RESUMEN
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.
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Óxidos N-Cíclicos , Liposomas , Espectroscopía de Resonancia por Spin del Electrón , Imagen por Resonancia MagnéticaRESUMEN
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.
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Pulmón , Imagen por Resonancia Magnética , Ratas , Animales , Espectroscopía de Resonancia Magnética , Imagen por Resonancia Magnética/métodos , Óxidos de Nitrógeno/químicaRESUMEN
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.
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Imagen por Resonancia Magnética/métodos , Músculo Esquelético/diagnóstico por imagen , Factores de Edad , Animales , Estimulación Eléctrica , Femenino , Músculo Esquelético/fisiología , Fantasmas de Imagen , Ratas , Ratas WistarRESUMEN
Lactate is an efficient neuronal energy source, even in presence of glucose. However, the importance of lactate shuttling between astrocytes and neurons for brain activation and function remains to be established. For this purpose, metabolic and hemodynamic responses to sensory stimulation have been measured by functional magnetic resonance spectroscopy and blood oxygen level-dependent (BOLD) fMRI after down-regulation of either neuronal MCT2 or astroglial MCT4 in the rat barrel cortex. Results show that the lactate rise in the barrel cortex upon whisker stimulation is abolished when either transporter is down-regulated. Under the same paradigm, the BOLD response is prevented in all MCT2 down-regulated rats, while about half of the MCT4 down-regulated rats exhibited a loss of the BOLD response. Interestingly, MCT4 down-regulated animals showing no BOLD response were rescued by peripheral lactate infusion, while this treatment had no effect on MCT2 down-regulated rats. When animals were tested in a novel object recognition task, MCT2 down-regulated animals were impaired in the textured but not in the visual version of the task. For MCT4 down-regulated animals, while all animal succeeded in the visual task, half of them exhibited a deficit in the textured task, a similar segregation into two groups as observed for BOLD experiments. Our data demonstrate that lactate shuttling between astrocytes and neurons is essential to give rise to both neurometabolic and neurovascular couplings, which form the basis for the detection of brain activation by functional brain imaging techniques. Moreover, our results establish that this metabolic cooperation is required to sustain behavioral performance based on cortical activation.
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Ácido Láctico/metabolismo , Imagen por Resonancia Magnética/métodos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Vibrisas/fisiología , Animales , Astrocitos/metabolismo , Aprendizaje , Espectroscopía de Resonancia Magnética , Masculino , Memoria , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Neuronas/metabolismo , Saturación de Oxígeno , Ratas , Ratas WistarRESUMEN
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.
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Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to technological developments over the last decade and the increase in magnetic field strength, it is now possible to obtain good resolution spectra in vivo in the rat brain. Neuroenergetics (i.e., the study of brain metabolism) and, especially, metabolic interactions between the different cell types have attracted more and more interest in recent years. Among these metabolic interactions, the existence of a lactate shuttle between neurons and astrocytes is still debated. It is, thus, of great interest to perform functional proton magnetic resonance spectroscopy (1H-MRS) in a rat model of brain activation and monitor lactate. However, the methyl lactate peak overlaps lipid resonance peaks and is difficult to quantify. The protocol described below allows metabolic and lactate fluctuations to be monitored in an activated brain area. Cerebral activation is obtained by whisker stimulation and 1H-MRS is performed in the corresponding activated barrel cortex, whose area is detected using blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI). All steps are fully described: the choice of anesthetics, coils, and sequences, achieving efficient whisker stimulation directly in the magnet, and data processing.
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Encéfalo/fisiología , Ácido Láctico/metabolismo , Imagen por Resonancia Magnética/métodos , Espectroscopía de Protones por Resonancia Magnética/métodos , Corteza Somatosensorial/fisiología , Vibrisas/fisiología , Animales , Masculino , Ratas , Ratas WistarRESUMEN
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 =â¯15⯱â¯2.9⯵M, kcat/Km =â¯930,000â¯s-1 M-1 and Km =â¯25⯱â¯5.4⯵M, kcat/Km =â¯640,000â¯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 1â¯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.
