Caractérisation multimodale des particules superparamagnétiques d'oxyde de fer pour l'imagerie de l'inflammation : application à l'ischémie cérébrale expérimentale

Several studies on small animals have shown that MRI enhanced with nanoparticles of iron oxide (USPIO) is able to detect the neuroinflammation. However, to our knowledge, no team had yet investigated the potential of this approach for monitoring an anti-inflammatory treatment. In this context, we have demonstrated the feasibility of this approach to monitor the effects of minocycline after cerebral ischemia in mice. MRI is a very sensitive technique for the detection of iron, but the precise location of USPIO as well as their quantification is difficult. We therefore propsed to complete the MRI approach by a new technique to our knowledge in the field of USPIO imaging in the brain : Synchrotron radiation tomography. We here present the first results showing the feasibility of this approach and a comparative study of the sensitivity of two techniques used for the detection of USPIO in the brain. In the last part of our work, we report our results on the biotransformation of USPIOs in the spleen of the mouse during the first 40 days after intravenous injection obtained by transmission electron microscopy (TEM).Plusieurs études menées chez le petit animal ont montré que l'IRM réhaussé avec les nanoparticules d'oxyde de fer (USPIO) permettait de détecter la neuroinflammation. Cependant, à notre connaissance, aucune équipe n'avait à ce jour étudié le potentiel de cette approche pour le suivi d'un traitement anti-inflammatoire. Dans ce contexte, nous avons démontré la faisabilité de cette approche pour monitorer les effets de la minocycline suite à une ischémie cérébrale chez la souris. L'IRM est une technique très sensible pour la détection du fer, mais la localisation précise des USPIO tout comme leur quantification est difficile. Nous avons donc proposé de compléter l'approche IRM par une technique inédite à notre connaissance dans le domaine de l'imagerie cérébrale des USPIO : la tomographie par rayonnement Synchrotron. Nous présentons ici les tous premiers résultats montrant la faisabilité de l'approche, ainsi qu'une étude comparée de la sensibilité, des deux techniques pour la détection des USPIO dans le cerveau. Dans la première partioe de notre travail , nous apportons nos résultats concernant la biotransformation des USPIOs dans la rate de la souris dans les 40 premiers jours suivant leur injection intraveineuse obtenus en microscopie électronique par transmission (MET)

Flaw shape reconstruction – an experimental approach

Flaws can be classified as acceptable and unacceptable flaws. As a result of nondestructive testing, one takes de decision Admit/Reject regarding the tested product related to some acceptability criteria. In order to take the right decision, one should know the shape and the dimension of the flaw. On the other hand, the flaws considered to be acceptable, develop in time, such that they can become unacceptable. In this case, the knowledge of the shape and dimension of the flaw allows determining the product time life. For interior flaw shape reconstruction the best procedure is the use of difference static magnetic field. We have a stationary magnetic field problem, but we face the problem given by the nonlinear media. This paper presents the results of the experimental work for control specimen with and without flaw

Clot injection technique affects thrombolytic efficacy in a rat embolic stroke model: implications for translaboratory collaborations

Current recommendations encourage the use of embolic stroke (ES) models and replication of results across laboratories in preclinical research. Since such endeavors employ different surgeons, we sought to ascertain the impact of injection technique on outcome and response to thrombolysis in an ES model. Embolic stroke was induced in Male Wistar Kyoto rats (n=166) by a fast or a slow clot injection (CI) technique. Saline or recombinant tissue plasminogen activator (rtPA) was given at 1 hour after stroke. Flow rate curves were assessed in 24 animals. Cerebral perfusion was assessed using laser Doppler flowmetry. Edema corrected infarct volume, hemispheric swelling, hemorrhagic transformation, and neurologic outcome were assessed at 24 hours after stroke. Clot burden was estimated in a subset of animals (n=40). Slow CI resulted in significantly smaller infarct volumes (P=0.024) and better neurologic outcomes (P=0.01) compared with fast CI at 24 hours. Unexpectedly, rtPA treatment attenuated infarct size in fast (P\u3c0.001) but not in slow CI experiments (P=0.382), possibly related to reperfusion injury as indicated by greater hemorrhagic transformation (P \u3c 0.001) and hemispheric swelling (P \u3c 0.05). Outcome and response to thrombolysis after ES are operator dependent, which needs to be considered when comparing results obtained from different laboratories

In vitro and in vivo models of cerebral ischemia show discrepancy in therapeutic effects of M2 macrophages.

