Dans le cadre du développement des nanotechnologies pour les sciences de la vie et de la santé, les nanovecteurs magnétiques connaissent un essor considérable. Ces structures composites constituées de sphères polymériques encapsulant des nanoparticules magnétiques ou d`un coeur nanoparticulaire magnétique entouré d`une couverture organique présentent une combinaison de propriétés physico-chimiques et magnétiques très performante pour le diagnostic en imageries par exemple, notamment Imagerie par Résonance Magnétique (IRM), ou la thérapie : vectorisation pharmaceutique ciblée, hyperthermie thérapeutique…L`obtention de tels vecteurs avec une taille nanométrique permet l`injection intraveineuse chez les patients et la propagation dans l`organisme, tout en augmentant l`action liée à la surface spécifique. Les présents travaux de doctorat ont porté sur deux familles importantes de nanovecteurs magnétiques : - des nanosphères de polymère biocompatible chargé en composé radioactivable et encapsulant des nanoparticules de magnétite, pour la thérapie tumorale - des agents de contraste pour l`IRM de l`inflammation vasculaire ou cérébrale chez la souris, constituée d`un coeur nanoparticulaire d`oxyde de fer (maghémite ou magnétite) entourée d`une enveloppe organique pour le ciblage de la région visée (ultrasmall superparamagnetic iron oxide – USPIO, en anglais) Pour cerner le comportement de ces nanovecteurs en interaction avec le milieu liquide de suspension, puis avec les régions ciblées dans l`organisme, une approche physique multiéchelle de leurs structure et propriétés a été développée. Les études structurales des nanovecteurs ont été menées à bien grâce à des développements innovants s`appuyant sur les microscopies électroniques à résolution nanométrique. Par l`application du mode Wet-STEM, un nouveau mode en transmission de microscopie électronique à balayage environnementale, l`image en transmission de la structure interne organique/nanoparticule(s) magnétiques(s) a été obtenue et les simulations d`images par méthode de Monte Carlo ont montré qu`une résolution nanométrique pouvait être obtenue. Pour les nanovecteurs en environnement tissulaire, on a utilisé la microscopie électronique en transmission (MET) pour laquelle on a fait varier le degré de coloration dans des préparations de tissus ex vivo inclus en résine ; on a ainsi obtenu les premières images MET en haute résolution (METHR) spatiale d`agents de contraste USPIO cristallisés dans les tissus de l`aorte ou la rate chez la souris athérotique. En combinant ces études structurales avec la mesure des propriétés magnétiques par SQUID, un suivi longitudinal d`agents USPIO injectés chez la souris pour l`IRM de la plaque d`athérome a été menée à bien dans l`aorte et la rate : les résultats ont été interprétés en terme d`agglomération de particules à taille variable en fonction du temps de séjour dans l`organisme et confrontés à un modèle in vitro de dégradation en milieu acide (métabolisme lysosomal). / As applications of nanotechnologies for life and health sciences get booming, magnetic nanovectors undergo a considerable development. Such composite structures made from polymer spheres encapsulating magnetic nanoparticles or from a nanoparticular magnetic core surrounded by an organic coverage exhibit a combination of physical, chemical and magnetic properties very appropriate for diagnostic by imaging such as Magnetic Resonance Imaging (MRI), or for therapy: targeted pharmaceutical vectorization, therapeutic hyperthermia... When such vectors exhibit a nanometric size, intravenous injection and easy spread in the body of the patients are allowed, while effects related to the specific surface area are increased. The present doctoral work was concerned by two important families of magnetic nanovectors: - nanospheres of biocompatible polymer having loaded a radioactivable compound for tumoral therapy and having encapsulated magnetite nanoparticles for diagnostic by MRI: a system for thera-diagnostic is thus obtained.- contrast agents for MRI of vascular or cerebral inflammation, consisting of a nanometric iron oxide (maghemite or magnetite) core i.e. ultrasmall superparamagnetic iron oxide – USPIO - surrounded by an organic coverage for targeting the affected region. These USPIO were used to study inflammation in the atherotic plaque of the aorta in a murine model.Most of the time, such nanovectors are administered to the patients in liquid suspensions by intravenous injection. It is thus crucial to characterize both the collective behaviour and the individual structure of the vectors in liquid suspension. On the other hand their interactions with the targeted regions in the body have to be understood. For this purposes, a multiscale approach of the structure and properties of such nanovectors has been developed, with structural