4-Amino-TEMPO loaded liposomes as sensitive EPR and OMRI probes for the detection of phospholipase A2 activity.

This work aims at developing a diagnostic method based on Electron Paramagnetic Resonance (EPR) measurements of stable nitroxide radicals released from "EPR silent" liposomes. The liposome destabilisation and consequent radical release is enzymatically triggered by the action of phospholipase A2 (PLA2) present in the biological sample of interest. PLA2 are involved in a broad range of processes, and changes in their activity may be considered as a unique valuable biomarker for early diagnoses. The minimum amount of PLA2 measured "in vitro" was 0.09 U/mL. Moreover, the liposomes were successfully used to perform Overhauser-enhanced Magnetic Resonance Imaging (OMRI) in vitro at 0.2 T. The amount of radicals released by PLA2 driven liposome destabilization was sufficient to generate a well detectable contrast enhancement in the corresponding OMRI image.

A system for in vivo on-demand ultra-low field Overhauser-enhanced 3D-Magnetic resonance imaging.

Development of very-low field MRI is an active area of research. It aims at reducing operating costs and improve portability. However, the signal-to-noise issue becomes prominent at ultra-low field (<1 mT), especially for molecular imaging purposes that addresses specific biochemical events. In the context of preclinical molecular MRI of abnormal proteolysis the paper describes a MRI system able to produce Overhauser-enhanced MR images in living rats through in situ Dynamic Nuclear Polarization at 206 µT using stable and non-toxic nitroxides. In parallel conventional images are generated at 206 µT following pre-polarization at 20 mT. Results show that nitroxides are visualized in 3D within a few minutes in the lungs, kidneys and bladder post-administration. This system will be used for molecular imaging of inflammation using protease-specific nitroxide probes.

New setup for multi-parametric MRI in young and old rat gastrocnemius at 4.7 and 7 T during muscle stimulation.

T1 and T2 relaxation times combined with 31 P spectroscopy have been proven efficient for muscular disease characterization as well as for pre- and post-muscle stimulation measurements. Even though 31 P spectroscopy can already be performed during muscle exercise, no method for T1 and T2 measurement enables this possibility. In this project, a complete setup and protocol for multi-parametrical MRI of the rat gastrocnemius before, during and after muscle stimulation at 4.7 and 7 T is presented. The setup is fully MRI compatible and is composed of a cradle, an electro-stimulator and an electronic card in order to synchronize MRI sequences with muscle stimulation. A 2D triggered radial-encoded Look-Locker sequence was developed, and enabled T1 measurements in less than 2 min on stimulated muscle. Also, a multi-slice multi-echo sequence was adapted and synchronized for T2 measurements as well as 31 P spectroscopy acquisitions in less than 4 min in both cases on stimulated muscle. Methods were validated on young rats using different stimulation paradigms. Then they were applied on older rats to compare quantification results, using the different stimulation paradigms, and allowed observation of metabolic changes related to aging with good reproducibility. The robustness of the whole setup shows wide application opportunities.

Magnetic Resonance Imaging of Protease-Mediated Lung Tissue Inflammation and Injury.

Pulmonary inflammation usually involves strong neutrophil recruitment with a marked release of proteases such as neutrophil elastase (NE). Noninvasive in vivo assessment of unregulated elastase activity in the lungs would provide a valuable diagnostic tool. Here, it is proposed to use Overhauser-enhanced magnetic resonance imaging (OMRI) in mice where inflammation was induced by the instillation of lipopolysaccharide (LPS). OMRI contrast in the lungs was generated by a dedicated NE free radical substrate. The free radical decayed more rapidly in LPS-treated mouse lungs than in control mice, indicating the occurrence of increased proteolysis under inflammation. Preclinical detection of abnormal proteolysis opens the way for new diagnosis modality and antiprotease testing in vivo.

An elastase activity reporter for Electronic Paramagnetic Resonance (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI) as a line-shifting nitroxide.