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Elastina/química , Elastasa de Leucocito/química , Pulmón/enzimología , Neumonía/enzimología , Animales , Líquido del Lavado Bronquioalveolar/química , Catepsina G/química , Elastina/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Elastasa de Leucocito/aislamiento & purificación , Pulmón/efectos de los fármacos , Pulmón/patología , Imagen por Resonancia Magnética , Ratones , Mieloblastina/química , Neutrófilos/enzimología , Oligopéptidos/síntesis química , Oligopéptidos/química , Oligopéptidos/farmacología , Neumonía/metabolismo , Neumonía/patología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Especificidad por SustratoRESUMEN
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.
RESUMEN
Inâ 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)=4â G) was observed. The enzymatic activities of several enzymes were conveniently monitored by electronic paramagnetic resonance (EPR). Using a 0.2â T MRI machine, inâ vitro and inâ vivo OMRI experiments were successfully performed, affording a 1200% enhanced MRI signal inâ vitro, and a 600% enhanced signal inâ vivo. These results highlight the enhanced imaging potential of these nitroxides upon specific enzymatic substrate-to-product conversion.
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Imagen por Resonancia Magnética , Óxidos de Nitrógeno/química , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Hidrólisis , Estructura Molecular , Óxidos de Nitrógeno/metabolismoRESUMEN
Two commercial statistical copolymers of ethylene oxide and propylene oxide, Jeffamine® M-2005 (PEO5-st-PPO37) and M-2070 (PEO46-st-PPO13), exhibiting lower critical solution temperature (LCST) in water, were grafted onto the surface of ultra-small superparamagnetic iron oxide nanoparticles (USPIOs) using silanization and amide-bond coupling reactions. The LCSTs of the polymers in solution were measured by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR). In accordance with the compositions of EO vs. PO, the transition temperature was measured to be 22 ± 2 °C for M-2005 by both DLS and NMR, while the LCST was much higher, 52 ± 2 °C, for M-2070 (a second transition was also detected above 80 °C by NMR in that case, ascribed to the full dehydration of chains at the molecular level). The resulting polymer-grafted USPIOs exhibit a temperature-responsive colloidal behaviour, their surface reversibly changing from hydrophilic below LCST to hydrophobic above it. This phenomenon was utilised to design thermo-sensitive contrast agents for MRI. Transverse relaxivities (r2) of the USPIO@PEO5-st-PPO37 core-shell nanoparticles were measured at 8.25, 20, 60, and 300 MHz. Nuclear magnetic resonance dispersion (NMRD) profiles, giving longitudinal relaxivities (r1) between 0.01 and 60 MHz, were acquired at temperatures ranging from 15 to 50 °C. For all tested frequencies except 300 MHz, both r1 and r2 decrease with temperature and show an inflection point at 25 °C, near the LCST. To illustrate the interest of such polymer-coated USPIOs for MRI thermometry, sample tubes were imaged on both low-field (8.25 MHz/0.194 Tesla) and high-field (300 MHz/7.05 Tesla) MRI scanners with either T1- or T2*-weighted spin echo sequences. The positive contrast on low-field MR images and the perfect linearity of the signal with a T2*-weighted sequence over the entire temperature range 15-50 °C render these LCST polymer coated USPIOs interesting positive contrast agents, also working as "nano-thermometers".
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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.
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Alcoholes/química , Alcoholes/farmacología , Aminas/química , Aminas/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Glioblastoma/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Imagen por Resonancia Magnética/métodos , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacosRESUMEN
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.
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Medios de Contraste/química , Elastina/química , Imagen por Resonancia Magnética/métodos , Óxidos de Nitrógeno/química , Animales , Espectroscopía de Resonancia por Spin del Electrón , Ratones , Proteolisis , Marcadores de SpinRESUMEN
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)NOË) and reactive alkyl radicals (R(3)Ë). 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.
