.976, p < .0001). There was robust inter- and intra-observer reproducibility (intra-class correlation coefficient > 0.998, p < .0001). CONCLUSIONS: We have developed a novel, noninvasive contrast imaging protocol which enables semi-automatic 3D volume quantification to analyze nonspherical tumor nodules and to follow up the growth of tumor nodules over time.

Hybrid ultrasound-MR guided HIFU treatment method with 3D motion compensation.

PURPOSE: Treatments using high-intensity focused ultrasound (HIFU) in the abdominal region remain challenging as a result of respiratory organ motion. A novel method is described here to achieve 3D motion-compensated ultrasound (US) MR-guided HIFU therapy using simultaneous ultrasound and MRI. METHODS: A truly hybrid US-MR-guided HIFU method was used to plan and control the treatment. Two-dimensional ultrasound was used in real time to enable tracking of the motion in the coronal plane, whereas an MR pencil-beam navigator was used to detect anterior-posterior motion. Prospective motion compensation of proton resonance frequency shift (PRFS) thermometry and HIFU electronic beam steering were achieved. RESULTS: The 3D prospective motion-corrected PRFS temperature maps showed reduced intrascan ghosting artifacts, a high signal-to-noise ratio, and low geometric distortion. The k-space data yielded a consistent temperature-dependent PRFS effect, matching the gold standard thermometry within approximately 1°C. The maximum in-plane temperature elevation ex vivo was improved by a factor of 2. Baseline thermometry acquired in volunteers indicated reduction of residual motion, together with an accuracy/precision of near-harmonic referenceless PRFS thermometry on the order of 0.5/1.0°C. CONCLUSIONS: Hybrid US-MR-guided HIFU ablation with 3D motion compensation was demonstrated ex vivo together with a stable referenceless PRFS thermometry baseline in healthy volunteer liver acquisitions. Magn Reson Med 79:2511-2523, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

4D cardiac imaging at clinical 3.0T provides accurate assessment of murine myocardial function and viability.

OBJECTIVES: We validate a 4D strategy tailored for 3T clinical systems to simultaneously quantify function and infarct size in wild type mice after ischemia/reperfusion, with improved spatial and temporal resolution by comparison to previous published protocols using clinical field MRI systems. METHODS: C57BL/6J mice underwent 60min ischemia/reperfusion (n=14) or were controls without surgery (n=6). Twenty-four hours after surgery mice were imaged with gadolinium injection and sacrificed for post-mortem MRI and histology with serum also taken for Troponin I levels. The double ECG- and respiratory-triggered 3D FLASH (Fast Low Angle Shot) gradient echo (GRE) cine sequence had an acquired isotropic resolution of 344µm, TR/TE of 7.8/2.9ms and acquisition time 25-35min. The conventional 2D FLASH cine sequence had the same in-plane resolution of 344µm, 1mm slice thickness and TR/TE 11/5.4ms for an acquisition time of 20-25min plus 5min for planning. Left ventricle (LV) and right ventricle (RV) volumes were measured and functional parameters compared 2D to 3D, left to right and for inter and intra observer reproducibility. MRI infarct volume was compared to histology. RESULTS: For the function evaluation, the 3D cine outperformed 2D cine for spatial and temporal resolution. Protocol time for the two methods was equivalent (25-35min). Flow artifacts were reduced (p=0.008) and epi/endo-cardial delineation showed good intra and interobserver reproducibility. Paired t-test comparing ejection volume left to right showed no significant difference for 3D (p=0.37), nor 2D (p=0.30) and correlation slopes of left to right EV were 1.17 (R2=0.75) for 2D and 1.05 (R2=0.50) for 3D. Quantifiable 'late gadolinium enhancement' infarct volume was seen only with the 3D cine and correlated to histology (R2=0.89). Left ejection fraction and MRI-measured infarct volume correlated (R2>0.3). CONCLUSIONS: The 4D strategy, with contrast injection, was validated in mice for function and infarct quantification from a single scan with minimal slice planning.

Treatment with anti-RANKL antibody reduces infarct size and attenuates dysfunction impacting on neutrophil-mediated injury.