Pulmonary inflammatory diseases are a major burden worldwide. They have in common an influx of neutrophils. Neutrophils secrete unchecked proteases at inflammation sites consequently leading to a protease/inhibitor imbalance. Among these proteases, neutrophil elastase is responsible for the degradation of the lung structure via elastin fragmentation. Therefore, monitoring the protease/inhibitor status in lungs non-invasively would be an important diagnostic tool. Herein we present the synthesis of a MeO-Suc-(Ala)2-Pro-Val-nitroxide, a line-shifting elastase activity probe suitable for Electron Paramagnetic Resonance spectroscopy (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI). It is a fast and sensitive neutrophil elastase substrate with Km = 15 ±â€¯2.9 µM, kcat/Km = 930,000 s-1 M-1 and Km = 25 ±â€¯5.4 µM, kcat/Km = 640,000 s-1 M-1 for the R and S isomers, respectively. These properties are suitable to detect accurately concentrations of neutrophil elastase as low as 1 nM. The substrate was assessed with broncho-alveolar lavages samples derived from a mouse model of Pseudomonas pneumonia. Using EPR spectroscopy we observed a clear-cut difference between wild type animals and animals deficient in neutrophil elastase or deprived of neutrophil Elastase, Cathepsin G and Proteinase 3 or non-infected animals. These results provide new preclinical ex vivo and in vivo diagnostic methods. They can lead to clinical methods to promote in time lung protection.

Selective On/Off-Nitroxides as Radical Probes to Investigate Non-radical Enzymatic Activity by Electron Paramagnetic Resonance.

A nitroxide carrying a peptide specific to the binding pocket of the serine proteases chymotrypsin and cathepsin G is prepared. This peptide is attached as an enol ester to the nitroxide. Upon enzymatic hydrolysis of the peptide, the enol ester moiety is transformed into a ketone moiety. This transformation affords a difference of 5 G in phosphorus hyperfine coupling constant between the electronic paramagnetic resonance (EPR) signals of each nitroxide. This property is used to monitor the enzymatic activity of chymotrypsin and cathepsin G by EPR. Michaelis constants were determined and match those reported for conventional optical probes.

Fast 3D ultrashort echo-time spiral projection imaging using golden-angle: A flexible protocol for in vivo mouse imaging at high magnetic field.

PURPOSE: To develop a fast three-dimensional (3D) k-space encoding method based on spiral projection imaging (SPI) with an interleaved golden-angle approach and to validate this novel sequence on small animal models. METHODS: A disk-like trajectory, in which each disk contained spirals, was developed. The 3D encoding was performed by tilting the disks with a golden angle. The sharpness was first calculated at different T2* values. Then, the sharpness was measured on phantom using variable undersampling ratios. Finally, the sampling method was validated by whole brain time-of-flight angiography and ultrasmall superparamagnetic iron oxide (USPIO) enhanced free-breathing liver angiography on mouse. RESULTS: The in vitro results demonstrated the robustness of the method for short T2* and high undersampling ratios. In vivo experiments showed the ability to properly detect small vessels in the brain with an acquisition time shorter than 1 min. Free-breathing mice liver angiography showed the insensitivity of this protocol toward motions and flow artifacts, and enabled the visualization of liver motion during breathing. CONCLUSIONS: The method implemented here allowed fast 3D k-space sampling with a high undersampling ratio. Combining the advantages of center-out spirals with the flexibility of the golden angle approach could have major implications for real-time imaging. Magn Reson Med 77:1831-1840, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

USPIO-enhanced 3D-cine self-gated cardiac MRI based on a stack-of-stars golden angle short echo time sequence: Application on mice with acute myocardial infarction.

PURPOSE: To develop and assess a 3D-cine self-gated method for cardiac imaging of murine models. MATERIALS AND METHODS: A 3D stack-of-stars (SOS) short echo time (STE) sequence with a navigator echo was performed at 7T on healthy mice (n = 4) and mice with acute myocardial infarction (MI) (n = 4) injected with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. In all, 402 spokes were acquired per stack with the incremental or the golden angle method using an angle increment of (360/402)° or 222.48°, respectively. A cylindrical k-space was filled and repeated with a maximum number of repetitions (NR) of 10. 3D cine cardiac images at 156 µm resolution were reconstructed retrospectively and compared for the two methods in terms of contrast-to-noise ratio (CNR). The golden angle images were also reconstructed with NR = 10, 6, and 3, to assess cardiac functional parameters (ejection fraction, EF) on both animal models. RESULTS: The combination of 3D SOS-STE and USPIO injection allowed us to optimize the identification of cardiac peaks on navigator signal and generate high CNR between blood and myocardium (15.3 ± 1.0). The golden angle method resulted in a more homogeneous distribution of the spokes inside a stack (P < 0.05), enabling reducing the acquisition time to 15 minutes. EF was significantly different between healthy and MI mice (P < 0.05). CONCLUSION: The method proposed here showed that 3D-cine images could be obtained without electrocardiogram or respiratory gating in mice. It allows precise measurement of cardiac functional parameters even on MI mice. J. Magn. Reson. Imaging 2016;44:355-365.

