Olivier Clément

Superparamagnetic iron oxides as positive MR contrast agents: In vitro and in vivo evidence

The ability of superparamagnetic iron oxides (SPIO) and ultrasmall superparamagnetic iron oxides (USPIO) to act as positive contrast enhancers due to a marked T1 relaxivity was investigated. At low concentrations, an important signal enhancement was observed in vitro, reaching 120% for SPIO and 140% for USPIO in a spin echo 500/22 sequence. The more heavily the sequence was T1-weighted the greater the enhancement. As the concentration increased, the signal dropped. The in vivo study of USPIO in the rat showed that at low doses (14 mumol Fe/kg), the myocardial signal was enhanced by 30%, whereas at high doses (77 mumol Fe/kg), it fell by -50%. These results indicate that in T1-weighted spin echo sequences, the MR signal can be enhanced by low concentrations of superparamagnetic compounds. This effect could be useful in perfusion imaging, and is also important for a better understanding of any possible paradoxical positive enhancement which could occur in perfused organs.

The neuronal network responsible for paradoxical sleep and its dysfunctions causing narcolepsy and rapid eye movement (REM) behavior disorder

Rapid eye movement (REM) sleep behavior disorder (RBD) is a parasomnia characterized by the loss of muscle atonia during paradoxical (REM) sleep (PS). Conversely, cataplexy, one of the key symptoms of narcolepsy, is a striking sudden episode of muscle weakness triggered by emotions during wakefulness, and comparable to REM sleep atonia. The neuronal dysfunctions responsible for RBD and cataplexy are not known. In the present review, we present the most recent results on the neuronal network responsible for PS. Based on these results, we propose an updated integrated model of the mechanisms responsible for PS and explore different hypotheses explaining RBD and cataplexy. We propose that RBD is due to a specific degeneration of a sub-population of PS-on glutamatergic neurons specifically responsible of muscle atonia, localized in the caudal pontine sublaterodorsal tegmental nucleus (SLD). Another possibility is the occurrence in RBD patients of a specific lesion of the glycinergic/GABAergic pre-motoneurons localized in the medullary ventral gigantocellular reticular nucleus. Conversely, cataplexy in narcoleptics would be due to the activation during waking of the caudal PS-on SLD neurons responsible for muscle atonia. A phasic glutamatergic excitatory pathway from the central amygdala to the SLD PS-on neurons activated during emotion would induce such activation. In normal conditions, the glutamate excitation would be blocked by the simultaneous excitation by the hypocretins of the PS-off GABAergic neurons localized in the ventrolateral periaqueductal gray and the adjacent deep mesencephalic reticular nucleus, gating the activation of the PS-on SLD neurons.

Localization of the brainstem GABAergic neurons controlling paradoxical (REM) sleep.

Paradoxical sleep (PS) is a state characterized by cortical activation, rapid eye movements and muscle atonia. Fifty years after its discovery, the neuronal network responsible for the genesis of PS has been only partially identified. We recently proposed that GABAergic neurons would have a pivotal role in that network. To localize these GABAergic neurons, we combined immunohistochemical detection of Fos with non-radioactive in situ hybridization of GAD67 mRNA (GABA synthesis enzyme) in control rats, rats deprived of PS for 72 h and rats allowed to recover after such deprivation. Here we show that GABAergic neurons gating PS (PS-off neurons) are principally located in the ventrolateral periaqueductal gray (vlPAG) and the dorsal part of the deep mesencephalic reticular nucleus immediately ventral to it (dDpMe). Furthermore, iontophoretic application of muscimol for 20 min in this area in head-restrained rats induced a strong and significant increase in PS quantities compared to saline. In addition, we found a large number of GABAergic PS-on neurons in the vlPAG/dDPMe region and the medullary reticular nuclei known to generate muscle atonia during PS. Finally, we showed that PS-on neurons triggering PS localized in the SLD are not GABAergic. Altogether, our results indicate that multiple populations of PS-on GABAergic neurons are distributed in the brainstem while only one population of PS-off GABAergic neurons localized in the vlPAG/dDpMe region exist. From these results, we propose a revised model for PS control in which GABAergic PS-on and PS-off neurons localized in the vlPAG/dDPMe region play leading roles.