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Aminas/uso terapéutico , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Profármacos/uso terapéutico , Aminas/química , Aminas/metabolismo , Diseño de Fármacos , Humanos , Profármacos/química , Profármacos/metabolismoRESUMEN
BACKGROUND: Magnetic resonance imaging can reveal exquisite anatomical details. However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations. In this context protease activity imaging is one of the most promising areas of research. METHODOLOGY/PRINCIPAL FINDINGS: We designed an elastase substrate by grafting stable nitroxide free radicals on soluble elastin. This substrate generates a high Overhauser magnetic resonance imaging (OMRI) contrast upon digestion by the target proteases through the modulation of its rotational correlation time. The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×10(4) cells per milliliter, well under the physiological neutrophils concentrations. CONCLUSIONS/SIGNIFICANCE: These ex-vivo experiments give evidence that OMRI is suitable for imaging elastase activity from neutrophil degranulation. Provided that a fast protease-substrate is used these results open the door to better diagnoses of a number of important pathologies (cystic fibrosis, inflammation, pancreatitis) by OMRI or Electron Paramagnetic Resonance Imaging in vivo. It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research.
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Degranulación de la Célula , Imagen por Resonancia Magnética/métodos , Neutrófilos/citología , Neutrófilos/enzimología , Elastasa Pancreática/metabolismo , Elastina/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Óxidos de Nitrógeno/metabolismo , RotaciónRESUMEN
Atherosclerosis is a chronic, progressive inflammatory disease that may develop into vulnerable lesions leading to thrombosis. To interrogate the molecular components involved in this process, single-chain variable fragments (scFvs) from a semi-synthetic human antibody library were selected on the lesions induced in a rabbit model of atherosclerosis after two rounds of in vivo phage display. Homing Phage-scFvs were isolated from (1) the injured endothelium, (2) the underlying lesional tissue and (3) the cells within the intima. Clones selected on the basis of their redundancy or the presence of key amino acids, as determined by comparing the distribution between the native and the selected libraries, were produced in soluble form, and seven scFvs were shown to specifically target the endothelial cell surface and inflamed intima-related regions of rabbit tissue sections by immunohistology approaches. The staining patterns differed depending on the scFv compartment of origin. This study demonstrates that large-scale scFv binding assays can be replaced by a sequence-based selection of best clones, paving the way for easier use of antibody libraries in in vivo biopanning experiments. Future investigations will be aimed at characterizing the scFv/target couples by mass spectrometry to set the stage for more accurate diagnostic of atherosclerosis and development of therapeutic strategies.
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Aterosclerosis/fisiopatología , Aterosclerosis/terapia , Anticuerpos de Cadena Única/química , Secuencia de Aminoácidos , Animales , Aorta Abdominal/patología , Células Espumosas/metabolismo , Humanos , Inmunohistoquímica , Inflamación/patología , Imagen por Resonancia Magnética , Masculino , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Biblioteca de Péptidos , Conejos , Homología de Secuencia de Aminoácido , Túnica Íntima/patologíaRESUMEN
Overhauser-enhanced MRI (OMRI) offers the potentiality of detecting low-concentrated species generated by specific biological processes. However molecular imaging applications of OMRI need significant improvement in spatial localization. Here it is shown that 3D-OMRI of a free radical injected in tumor-bearing mice can be performed at high anatomical resolution at a constant field. A 30 mm cavity operating at 5.43 GHz was inserted in a C-shaped magnet for proton MRI at 0.194 T. Nude mice with or without brain-implanted C6 rat glioma were positioned in the cavity and injected with TOPCA (1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole-3-carboxylic acid). OMRI was performed in 3D within several minutes in the brain region without high overheating of the animals. Voxel size was 0.5 × 0.5 × 1 mm³ , providing good delineation of brain regions. Signal amplifications ranged from 2 in tumors to 10 in vessels several minutes after TOPCA injection. Time-course of signal enhancement could be measured by 2D OMRI at 15 s time intervals in a localized thin slice. The method opens the way for molecular imaging of biological activities able to generate OMRI-visible free radicals.