studies carried out through innovative developments based on electron microscopies down to subnanometric resolution and correlated with physical properties. To achieve characterization of nanovectors in liquid media we have developed the application of Wet-STEM, a new mode in transmission of environmental scanning electron microscopy (ESEM), to image the internal structure of the magnetic nanoparticles in liquid suspension and image calculations by Monte Carlo simulations have shown that a nanometric resolution could be theoretically achieved. By the same technique, stability or tendency to flocculation in suspensions can be evidenced with respect to the collective behavior of different nanovectors.In a second step we have investigated the interactions of the nanoparticles with targeted regions. The biodistribution and biotransformation of the USPIO contrast agents in the tissular and cellular environments were investigated at increasing spatial resolution using different techniques. The biodistribution of a MRI contrast agent grafted with a fluorophore, in ex vivo samples from atherotic aorta and spleen were revealed by biphoton microscopy with a resolution of a few hundred nanometers, down to macrophage scale. Then preparation of ex vivo samples for transmission electron microscopy (TEM) was adapted from standard protocols especially with respect to staining after inclusion in resin. This way, the first high resolution HR(TEM) images and electron diffraction patterns of crystallized USPIO contrast agents in the aorta or the spleen of an atheromatous mouse were obtained. Combining such structural studies with measurement (using a SQUID setup) of magnetic properties, a longitudinal follow-up of USPIO nanoparticles injected in mice for MRI of the atherotic plaque has been completed for USPIO particles embedded in the aorta and the spleen: the results were interpreted in terms of agglomeration of the particles with a decreasing size depending on time after injection and found consistent with a model of in vitro degradation in acidic environment proposed to mimick the lysosomal metabolism.

La Sclérose en Plaques (SEP) est la plus fréquente des maladies neurologiques acquises conduisant à un handicap chez l’adulte jeune. Le repérage précoce des patients susceptibles d’évoluer vers un handicap est un enjeu majeur afin de proposer un traitement adapté. Dans ce domaine, l’imagerie par résonnance magnétique (IRM) a complètement modifié la prise en charge des patients. Dans cette thèse, deux pistes sont explorées en vue d’améliorer le caractère pronostique de l’imagerie réalisée en début de maladie. Une première partie est consacrée aux apports potentiels de l’imagerie médullaire. Une deuxième partie s’intéresse à l’imagerie spécifique de l’inflammation par USPIO. La première partie de cette thèse correspond à un projet longitudinal et multicentrique intitulé EMISEP. Il vise à mesurer l’atteinte tissulaire médullaire en IRM conventionnelle et quantitative chez les patients atteints de SEP rémittente débutante, puis à étudier son lien avec le handicap ambulatoire à 5 ans. Aucune donnée n’était disponible concernant la variabilité en multicentrique des métriques issues de l’IRM médullaire quantitative. Notre première étude est donc consacrée à l’évaluation de la variabilité de la mesure du ratio de transfert d’aimantation (MTR) médullaire chez les sujets sains. Nous avons montré que la variabilité globale de la mesure du MTR médullaire était modérée avec un coefficient de variation de l’ordre de 3 %. Dans une deuxième étude, nous avons analysé les mesures de MTR initiales chez les patients SEP rémittents, comparativement à une population contrôle, afin de détecter une atteinte médullaire précoce. Nous avons mis en évidence une diminution significative des valeurs de MTR chez les patients comparés aux contrôles, mesurable dans la moelle épinière d’apparence normale et plus marquée proche de son centre et de sa périphérie. Puis nous avons réalisé une troisième étude centrée sur les voies motrices, particulièrement fonctionnelles. Nous avons quantifié spécifiquement l’atteinte du tractus cortico-spinal encéphalique et médullaire en IRM et évalué ses conséquences fonctionnelles cliniquement et en électrophysiologie. Nous avons montré que cette atteinte est fréquente, d’étendue très variable selon les patients, et qu’elle prédomine sur le segment médullaire des voies motrices. La sévérité de l’atteinte focale mesurée en imagerie en début de la maladie est déjà corrélée à ses conséquences cliniques et électrophysiologiques. Enfin, l’étude longitudinale, permettant d’évaluer le caractère pronostique de l’atteinte médullaire focale et diffuse précoce est en cours. La deuxième partie de cette thèse correspond à une autre étude longitudinale intitulée USPIO-CIS. Ses objectifs étaient d’étudier les prises