Selective pharmacological treatments targeting reperfusion injury produced modest protective effects and might be associated with immunosuppression. In order to identify novel and better-tolerated approaches, we focused on the neutralization of receptor activator of nuclear factor kappa-B ligand [RANKL], a cytokine recently shown to activate inflammatory cells (i.e. neutrophils) orchestrating post-infarction injury and repair. Myocardial ischemia (60min) and reperfusion injury was surgically induced in C57Bl/6 mice. In hearts and serum, RANKL was early upregulated during reperfusion. A "one-shot" injection with neutralizing anti-RANKL IgG during ischemia ameliorated myocardial infarct size and function, but not adverse remodeling (determined by Magnetic Resonance Imaging [MRI]) as compared to Vehicle or control IgG. These beneficial effects were accompanied in vivo by reduction in cardiac neutrophil infiltration, reactive oxygen species (ROS) and MMP-9 release. Anti-RANKL IgG treatment suppressed sudden peak of neutrophil granule products in mouse serum early after reperfusion onset. In vitro, RANK mRNA expression was detected in isolated mouse neutrophils. Co-incubation with neutralizing anti-RANKL IgG abrogated RANKL-induced mouse neutrophil degranulation and migration, suggesting a critical role of RANKL in neutrophil-mediated injury. Conversely, anti-RANKL IgG did not affect salvage pathways in cardiac cells (i.e. ERK p42/p44, Akt and STAT-3) or macrophage cardiac infiltration. Finally, treatment with anti-RANKL IgG showed no effect on B and T lymphocyte polarization (in serum, spleen and infarcted myocardium) and circulating chemokines as compared with Vehicle or control IgG. In conclusion, acute treatment with anti-RANKL IgG improved cardiac infarct size and function by potentially impacting on neutrophil-mediated injury and repair.

Accumulation of amino-polyvinyl alcohol-coated superparamagnetic iron oxide nanoparticles in bone marrow: implications for local stromal cells.

AIMS: First, it will be investigated if amino-polyvinyl alcohol-coated superparamagnetic iron oxide nanoparticles (A-PVA-SPIONs) are suitable for MRI contrast enhancement in bone marrow. Second, the impact of A-PVA-SPION exposure in vivo on the viability and key functions of local bone marrow stromal cells (BMSCs) will be investigated. MATERIAL & METHODS: Animals were systemically injected with A-PVA-SPIONs, followed by a 7-day survival time. Accumulation of A-PVA-SPIONs was confirmed by MRI, histology and inductively coupled plasma optical emission spectrometry. BMSCs were isolated from bone marrow for in vitro assessment of their viability and regenerative key functions. RESULTS: In this study, A-PVA-SPIONs were found to accumulate in bone marrow and increase the BMSCs' metabolic activity and migration rate. CONCLUSION: A-PVA-SPIONs appear suitable for contrast enhancement in bone marrow while our data suggest an influence on the BMSCs biology that necessitates future research.

The in-vivo use of superparamagnetic iron oxide nanoparticles to detect inflammation elicits a cytokine response but does not aggravate experimental arthritis.

BACKGROUND: Superparamagnetic Iron Oxide Nanoparticles (SPION) are used in diagnostic imaging of a variety of different diseases. For such in-vivo application, an additional coating with a polymer, for example polyvinyl alcohol (PVA), is needed to stabilize the SPION and prevent aggregation. As the particles are foreign to the body, reaction against the SPION could occur. In this study we investigated the effects that SPION may have on experimental arthritis after intra-articular (i.a.) or intravenous (i.v.) injection. METHODS: PVA-coated SPION were injected either i.a. (6 or 24 µg iron) or i.v. (100 µg or 1 mg iron) into naïve Toll-like receptor-4 deficient (TLR4-/-) or wild-type C57Bl/6 mice, or C57Bl/6 mice with antigen-induced arthritis. As control, some mice were injected with PVA or PBS. MR imaging was performed at 1 and 7 days after injection. Mice were sacrificed 2 hours and 1, 2, 7, 10 and 14 days after injection of the SPION, and RNA from synovium and liver was isolated for pro-inflammatory gene expression analysis. Serum cytokine measurements and whole knee joint histology were also performed. RESULTS: Injection of a high dose of SPION or PVA into naïve knee joints resulted in an immediate upregulation of pro-inflammatory gene expression in the synovium. A similar gene expression profile was observed after SPION or PVA injection into knee joints of TLR4-/- mice, indicating that this effect is not due to LPS contamination. Histological analysis of the knee joints also revealed synovial inflammation after SPION injection. Two hours after i.v. injection of SPION or PVA into naïve mice, an upregulation of pro-inflammatory gene expression was detected in the liver. Administration of SPION or PVA into arthritic mice via i.a. injection did not result in an upregulation in gene expression and also no additional effects were observed on histology. MR imaging and histology showed long-term retention of SPION in the inflamed joint. However, 14 days after the injections no long-term effects were evident for gene expression, histology or serum cytokine concentrations. CONCLUSIONS: Injection of SPION, either locally or systemically, gives an acute inflammatory response. In the long term, up to 14 days after the injection, while the SPION reside in the joint, no further activating effects of SPION were observed. Hence, we conclude that SPION do not aggravate arthritis and can therefore be used safely to detect joint inflammation by MR imaging.