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Neoplasias Encefálicas/patología , Medios de Contraste/análisis , Óxidos N-Cíclicos , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Glioma/patología , Imagenología Tridimensional/métodos , Imagen por Resonancia Magnética/métodos , Pirroles , Animales , Temperatura Corporal , Neoplasias Encefálicas/química , Línea Celular Tumoral/trasplante , Medios de Contraste/administración & dosificación , Medios de Contraste/farmacocinética , Óxidos N-Cíclicos/administración & dosificación , Óxidos N-Cíclicos/análisis , Óxidos N-Cíclicos/farmacocinética , Electrones , Estudios de Factibilidad , Radicales Libres/administración & dosificación , Radicales Libres/análisis , Radicales Libres/farmacocinética , Glioma/química , Inyecciones Intravenosas , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Protones , Pirroles/administración & dosificación , Pirroles/análisis , Pirroles/farmacocinética , Ratas , Trasplante Heterólogo , AguaRESUMEN
The noninvasive imaging of atherosclerotic plaques at an early stage of atherogenesis remains a major challenge for the evaluation of the pathologic state of patients at high risk of acute coronary syndromes. Recent studies have emphasized the importance of platelet-endothelial cell interactions in atherosclerosis-prone arteries at early stages, and the prominent role of P-selectin in the initial loose contact between platelets and diseased vessel walls. A specific MR contrast agent was developed here for the targeting, with high affinity, of P-selectin expressed in large amounts on activated platelets and endothelial cells. For this purpose, PEGylated dextran/iron oxide nanoparticles [PEG, poly(ethylene glycol)], named versatile ultrasmall superparamagnetic iron oxide (VUSPIO) particles, labeled with rhodamine were coupled to an anti-human P-selectin antibody (VH10). Flow cytometry and microscopy experiments on human activated platelets were highly correlated with MRI (performed at 4.7 and 0.2 T), with a 50% signal decrease in T(2) and T(1) values corresponding to the strong labeling of activated vs resting platelets. The number of 1000 VH10-VUSPIO nanoparticles attained per activated platelet appeared to be optimal for the detection of hypo- and hyper-signals in the platelet pellet on T(2) - and T(1) -weighted MRI. Furthermore, in vivo imaging of atherosclerotic plaques in ApoE mice at 4.7 T showed a spatial resolution adapted to the imaging of intimal thickening and a hypo-signal at 4.7 T, as a result of the accumulation of VH10-VUSPIO nanoparticles in the plaque. Our work provides support for the further assessment of the use of VH10-VUSPIO nanoparticles as a promising imaging modality able to identify the early stages of atherosclerosis with regard to the pertinence of both the target and the antibody-conjugated contrast agent used.
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Aterosclerosis/sangre , Aterosclerosis/diagnóstico , Plaquetas/metabolismo , Imagen por Resonancia Magnética/métodos , Selectina-P/sangre , Activación Plaquetaria , Animales , Anticuerpos/metabolismo , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/patología , Apolipoproteínas E/deficiencia , Plaquetas/efectos de los fármacos , Dextranos/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Citometría de Flujo , Humanos , Nanopartículas de Magnetita , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Activación Plaquetaria/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Receptores de Trombina/metabolismo , Trombina/farmacologíaRESUMEN
Levosimendan is a calcium sensitizer developed for the treatment of heart failure. It increases contractile force by enhancing the sensitivity of myofilaments to calcium. Besides this sensitizing effect, the drug has also been reported to show some inhibitory action on phosphodiesterase 3 (PDE3). The inotropic effects of levosimendan have been studied on guinea pig paced perfused hearts by using modular control analysis (MoCA) (Diolez P, Deschodt-Arsac V, Raffard G, Simon C, Santos PD, Thiaudiere E, Arsac L, Franconi JM. Am J Physiol Regul Integr Comp Physiol 293: R13-R19, 2007.), an integrative approach of heart energetics using noninvasive (31)P NMR. The aim was to evaluate quantitatively the respective effects of this drug on energy supply and demand modules. Under our experimental conditions, 0.7 muM levosimendan induced a 45% increase in paced heart output associated with a 7% decrease in phosphocreatine and a negligible increase in oxygen consumption. Because MoCA allows in situ study of the internal regulations in intact beating heart energetics, it was applied to describe quantitatively by which routes levosimendan exerts its inotropic action. MoCA demonstrated the absence of any significant effect of the drug on the supply module, which is responsible for the lower increase in oxygen consumption, compared with epinephrine, which increases the ratio between myocardial oxygen consumption and cardiac contraction. This result evidences that, under our conditions, a possible effect of levosimendan on PDE3 activity and/or intracellular calcium remains very low on mitochondrial activity and insignificant on integrated cardiac energetics. Thus, levosimendan inotropic effect on guinea pig heart depends almost entirely on the calcium-sensitizing properties leading to myofilament activation and the concomitant activation of energy supply by the decrease in PCr, therefore improving energetic efficiency of contraction.