de contraste USPIO comparativement au gadolinium dans une population de patients ayant un syndrome cliniquement isolé et d’évaluer leur rôle pronostique à moyen terme (3 ans). Nous avons montré que les prises de contraste USPIO sont rarement visualisées comparativement aux prises de contraste après injection de gadolinium dans cette population, mais sont associées à une déstructuration tissulaire initiale majeure, qui diminue progressivement au cours de la première année. Elle reste cependant plus marquée dans les lésions initialement USPIO positives comparées aux lésions seulement positives pour le gadolinium à 3 ans. En conclusion, nos travaux ont montré que l’atteinte médullaire est précoce chez les patients ayant une SEP rémittente, de sévérité variable selon les patients et intéresse notamment des zones très fonctionnelles comme les voies motrices. Son rôle pronostique, prometteur, est en cours d’évaluation. L’imagerie spécifique de l’inflammation par USPIO permet quant à elle d’expliquer les différents degrés de sévérité des lésions focales à moyen terme, mais les risques liés à son utilisation chez l’homme compromettent son potentiel comme outil pronostique en routine clinique. / Multiple Sclerosis (MS) is the most frequent acquired neurological disease leading to disability in young adults. In clinical practice, the identification of patients at risk of disability is a major issue, in order to adapt the treatment. Thus, prognostic factors are needed from the diagnosis and the very first years of the disease. In this area, MRI potentially has a crucial role. Two main avenue will be investigated in this work. The first part will be dedicated to spinal cord quantitative MRI and the second part to inflammation imaging using ultra-small paramagnetic iron oxides (USPIO). The first part of this work is a longitudinal, multicenter project called EMISEP. Its objectives are to quantify the structural damage of the spinal cord in early relapsing remitting MS patients, and to describe the link between spinal cord damage and walking disability at 5 years. In a first study, we assess the multicenter variability of magnetization transfer ratio (MTR) measurements in the spinal cord of healthy controls, before investigating this metric in MS patient. We demonstrate that the overall variability of the MTR measurements is low, with a coefficient of variation of 2.9%. The between-session variability represents the major part of the overall variability, compared to between-scanner variability. These results pave the way for multicenter analyses in MS patients. In a second study, we analyze MTR measurement in the spinal cord of early relapsing-remitting MS compared to controls. The MTR values are significantly lower in patients than controls, even after excluding lesions. We observe a greater mean reduction in MTR for vertebral levels displaying the highest lesion loads (C2-C4) and at the spinal cord periphery and barycentre. A third study focuses on the motor tracts. We specifically evaluate the cortico-spinal tracts damage in it brain and spinal cord portion and the functional consequences from the electrophysiological and the clinical point of view. The focal damage on CSTs is very frequent in our cohort of early RRMS patients. The spinal cord portion of the CSTs is the most affected by lesions, with a substantial variability between patients. We already find significant associations between the lesion volume fraction in the CSTs and clinical and electrophysiological motor tracts related abnormalities. Finally, longitudinal studies are ongoing to evaluate the prognostic value of initial spinal cord and cortico-spinal tract damage on subsequent ambulatory disability 5 years later. The second part of this work is another longitudinal study called USPIO-CIS. Its objectives are to describe the USPIO enhancement compared to gadolinium enhancement in a population of clinically isolated syndrome patients and to study their 3-years prognostic value. We demonstrate that USPIO enhancements are rare compared to gadolinium enhancement, and transient. USPIO positive lesions are associated with greater damage than gadolinium positive only lesions at baseline and throughout the 3-year follow up. In conclusion, our work demonstrates that spinal cord involvement is measurable using quantitative MRI in early relapsing remitting MS, even in a multicenter context. This early spinal cord damage is highly variable among patients, and is present in very functional tracts such as the cortico-spinal tracts. The prognostic value of this early damage is promising and will be evaluated in ongoing longitudinal studies. Inflammation imaging using USPIO is promising to explain the various degree of MS lesion severity. However, the risks associated with USPIO compromise it used as a prognostic tool in clinical routine.