THE INFLAMMATORY RESPONSE FOLLOWING ISCHEMIC STROKE IS DOMINATED BY INNATE IMMUNE CELLS: resident microglia and blood-derived macrophages. The ambivalent role of these cells in stroke outcome might be explained in part by the acquisition of distinct functional phenotypes: classically (M1) and alternatively activated (M2) macrophages. To shed light on the crosstalk between hypoxic neurons and macrophages, an in vitro model was set up in which bone marrow-derived macrophages were co-cultured with hippocampal slices subjected to oxygen and glucose deprivation. The results showed that macrophages provided potent protection against neuron cell loss through a paracrine mechanism, and that they expressed M2-type alternative polarization. These findings raised the possibility of using bone marrow-derived M2 macrophages in cellular therapy for stroke. Therefore, 2 million M2 macrophages (or vehicle) were intravenously administered during the subacute stage of ischemia (D4) in a model of transient middle cerebral artery occlusion. Functional neuroscores and magnetic resonance imaging endpoints (infarct volumes, blood-brain barrier integrity, phagocytic activity assessed by iron oxide uptake) were longitudinally monitored for 2 weeks. This cell-based treatment did not significantly improve any outcome measure compared with vehicle, suggesting that this strategy is not relevant to stroke therapy

M2 macrophages do not improve functional outcome after ischemic stroke.

<p><b>A-</b> mNSS test. <b>B-</b> Adhesive removal test. There was no statistical difference in rat performance before treatment (D2) for either behavioral test. After treatment (D11 and D16), there was an improvement in neuroscores in both treatment groups, but no statistically significant difference between them (analysis of variance for repeated measures, N = 8 in each group, p = 0.505). Similarly, time to remove the adhesive was slightly improved in both groups, without any significant difference (analysis of variance for repeated measures, N = 8 in each group, p = 0.699). PBS: phosphate-buffered saline (used as vehicle for cell delivery).</p

Experimental protocol and diagram of MRI session.

<p>(<b>A</b>) Experimental protocol. Surgery consisted in either transient middle cerebral artery occlusion (tMCAO) or sham operation. Treatment consisted in intravenous injection of either 2 million M2 macrophages diluted in 1 ml phosphate-buffered saline (PBS) or the same volume of pure PBS. The number of animals included is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067063#pone-0067063-t001" target="_blank">Table 1</a>. (<b>B</b>) Diagram of one MRI session. T1/T2-WI: T1/T2-weighted imaging; MSME: Multi-Slice Multi-Echo; USPIO: ultrasmall superparamagnetic particles of iron oxide. BBB: blood-brain barrier. The sequence parameters are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067063#pone-0067063-t003" target="_blank">Table 3</a>.</p

Hypoxic hippocampal slices trigger an alternatively-activated program in macrophages.

<p>Bone marrow-derived macrophages were cultured in the presence of control (Control) or oxygen/glucose-deprived OHCs (Hypoxia) in 6-well culture plates. Macrophages from each experimental condition were pooled and assessed by Q-RTPCR for the expression of the following genes: inductible nitric oxide synthase (iNOS), IL-6, TNF-α and Arginase-1. As controls, analyses were performed in parallel on mRNAs extracted from M1 macrophages (stimulated with IFN-γ), M2 macrophages (stimulated with IL-4) or M0 macrophages (non-stimulated). Data showed that, as expected, iNOS and the pro-inflammatory cytokines IL-6 and TNF-α were highly expressed in M1 macrophages as compared to M0 or M2 macrophages. With regard to these M1-type pro-inflammatory genes, bone marrow-derived macrophages co-cultured with oxygen/glucose-deprived OHCs (Hypoxia) exhibited low levels of mRNA expression and a similar profile to bone marrow-derived macrophages co-cultured with control OHCs (Control). In contrast, the M2-type gene Arginase-1 was highly expressed in hypoxic macrophages (co-cultured with oxygen/glucose-deprived OHCs) as compared to control macrophages (co-cultured with control OHCs). As expected, Arginase-1 was also highly expressed in M2 macrophages as compared to M0 or M1 macrophages. Data shown are representative of two independent experiments.</p