Free-breathing 3D diffusion MRI for high-resolution hepatic metastasis characterization in small animals.

The goal of this study was to develop a 3D diffusion weighted sequence for free breathing liver imaging in small animals at high magnetic field. Hepatic metastases were detected and the apparent diffusion coefficients (ADC) were measured. A 3D SE-EPI sequence was developed by (i) inserting a water-selective excitation radiofrequency pulse to suppress adipose tissue signal and (ii) bipolar diffusion gradients to decrease the sensitivity to respiration motion. Mice with hepatic metastases were imaged at 7T by applying b values from 200 to 1100 s/mm(2). 3D images with high spatial resolution (182 × 156 × 125 µm) were obtained in only 8 min 32 s. The modified DW-SE-EPI sequence allowed to obtain 3D abdominal images of healthy mice with fat SNR 2.5 times lower than without any fat suppression method and sharpness 2.8 times higher than on respiration-triggered images. Due to the high spatial resolution, the core and the periphery of disseminated hepatic metastases were differentiated at high b-values only, demonstrating the presence of edema and proliferating cells (with ADC of 2.65 × 10(-3) and 1.55 × 10(-3) mm(2)/s, respectively). Furthermore, these metastases were accurately distinguished from proliferating ones within the same animal at high b-values (mean ADC of 0.38 × 10(-3) mm(2)/s). Metastases of less than 1.7 mm(3) diameter were detected. The new 3D SE-EPI sequence enabled to obtain diffusion information within liver metastases. In addition of intra-metastasis heterogeneity, differences in diffusion were measured between metastases within an animal. This sequence could be used to obtain diffusion information at high magnetic field.

Water Selective Imaging and bSSFP Banding Artifact Correction in Humans and Small Animals at 3T and 7T, Respectively.

INTRODUCTION: The purpose of this paper is to develop an easy method to generate both fat signal and banding artifact free 3D balanced Steady State Free Precession (bSSFP) images at high magnetic field. METHODS: In order to suppress fat signal and bSSFP banding artifacts, two or four images were acquired with the excitation frequency of the water-selective binomial radiofrequency pulse set On Resonance or shifted by a maximum of 3/4TR. Mice and human volunteers were imaged at 7 T and 3 T, respectively to perform whole-body and musculoskeletal imaging. "Sum-Of-Square" reconstruction was performed and combined or not with parallel imaging. RESULTS: The frequency selectivity of 1-2-3-2-1 or 1-3-3-1 binomial pulses was preserved after (3/4TR) frequency shifting. Consequently, whole body small animal 3D imaging was performed at 7 T and enabled visualization of small structures within adipose tissue like lymph nodes. In parallel, this method allowed 3D musculoskeletal imaging in humans with high spatial resolution at 3 T. The combination with parallel imaging allowed the acquisition of knee images with ~500 µm resolution images in less than 2 min. In addition, ankles, full head coverage and legs of volunteers were imaged, demonstrating the possible application of the method also for large FOV. CONCLUSION: In conclusion, this robust method can be applied in small animals and humans at high magnetic fields. The high SNR and tissue contrast obtained in short acquisition times allows to prescribe bSSFP sequence for several preclinical and clinical applications.

Enzymatically Shifting Nitroxides for EPR Spectroscopy and Overhauser-Enhanced Magnetic Resonance Imaging.