Background Ultrasmall superparamagnetic particles of iron oxide (USPIO)- enhanced magnetic resonance imaging (MRI) can detect tissue-resident inflammatory macrophages and identify cellular inflammation. Clinical studies using this non-invasive technique are now emerging. Objectives The aims of this thesis were (i) to determine whether USPIO-enhanced MRI can detect and serially monitor myocardial inflammation after myocardial infarction (MI) using single and repeated USPIO administration, (ii) to report a range of normal R2* (1/T2*) values at 1.5 tesla (T) and 3 T in healthy myocardium and other tissues before and after USPIO administration, (iii) to determine whether USPIO-enhanced MRI can detect myocardial inflammation in acute myocarditis, and (iv) to determine whether USPIO-enhanced MRI can detect myocardial inflammation in patients with a prior cardiac transplant. Methods Thirty-one patients were recruited following acute MI and followed up for 3 months with repeated T2 and USPIO-enhanced T2* mapping 3 T MRI. Twenty healthy volunteers were recruited: 10 imaged each at 1.5 T and 3 T. T2 and USPIO-enhanced T2* mapping MRI was conducted. Fourteen patients with suspected acute myocarditis underwent T2 and USPIO-enhanced T2* mapping 3 T MRI, with further imaging at 3 months. Eleven patients with prior cardiac transplant underwent T2 and USPIO-enhanced T2* mapping 1.5 T MRI with further imaging at 3 months. Regions of interest within the myocardium, along with other tissues, were selected for analysis. Pre-contrast T2 values, and the change in R2* due to USPIO from baseline to 24 hours after USPIO were compared for each region of interest. Results In patients with MI, USPIO uptake in the infarct zone peaked at days 2-3, and greater USPIO uptake was detected in the infarct zone compared to remote myocardium in the first 2 weeks after myocardial infarction. In contrast, T2-defined myocardial oedema peaked at days 3-9 and remained increased in the infarct zone throughout the 3-month follow up period. Histology confirmed colocalisation of iron and macrophages within the infarcted, but not the non-infarcted, myocardium. In healthy volunteers, we reported a range of normal myocardial and tissue R2* values at baseline, and following USPIO. Tissues showing greatest USPIO enhancement were organs of the reticuloendothelial system: the liver, spleen and bone marrow. Myocarditis was confirmed in 9 of the 14 suspected cases of myocarditis. There was greater myocardial oedema, but no demonstrable difference in USPIO enhancement, in inflamed myocardial regions in patients with myocarditis when compared to healthy myocardium. We recorded an improvement in cardiac function and reduced imaging measures of inflammation after 3 months. Ten patients with cardiac transplant were retained for analysis. Measures of myocardial oedema were greater in patients with cardiac transplant than healthy volunteers. There was no difference in the change in R2* due to USPIO between patients with transplantation and healthy volunteers. Imaging recordings did not change when repeated at 3 months. Conclusions Myocardial macrophage activity can be detected using USPIO-enhanced MRI in the first 2 weeks following acute MI. This observed pattern of cellular inflammation is distinct, and provides complementary information to, the more prolonged myocardial oedema detectable using T2 mapping. In patients with acute myocarditis, USPIO-enhanced MRI does not provide additional clinically relevant information to standard clinical MRI sequences. This suggests that tissue-resident macrophages do not provide a substantial contribution to the myocardial inflammation in this condition. Stable patients with cardiac transplantation have increased myocardial T2 values, consistent with resting myocardial oedema or fibrosis. In contrast, USPIO-enhanced MRI is normal and stable over time suggesting the absence of chronic macrophage-driven cellular inflammation. In conclusion, this imaging technique holds promise as a non-invasive method of assessing and monitoring macrophage-driven myocardial inflammation after myocardial infarction with potential application to diagnosis, risk stratification and assessment of novel anti-inflammatory therapeutic interventions. It remains to be determined whether USPIO-enhanced MRI may be able to identify myocardial inflammation in other myocardial inflammatory conditions including acute cardiac transplant rejection.