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Antiarrítmicos/farmacología , Corazón/fisiología , Hidrazonas/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/fisiología , Piridazinas/farmacología , Animales , Cobayas , Masculino , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Consumo de Oxígeno/fisiología , SimendánRESUMEN
The purpose of this study was to demonstrate the feasibility of steady-state True fast imaging with steady precession (TrueFISP) four-dimensional imaging of mouse heart at high resolution and its efficiency for cardiac volumetry. Three-dimensional cine-imaging of control and hypoxic mice was carried out at 4.7 T without magnetization preparation or ECG-triggering. The k-space lines were acquired with the TrueFISP sequence (pulse repetition time/echo time = 4/2 ms) in a repeated sequential manner. Retrospective reordering of raw data allowed the reconstruction of 10 three-dimensional images per cardiac cycle. The acquisition scheme used an alternating radiofrequency phase and sum-of-square reconstruction method. Black-blood three-dimensional images at around 200 mum resolution were produced without banding artifact throughout the cardiac cycle. High contrast to noise made it possible to estimate cavity volumes during diastole and systole. Right and left ventricular stroke volume was significantly higher in hypoxic mice vs controls (20.2 +/- 2 vs 15.1 +/- 2; P < 0.05, 24.9 +/- 2 vs 20.4 +/- 2; P < 0.05, respectively). In conclusion, four-dimensional black-blood TrueFISP imaging in living mice is a method of choice to investigate cardiac abnormalities in mouse models.
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Algoritmos , Técnicas de Imagen Sincronizada Cardíacas/métodos , Corazón/anatomía & histología , Interpretación de Imagen Asistida por Computador/métodos , Imagenología Tridimensional/métodos , Imagen por Resonancia Cinemagnética/métodos , Animales , Femenino , Aumento de la Imagen/métodos , Ratones , Ratones Endogámicos C57BL , Reproducibilidad de los Resultados , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Proteolysis, involved in many processes in living organisms, is tightly regulated in space and time under physiological conditions. However deregulation can occur with local persistent proteolytic activities, e.g. in inflammation, cystic fibrosis, tumors, or pancreatitis. Furthermore, little is known about the role of many proteases, hence there is a need of new imaging methods to visualize specifically normal or disease-related proteolysis in intact bodies. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, a new concept for non invasive proteolysis imaging is proposed. Overhauser-enhanced Magnetic Resonance Imaging (OMRI) at 0.2 Tesla was used to monitor the enzymatic hydrolysis of a nitroxide-labeled protein. In vitro, image intensity switched from 1 to 25 upon proteolysis due to the associated decrease in the motional correlation time of the substrate. The OMRI experimental device used in this study is consistent with protease imaging in mice at 0.2 T without significant heating. Simulations show that this enzymatic-driven OMRI signal switch can be obtained at lower frequencies suitable for larger animals or humans. CONCLUSIONS/SIGNIFICANCE: The method is highly sensitive and makes possible proteolysis imaging in three dimensions with a good spatial resolution. Any protease could be targeted specifically through the use of taylor-made cleavable macromolecules. At short term OMRI of proteolysis may be applied to basic research as well as to evaluate therapeutic treatments in small animal models of experimental diseases.