Amino-polyvinyl alcohol coated superparamagnetic iron oxide nanoparticles are suitable for monitoring of human mesenchymal stromal cells in vivo.

Mesenchymal stromal cells (MSCs) are promising candidates in regenerative cell-therapies. However, optimizing their number and route of delivery remains a critical issue, which can be addressed by monitoring the MSCs' bio-distribution in vivo using super-paramagnetic iron-oxide nanoparticles (SPIONs). In this study, amino-polyvinyl alcohol coated (A-PVA) SPIONs are introduced for cell-labeling and visualization by magnetic resonance imaging (MRI) of human MSCs. Size and surface charge of A-PVA-SPIONs differ depending on their solvent. Under MSC-labeling conditions, A-PVA-SPIONs have a hydrodynamic diameter of 42 ± 2 nm and a negative Zeta potential of 25 ± 5 mV, which enable efficient internalization by MSCs without the need to use transfection agents. Transmission X-ray microscopy localizes A-PVA-SPIONs in intracellular vesicles and as cytosolic single particles. After identifying non-interfering cell-assays and determining the delivered and cellular dose, in addition to the administered dose, A-PVA-SPIONs are found to be non-toxic to MSCs and non-destructive towards their multi-lineage differentiation potential. Surprisingly, MSC migration is increased. In MRI, A-PVA-SPION-labeled MSCs are successfully visualized in vitro and in vivo. In conclusion, A-PVA-SPIONs have no unfavorable influences on MSCs, although it becomes evident how sensitive their functional behavior is towards SPION-labeling. And A-PVA-SPIONs allow MSC-monitoring in vivo.

Monitoring the effects of dexamethasone treatment by MRI using in vivo iron oxide nanoparticle-labeled macrophages.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic disease causing recurring inflammatory joint attacks. These attacks are characterized by macrophage infiltration contributing to joint destruction. Studies have shown that RA treatment efficacy is correlated to synovial macrophage number. The aim of this study was to experimentally validate the use of in vivo superparamagnetic iron oxide nanoparticle (SPION) labeled macrophages to evaluate RA treatment by MRI. METHODS: The evolution of macrophages was monitored with and without dexamethasone (Dexa) treatment in rats. Two doses of 3 and 1 mg/kg Dexa were administered two and five days following induction of antigen induced arthritis. SPIONs (7 mg Fe/rat) were injected intravenously and the knees were imaged in vivo on days 6, 10 and 13. The MR images were scored for three parameters: SPION signal intensity, SPION distribution pattern and synovial oedema. Using 3D semi-automated software, the MR SPION signal was quantified. The efficacy of SPIONs and gadolinium chelate (Gd), an MR contrast agent, in illustrating treatment effects were compared. Those results were confirmed through histological measurements of number and area of macrophages and nanoparticle clusters using CD68 immunostaining and Prussian blue staining respectively. RESULTS: Results show that the pattern and the intensity of SPION-labeled macrophages on MRI were altered by Dexa treatment. While the Dexa group had a uniform elliptical line surrounding an oedema pocket, the untreated group showed a diffused SPION distribution on day 6 post-induction. Dexa reduced the intensity of SPION signal 50-60% on days 10 and 13 compared to controls (P = 0.00008 and 0.002 respectively). Similar results were found when the signal was measured by the 3D tool. On day 13, the persisting low grade arthritis progression could not be demonstrated by Gd. Analysis of knee samples by Prussian blue and CD68 immunostaining confirmed in vivo SPION uptake by macrophages. Furthermore, CD68 immunostaining revealed that Dexa treatment significantly decreased the area and number of synovial macrophages. Prussian blue quantification corresponded to the macrophage measurements and both were in agreement with the MRI findings. CONCLUSIONS: We have demonstrated the feasibility of MRI tracking of in vivo SPION-labeled macrophages to assess RA treatment effects.

Pre-retrieval reperfusion decreases cancer recurrence after rat ischemic liver graft transplantation.