An essential component of developing successful neural stem cell (NSC)-based therapies involves the establishment of methodologies to noninvasively monitor grafted NSCs within brain tissues in real time. In this context, ex vivo labeling with ultrasmall superparamagnetic iron oxide (USPIO) particles has been shown to enable efficient tracking of transplanted NSCs via magnetic resonance imaging (MRI). However, whether and how USPIO labeling affects the intrinsic biology of NSCs is not thoroughly understood, and remains an active area of investigation. Here, we perform a comprehensive examination of rat NSC survival and regenerative function upon labeling with the USPIO, Molday ION Rhodamine B (MIRB), which allows for dual magnetic resonance and optical imaging. After optimization of labeling efficiency, two specific doses of MIRB (20 and 50 mu g/mL) were chosen and were followed for the rest of the study. We observed that both MIRB doses supported the robust detection of NSCs, over an extended period of time in vitro and in vivo after transplantation into the striata of host rats, using MRI and post hoc fluorescence imaging. Both in culture and after neural transplantation, the higher 50 mu g/mL MIRB dose significantly reduced the survival, proliferation, and differentiation rate of the NSCs. Interestingly, although the lower 20 mu g/mL MIRB labeling did not produce overtly negative effects, it increased the proliferation and glial differentiation of the NSCs. Additionally, application of this dose also changed the morphological characteristics of neurons and glia produced after NSC differentiation. Importantly, the transplantation of NSCs labeled with either of the two MIRB doses upregulated the immune response in recipient animals. In particular, in animals receiving the 50 mu g/mL MIRB-labeled NSCs, this immune response consisted of an increased number of CD68(+)-activated microglia, which appeared to have phagocytosed MIRB particles and cells contributing to an exaggerated MRI signal dropout in the animals. Overall, these results indicate that although USPIO particles, such as MIRB, may have advantageous labeling and magnetic resonance-sensitive features for NSC tracking, a further examination of their effects might be necessary before they can be used in clinical scenarios of cell-based transplantation.

Coronary atherosclerosis and plaque rupture leads to acute coronary thrombosis and myocardial infarction. Current treatment involves re-establishing vessel patency, but no treatments have been developed to target post-infarction inflammatory pathways. Such treatments may reduce cardiomyocyte injury, attenuate adverse remodelling and improve clinical outcome. Inflammation within the infarcted myocardium is associated with chemotaxis of neutrophils and monocytes to the site of injury. Early reperfusion therapy amplifies this inflammatory cell influx. Neutrophil release a variety of pro-inflammatory factors, including human neutrophil elastase (HNE). HNE has a wide range of substrates. Preclinical studies have demonstrated that neutrophil depletion or inhibition of neutrophil elastase attenuates post-ischemic inflammatory reperfusion injury within the myocardium. Recruitment of monocytes into the infarcted myocardium is followed by maturation and differentiation into macrophages. Macrophages play a key role in orchestrating inflammation and repair. Therapeutic manipulation of this healing process will only come from understanding mechanisms and targeting reparative pathways. “Ultrasmall superparamagnetic iron oxide particles” (USPIOs) extravasate through capillaries and are phagocytosed by tissue inflammatory cells. These cells are predominately macrophages, but neutrophils have also been shown to take up USPIOs. USPIO-enhanced MRI can identify areas of inflammation in models inflammation in various tissues. Therefore we hypothesised that USPIO enhanced MRI could identify and assess cellular inflammation of the myocardium. During coronary artery bypass graft surgery (CABG), the myocardium receives an immediate ischaemic insult that is exacerbated by post-ischaemic reperfusion inflammatory responses leading to increased myocardial injury. CABG surgery can therefore be used as a clinical model of myocardial infarction and inflammation. We investigated this with blood markers of inflammation, MRI scanning and USPIO. Elafin inhibits the destructive and inflammatory HNE enzyme. Beyond this elafin inhibits inflammatory cytokines and modulates the innate and adaptive immune systems. In preclinical studies elafin treatment is associated with reduced myocardial injury. As such, elafin has a marked potential for the treatment of cardiovascular disease involving inflammation. Therefore, we hypothesised that elafin will reduce perioperative ischaemic myocardial injury and inflammation in patients undergoing elective coronary artery bypass graft surgery. We demonstrated