In vivo investigations of enzymatic processes using non-invasive approaches are a long-lasting challenge. Recently, we showed that Overhauser-enhanced MRI is suitable to such a purpose. A ß-phosphorylated nitroxide substrate prototype exhibiting keto-enol equilibrium upon enzymatic activity has been prepared. Upon enzymatic hydrolysis, a large variation of the phosphorus hyperfine coupling constant (Δa(P)=4 G) was observed. The enzymatic activities of several enzymes were conveniently monitored by electronic paramagnetic resonance (EPR). Using a 0.2 T MRI machine, in vitro and in vivo OMRI experiments were successfully performed, affording a 1200% enhanced MRI signal in vitro, and a 600% enhanced signal in vivo. These results highlight the enhanced imaging potential of these nitroxides upon specific enzymatic substrate-to-product conversion.

The ß-phosphorus hyperfine coupling constant in nitroxide: part 3: titration of water by electron paramagnetic resonance.

Recently, we showed that the phosphorus hyperfine coupling constant aPß of persistent cyclic nitroxides decreased with the normalized polarity Reichardt's constant E. Thus, we investigated the changes in aPß in binary mixtures of solvents. The sensitivity of aPß to the solvent was high enough to allow us to perform water titration in THF, 1,4-dioxane, and acetonitrile by EPR. Accuracies of a few percent were achieved.

Positive contrast high-resolution 3D-cine imaging of the cardiovascular system in small animals using a UTE sequence and iron nanoparticles at 4.7, 7 and 9.4 T.

BACKGROUND: To show that 3D sequences with ultra-short echo times (UTEs) can generate a positive contrast whatever the magnetic field (4.7, 7 or 9.4 T) and whatever Ultra Small Particles of Iron Oxide (USPIO) concentration injected and to use it for 3D time-resolved imaging of the murine cardiovascular system with high spatial and temporal resolutions. METHODS: Three different concentrations (50, 200 and 500 µmol Fe/kg) of USPIO were injected in mice and static images of the middle part of the animals were acquired at 4.7, 7 and 9.4 T pre and post-contrast with UTE (TE/TR = 0.05/4.5 ms) sequences. Signal-to-Noise Ratio (SNR) and Contrast-to-Noise Ratio (CNR) of blood and static tissus were evaluated before and after contrast agent injection. 3D-cine images (TE/TR = 0.05/3.5 ms, scan time < 12 min) at 156 µm isotropic resolution of the mouse cardiopulmonary system were acquired prospectively with the UTE sequence for the three magnetic fields and with an USPIO dose of 200 µmol Fe/kg. SNR, CNR and signal homogeneity of blood were measured. High spatial (104 µm) or temporal (3.5 ms) resolution 3D-cine imaging (scan time < 35 min) isotropic resolution were also performed at 7 T with a new sequence encoding scheme. RESULTS: UTE imaging generated positive contrast and higher SNR and CNR whatever the magnetic field and the USPIO concentration used compared to pre-contrast images. Time-resolved 3D acquisition enables high blood SNR (66.6 ± 4.5 at 7 T) and CNR (33.2 ± 4.2 at 7 T) without flow or motion artefact. Coronary arteries and aortic valve were visible on images acquired at 104 µm resolution. CONCLUSIONS: We have demonstrated that by combining the injection of iron nanoparticles with 3D-cine UTE sequences, it was possible to generate a strong positive contrast between blood and surrounding tissues. These properties were exploited to produce images of the cardiovascular system in small animals at high magnetic fields with a high spatial and temporal resolution. This approach might be useful to measure the functional cardiac parameters or to assess anatomical modifications to the blood vessels in cardio-vascular disease models.

Fast and robust 3D T1 mapping using spiral encoding and steady RF excitation at 7 T: application to cardiac manganese enhanced MRI (MEMRI) in mice.

Mapping longitudinal relaxation times in 3D is a promising quantitative and non-invasive imaging tool to assess cardiac remodeling. Few methods are proposed in the literature allowing us to perform 3D T1 mapping. These methods often require long scan times and use a low number of 3D images to calculate T1 . In this project, a fast 3D T1 mapping method using a stack-of-spirals sampling scheme and regular RF pulse excitation at 7 T is presented. This sequence, combined with a newly developed fitting procedure, allowed us to quantify T1 of the whole mouse heart with a high spatial resolution of 208 × 208 × 315 µm(3) in 10-12 min acquisition time. The sensitivity of this method for measuring T1 variations was demonstrated on mouse hearts after several injections of manganese chloride (doses from 25 to 150 µmol kg(-1) ). T1 values were measured in vivo in both pre- and post-contrast experiments. This protocol was also validated on ischemic mice to demonstrate its efficiency to visualize tissue damage induced by a myocardial infarction. This study showed that combining spiral gradient shape and steady RF excitation enabled fast and robust 3D T1 mapping of the entire heart with a high spatial resolution.