Gadolinium-Ethoxybenzyl-DTPA, a New Liver-Specific Magnetic Resonance Contrast Agent: Kinetic and Enhancement Patterns in Normal and Cholestatic Rats

OBJECTIVES Gadolinium-ethoxybenzyl-DTPA (Gd-EOB-DTPA) is a new hepatobiliary magnetic resonance imaging (MRI) contrast agent with a dual elimination: 70% via the liver and bile and 30% via the kidney in normal rats. The abdominal enhancement patterns of this new compound and the uptake mechanism by the liver were studied in rats using tissue relaxometry and MRI. METHODS Twelve normal rats, 33 rats treated with agents designed to inhibit biliary excretion of the agent, and 6 rats with surgically ligated common bile ducts received Gd-EOB-DTPA intravenously. Distribution and excretion were measured by MR relaxometry. MR signal intensity was measured over time for liver, kidney, and bowel. RESULTS In normal animals, 0.1 mmol/kg Gd-EOB-DTPA induced a significantly greater (200%) and more prolonged liver signal enhancement (100% at 30 minutes) than Gd-DTPA at the same dose. Either hyperbilirubinemia, induced by common bile duct ligation, or bromosulfophtalein (BSP) infusion inhibited liver uptake of Gd-EOB-DTPA, resulting in a preferential elimination via the kidney. Taurocholate (TC), an inhibitor of the bile acid transporter, was unable to block the liver uptake of Gd-EOB-DTPA. Blood half-lives of Gd-EOB-DTPA in rats were 2.4 minutes for the first component and 8.2 minutes for the second. CONCLUSIONS Data indicate that transport of Gd-EOB-DTPA through the liver into bile is driven by the organic anion transporter. The relation between enhancement of liver and kidney may be diagnostically useful to indirectly evaluate liver excretory function. Yet, persistent enhancement of liver, even in the presence of severe hyperbilirubinemia, should be sufficient to identify focal mass lesions.

Ultra magnetic liposomes for MR imaging, targeting, and hyperthermia.

Magnetic liposomes offer opportunities as theranostic systems. The prerequisite for efficient imaging, tissue targeting or hyperthermia is high magnetic load of these vesicles. Here we describe the preparation of Ultra Magnetic Liposomes (UMLs), which may encapsulate iron oxide nanoparticles in a volume fraction of up to 30%. This remarkable magnetic charge provides UMLs with high magnetic mobilities, MRI relaxivities, and heating capacities for magnetic hyperthermia. Moreover, these UMLs are rapidly and efficiently internalized by cultured tumor cells and, when they are administered to mice, they can be vectorized to tumors by an external magnet.

Combining Magnetic Hyperthermia and Photodynamic Therapy for Tumor Ablation with Photoresponsive Magnetic Liposomes

The ongoing nanotech revolution has the potential to transform diagnostic and therapeutic methods. Stimuli-triggered nanotherapies based on remotely activated agents have become attractive alternatives to conventional chemotherapy. Herein, we designed an optimized smart nanoplatform based on dually loaded hybrid liposomes to achieve enhanced tumor therapy. The aqueous core was highly loaded with iron oxide nanoparticles, while the lipid bilayer was supplied with a photosensitizer payload. The double cargo translated into double functionality: generation of singlet oxygen under laser excitation and heat production under alternating magnetic field stimulation, coupling photodynamic therapy (PDT) to magnetic hyperthermia (MHT). These liposomes address both therapeutic agents within tumor cells, and the combined PDT/MHT therapy resulted in complete cancer cell death in vitro while total solid-tumor ablation was achieved in an in vivo rodent model.

Liver positive enhancement after injection of superparamagnetic nanoparticles: Respective role of circulating and uptaken particles