BACKGROUND & AIMS: Liver transplantation from marginal donors is associated with ischemia/reperfusion (I/R) lesions, which may increase the risk of post-transplant hepatocellular carcinoma (HCC) recurrence. Graft reperfusion prior to retrieval (as for extracorporeal membrane oxygenation--ECMO) can prevent I/R lesions. The impact of I/R on the risk of cancer recurrence was assessed on a syngeneic Fischer-rat liver transplantation model. METHODS: HCC cells were injected into the vena porta of all recipients at the end of an orthotopic liver transplantation (OLT). Control donors were standard heart-beating, ischemic ones (ISC), underwent 10 min or 30 min inflow liver clamping prior to retrieval, and ischemic/reperfused (ISC/R) donors underwent 2h liver reperfusion after the clamping. RESULTS: I/R lesions were confirmed in the ISC group, with the presence of endothelial and hepatocyte injury, and increased liver function tests. These lesions were in part reversed by the 2h reperfusion in the ISC/R group. HCC growth was higher in the 10 min and 30 min ISC recipients (p = 0.018 and 0.004 vs. control, as assessed by MRI difference between weeks one and two), and was prevented in the ISC/Rs (p = 0.04 and 0.01 vs. ISC). These observations were associated with a stronger pro-inflammatory cytokine profile in the ISC recipients only, and the expression of hypoxia and HCC growth-enhancer genes, including Hmox1, Hif1a and Serpine1. CONCLUSIONS: This experiment suggests that ischemia/reperfusion lesions lead to an increased risk of post-transplant HCC recurrence and growth. This observation can be reversed by graft reperfusion prior to retrieval.

Manganese kinetics demonstrated double contrast in acute but not in chronic infarction in a mouse model of myocardial occlusion reperfusion.

Manganese (Mn(2+)) is considered as a specific MRI contrast agent that enters viable cardiomyocytes through calcium pathways. Compared to extracellular gadolinium based contrast agents, it has the potential to assess cell viability. To date, only information from the washout phase after recirculation has been used for the detection and characterization of myocardial infarct. This study showed for the first time that in a mouse model of coronary occlusion-reperfusion, Mn(2+) wash-in kinetics are different at 24 h after surgery (acute infarction) than at eight days after surgery (chronic infarction). A fast but transient entry of Mn(2+) into the acute infarct area led to a double contrast between infarct and remote areas, whereas entry of Mn(2+) into the chronic infarct area remained reduced compared to remote regions during both wash-in and washout phases. The main hypothesis is that extracellular space is largely enhanced in acute infarction due to cell membrane rupture and interstitial edema, whereas scar tissue is densely composed of collagen fibers that reduce the distribution volume of free Mn(2+) ions. In addition to its ability to accurately depict the infarct area during the redistribution phase, Mn(2+) is also able to discriminate acute versus chronic injury by the observation of double-contrast kinetics in a mouse model of ischemia reperfusion.

Assessment of human islet labeling with clinical grade iron nanoparticles prior to transplantation for graft monitoring by MRI.

Ex vivo labeling of islets with superparamagnetic iron oxide (SPIO) nanoparticles allows posttransplant MRI imaging of the graft. In the present study, we compare two clinical grade SPIOs (ferucarbotran and ferumoxide) in terms of toxicity, islet cellular uptake, and MRI imaging. Human islets (80-90% purity) were incubated for 24 h with various concentrations of SPIOs (14-280 µg/ml of iron). Static incubations were performed, comparing insulin response to basal (2.8 mM) or high glucose stimulation (16.7 mM), with or without cAMP stimulation. Insulin and Perl's (assessment of iron content) staining were performed. Electronic microscopy analysis was performed. Labeled islets were used for in vitro or in vivo imaging in MRI 1.5T. Liver section after organ removal was performed in the same plane as MRI imaging to get a correlation between histology and radiology. Postlabeling islet viability (80 ± 10%) and function (in vitro static incubation and in vivo engraftment of human islets in nude mice) were similar in both groups. Iron uptake assessed by electron microscopy showed iron inclusions within the islets with ferucarbotran, but not with ferumoxide. MRI imaging (1.5T) of phantoms and of human islets transplanted in rats, demonstrated a strong signal with ferucarbotran, but only a weak signal with ferumoxide. Signal persisted for >8 weeks in the absence of rejection. An excellent correlation was observed between radiologic images and histology. The hepatic clearance of intraportally injected ferucarbotran was faster than that of ferumoxide, generating less background. A rapid signal decrease was observed in rejecting xenogeneic islets. According to the present data, ferucarbotran is the most appropriate of available clinical grade SPIOs for human islet imaging.