Time-resolved TOF MR angiography in mice using a prospective 3D radial double golden angle approach.

PURPOSE: To develop an undersampled anatomical, three-dimensional (3-D) time-resolved magnetic resonance angiography (MRA) method for small animals based on time-of-flight (TOF) effect and radial sampling. METHODS: Mouse carotid arteries and Circle of Willis images were acquired on a 7T scanner with an electrocardiogram (ECG)-triggered sequence. Preliminary experiments were used to generate an approximately uniform distribution of radial projections with a first golden angle and to produce anatomical TOF images. A second golden angle ratio between consecutive projections of cine acquisitions was added to make it possible to use a temporal filter during reconstruction of time-resolved angiography. A decreasing number of projections were tested, and their impact on signal-to-noise ratio (SNR) and spatial resolution was assessed. RESULTS: In anatomical MRA, the undersampled radial approach efficiently allows fast acquisition of mouse angiogram in 3D (22 sec). It was also only slightly sensitive to motion and flow artifacts. The time-resolved sequence can be performed with only 2,500 projections per cine and a temporal resolution under 4 ms in a relatively short acquisition time (less than 5 min). CONCLUSION: This technique simultaneously provided high 3D isotropic spatial resolution and excellent temporal resolution with a good SNR level, allowing blood flow to be visualized in a restricted acquisition time.

Self-gated bSSFP sequences to detect iron-labeled cancer cells and/or metastases in vivo in mouse liver at 7 Tesla.

BACKGROUND: To develop and evaluate three-dimensional (3D) self-gated balanced steady state free precession (bSSFP) imaging at high magnetic fields to track iron-labeled cells and metastases in murine abdomens. METHODS: Mice were injected intravenously with iron-labeled melanoma cells and imaged at 7 Tesla (T). Respiration peaks were identified using Free Induction Decay acquired immediately after the radiofrequency pulse. Respiration-corrupted k-space lines were deleted. Four images were acquired to reconstruct final images using the Sum-Of-Square technique. Image sharpness, metastasis contrast and iron-labeled cell detection with SG-bSSFP sequence (acquired with echo time [TE] = 3 ms or TE = 6 ms) were compared with standard methods (gradient echo (GRE) and RARE). RESULTS: After reconstruction, the 3D SG-bSSFP images were 75-80% sharper, free from banding (75% liver signal-to-noise ratio recovery) and respiratory motion (26-42% improvement in signal homogeneity) artifacts. Metastasis contrast was twice higher on SG-bSSFP with TE = 3 ms than on RARE images. Iron-labeled cells and metastases were simultaneously detected on SG-bSSFP images with TE = 6 ms, with similar void intensity and tumor contrast to GRE and RARE, respectively. Halving acquisition time preserved iron sensitivity and metastasis contrast, allowing for 3D abdomen imaging in 13 min (TE = 3 ms) or 26 min (TE = 6 ms). CONCLUSION: Combining a self-gating technique with bSSFP sequences at 7T provides high-resolution 3D artifact-free abdominal images of small animals.

Alkoxyamines: toward a new family of theranostic agents against cancer.

Theranostics combines therapeutic and diagnostic or drug deposition monitoring abilities of suitable molecules. Here we describe the first steps of building an alkoxyamine-based theranostic agent against cancer. The labile alkoxyamine ALK-1 (t(1/2) = 50 min at 37 °C) cleaves spontaneously to generate (1) a highly reactive free alkyl radical used as therapeutic agents to induce cell damages leading to cell death and (2) a stable nitroxide used as contrast agent for Overhauser-enhanced magnetic resonance imaging (OMRI). The ALK-1 toxicity was studied extensively in vitro on the glioblastoma cell line U87-MG. Cell viability appeared to be dependent on ALK-1 concentration and on the time of the observation following alkoxyamine treatment. For instance, the LC50 at 72 h was 250 µM. Data showed that cell toxicity was specifically due to the in situ released alkyl radical. This radical induced oxidative stress, mitochondrial changes, and ultimately the U87 cell apoptosis. The nitroxide production, during the alkoxyamine homolysis, was monitored by OMRI, showing a progressive MRI signal enhancement to 6-fold concomitant to the ALK-1 homolysis. In conclusion, we have demonstrated for the first time that the alkoxyamines are promising molecules to build theranostic tools against solid tumors.