Superparamagnetic nanoparticles have both high r1 and r2 relaxivities responsible for positive or negative enhancement properties. The aim of this study was to investigate to what extent perfusion (circulating particles) and uptake (clustered particles) mechanisms contribute to liver positive or negative enhancement using two different particles, superparamagnetic iron oxides (ferumoxides, AMI 25) and ultrasmall superparamagnetic iron oxides (ferumoxtran, AMI-227). Uptake kinetics were studied after intravenous injection of 20 micromol Fe/kg ferumoxtran on a washout liver model. Livers of 82 rats were surgically isolated and washed with saline infusion. Imaging was performed ex vivo at 0.5T with T1- and T2-weighted sequences. Enhancement kinetics of the liver were studied in vivo using MRI up to 180 min post injection of 20 micromol Fe/kg ferumoxtran (time response study) or 10, 20, 40 micromol Fe/kg ferumoxtran and 20 micromol Fe/kg ferumoxides (dose response study.) Particle uptake occurred early and resulted in a negative enhancement of the washed livers 15 min after injection of both T1 and T2 sequences. In vivo, a positive enhancement was only seen during the first five min with the lowest dose of ultrasmall superparamagnetic iron oxides and the T1 sequence. Uptake and clustering of the particles induced a negative liver enhancement. During the first minutes after injection, when uptake has not significantly occurred, perfusion imaging of the liver at a dose of 10 micromol Fe/kg results in a positive enhancement with T1-weighted sequences.

Brainstem mechanisms of paradoxical (REM) sleep generation

Paradoxical sleep (PS) is characterized by EEG activation with a disappearance of muscle tone and the occurrence of rapid eye movements (REM) in contrast to slow-wave sleep (SWS, also known as non-REM sleep) identified by the presence of delta waves. Soon after the discovery of PS, it was demonstrated that the structures necessary and sufficient for its genesis are restricted to the brainstem. We review here recent results indicating that brainstem glutamatergic and GABAergic, rather than cholinergic and monoaminergic, neurons play a key role in the genesis of PS. We hypothesize that the entrance to PS from SWS is due to the activation of PS-on glutamatergic neurons localized in the pontine sublaterodorsal tegmental nucleus. The activation of these neurons would be due to a permanent glutamatergic input arising from the lateral and ventrolateral periaqueductal gray (vlPAG) and the removal at the onset of PS of a GABAergic inhibition present during W and SWS. Such inhibition would be coming from PS-off GABAergic neurons localized in the vlPAG and the adjacent deep mesencephalic reticular nucleus. The cessation of activity of these PS-off GABAergic neurons at the onset and during PS would be due to direct projections from intermingled GABAergic PS-on neurons. Activation of PS would depend on the reciprocal interactions between the GABAergic PS-on and PS-off neurons, intrinsic cellular and molecular events, and integration of multiple physiological parameters.

In vivo cellular imaging of lymphocyte trafficking by MRI: a tumor model approach to cell-based anticancer therapy.

The aim of this study was to demonstrate the feasibility of in vivo cell tracking to monitor anticancer cell therapy by means of a high‐resolution noninvasive MRI method. Ovalbumin‐specific splenocytes (OT‐1) labeled with anionic γ‐Fe2O3 superparamagnetic iron oxide (SPIO) nanoparticles were adoptively transferred into C57BL/6 mice with growing ovalbumin‐expressing tumors. OT‐1 cells were tracked in vivo by 7 T MRI 24, 48, and 72 hr after they were injected. The results showed significant negative enhancement of the spleen at 24 hr, and of the tumor at 48 and 72 hr, after labeled cell injection. This suggests that the lymphocytes initially homed toward the spleen and were then recruited by the tumor. The presence of labeled cells was confirmed in ex vivo by 9.4 T microimaging of tumors and magnetic sorting of spleen cells. These results confirm that MR tracking of lymphocytes is feasible in vivo. This high‐resolution imaging method could be used to improve the monitoring of immune cell therapy. Magn Reson Med, 2006.

Reduced capillary perfusion and permeability in human tumour xenografts treated with the VEGF signalling inhibitor ZD4190: an in vivo assessment using dynamic MR imaging and macromolecular contrast media

We describe the use of perfusion-permeability magnetic resonance imaging (ppMRI) to study hemodynamic parameters in human prostate tumor xenografts, following treatment with the vascular endothelial growth factor-A (VEGF) receptor tyrosine kinase inhibitor, ZD4190. Using a macromolecular contrast agent (P792), a fast MR imaging protocol and a compartmental data analysis, we were able to demonstrate a significant simultaneous reduction in tumor vascular permeability, tumor vascular volume and tumor blood flow (43%, 30% and 42%, respectively) following ZD4190 treatment (100 mg/kg orally, 24 h and 2 h prior to imaging). This study indicates that MR imaging can be used to measure multiple hemodynamic parameters in tumors, and that tumor vascular permeability, volume and flow, can change in response to acute treatment with a VEGF signaling inhibitor.