In vivo Overhauser-enhanced MRI of proteolytic activity.

There is an increasing interest in developing novel imaging strategies for sensing proteolytic activities in intact organisms in vivo. Overhauser-enhanced MRI (OMRI) offers the possibility to reveal the proteolysis of nitroxide-labeled macromolecules thanks to a sharp decrease of the rotational correlation time of the nitroxide moiety upon cleavage. In this paper, this concept is illustrated in vivo at 0.2 T using nitroxide-labeled elastin orally administered in mice. In vitro, this elastin derivative was OMRI-visible and gave rise to high Overhauser enhancements (19-fold at 18 mm nitroxide) upon proteolysis by pancreatic porcine elastase. In vivo three-dimensional OMRI detection of proteolysis was carried out. A keyhole fully balanced steady-state free precession sequence was used, which allowed 3D OMRI acquisition within 20 s at 0.125 mm(3) resolution. About 30 min after mouse gavage, proteolysis was detected in the duodenum, where Overhauser enhancements were 7.2 ± 2.4 (n = 7) and was not observed in the stomach. Conversely, orally administered free nitroxides or pre-digested nitroxide-labeled elastin were detected in the mouse's stomach by OMRI. Combined with specific molecular probes, this Overhauser-enhanced MRI technique can be used to evaluate unregulated proteolytic activities in various models of experimental diseases and for drug testing.

Mitochondrial energetics is impaired in vivo in aged skeletal muscle.

With aging, most skeletal muscles undergo a progressive loss of mass and strength, a process termed sarcopenia. Aging-related defects in mitochondrial energetics have been proposed to be causally involved in sarcopenia. However, changes in muscle mitochondrial oxidative phosphorylation with aging remain a highly controversial issue, creating a pressing need for integrative approaches to determine whether mitochondrial bioenergetics are impaired in aged skeletal muscle. To address this issue, mitochondrial bioenergetics was first investigated in vivo in the gastrocnemius muscle of adult (6 months) and aged (21 months) male Wistar rats by combining a modular control analysis approach with (31) P magnetic resonance spectroscopy measurements of energetic metabolites. Using this innovative approach, we revealed that the in vivo responsiveness ('elasticity') of mitochondrial oxidative phosphorylation to contraction-induced increase in ATP demand is significantly reduced in aged skeletal muscle, a reduction especially pronounced under low contractile activities. In line with this in vivo aging-related defect in mitochondrial energetics, we found that the mitochondrial affinity for ADP is significantly decreased in mitochondria isolated from aged skeletal muscle. Collectively, the results of this study demonstrate that mitochondrial bioenergetics are effectively altered in vivo in aged skeletal muscle and provide a novel cellular basis for this phenomenon.

Alkoxyamines: a new family of pro-drugs against cancer. Concept for theranostics.

Development of anti-cancerous theranostic agents is a vivid field. This article describes a theranostic approach that relies on the triggering of cancer cell death by generation of alkyl radicals at the right place and at the right time using the presence of active proteases in the tumour environment. Alkoxyamines (R(1)R(2)NOR(3)) are labile molecules that homolyze into nitroxides (R(1)R(2)NO˙) and reactive alkyl radicals (R(3)˙). They are used as a source of active alkyl radicals for curing and nitroxides for monitoring by Overhauser-enhanced magnetic resonance imaging (OMRI). Herein, the requirements needed for applying alkoxyamines are described: (i) highly selective activation of the alkoxyamine by specific proteases; (ii) fast homolysis of the alkoxyamine C-ON bond at physiological temperature; (iii) activation of cell death processes through an increase of the local oxidative stress or potential re-activation of the immune system due to short-lived alkyl radicals; and (iv) imaging of the tumor and the drug release by sensing the nitroxide by OMRI.