for the first time that USPIOs are taken up by the infarct tissue in patients with recent myocardial infarction and by the peri-infarct myocardium to a lesser degree. This represents a novel non-invasive method to further study cardiac inflammation and therapeutic interventions. All patients undergoing CABG surgery demonstrated >10-fold elevation above the 99th centile of cardiac troponin by high sensitivity assay (hs-cTnI) indicating the current universal definition of type 5 myocardial infarction lacks specificity. A peak hs-cTnI at 6 hours following CABG surgery appears to be related to the surgical process and non-specific myocardial injury whilst a continuing increase at 24 hours suggests myocardial infarction. We would suggest hs-cTnI sampling at 6 and 24 hours post CABG surgery together with ECG assessment for the routine detection and diagnosis of type 5 MI. Differing levels of humoral makers inflammation post CABG surgery occurred, and did not correlate directly with the length of cardiopulmonary bypass time or hs-cTnI release. For the first time we identified differing levels of inflammatory cell infiltrate into the myocardium post CABG. This varied from none to levels similar to infarcted myocardial tissues. Elafin did not attenuate myocardial ischemia-reperfusion injury and inflammation. Post-hoc analysis identified reduced cTnI concentrations at 6 hours in Elafin treated patients and it is possible that a bigger dose would have conferred protection out to 48 hours. Elafin did not attenuate the cellular infiltration into the myocardium post CABG surgery, but did appear to reduce inflammation in renal tissue. USPIO enhanced CMR holds major promise in the non-invasive assessment of myocardial inflammation post surgery.

No description available.

La réaction inflammatoire post-ischémique est dominée par les cellules mononuclées phagocytaires : la microglie, cellules immunocompétentes du parenchyme cérébral, et les macrophages dérivant du sang et infiltrant le tissu cérébral lésé. L’imagerie cellulaire par IRM avec injection de nanoparticules superparamagnétiques d’oxydes de fer (USPIO) a contribué à la description dynamique de cette infiltration macrophagique tardive. Cependant, la séquence spatio-temporelle de l’activation microgliale et du recrutement macrophagique, intriqués avec des altérations de la barrière hématoencéphalique au cours des premières heures post-ischémie, reste mal élucidée. Nous avons tenté de mieux comprendre la relation entre les signaux IRM après injection d’USPIO et la réaction phagocytaire aux temps précoces post-ischémie dans un modèle d’accident vasculaire cérébral chez la souris. Nos résultats suggèrent que les modifications précoces de signal après injection d’USPIO reflètent principalement des mécanismes non spécifiques d’entrée des particules dans le tissu lésé. Pour étudier les interactions entre les cellules neurales et les macrophages périphériques au cours de la réaction inflammatoire post-ischémique précoce, nous avons développé un modèle in vivo d’ischémie globale chez la souris et un modèle in vitro d’hypoxie sur cultures organotypiques d’hippocampe. Ce dernier modèle nous a permis d’analyser les effets de co-cultures macrophagiques sur la survie des cellules neurales au sein du parenchyme cérébral ischémié et d’analyser les profils d’expressions cytokiniques impliqués dans ces interactions à médiation humorale. Nous avons observé un effet neuroprotecteur sur la perte neurale post-hypoxique des co-cultures macrophagiques. Cet effet à médiation humorale est associé à un profil d’activation macrophagique de type alternatif (M2). / Clinical outcome in cerebral ischemia may be influenced by innate immune cells of myeloid lineage : central nervous system (CNS)-infiltrating peripheral macrophages and CNS-resident microglia. Noninvasive monitoring of these cells may improve the understanding of postischemic inflammation. Accumulation of Ultrasmall Superparamagnetic Particles of Iron Oxide (USPIO) has been observed in infarcted areas at the subacute stage of experimental stroke. However, the exact route of USPIO uptake and early brain distribution remain elusive, hampering the interpretation of USPIO-relatedsignals. Therefore, we compared MRI signal changes after intravenous USPIO injection with the histological distribution of iron particles and macrophagic cells 6 to 24 hours after permanent middle cerebral artery occlusion in mice. Our results suggest that in this model, early USPIO-related MR signal changes are mainly related to passive diffusion of free USPIO through a damaged blood-brain barrier and to intravascular trapping rather than peripheral phagocyte infiltration. To understand the complex interactions between microglia, hypoxic neurons and CNS-infiltrating macrophages, we setup an in vitro model where primary macrophages were co-cultured with hippocampal slices submitted to hypoxia and glucose deprivation (OGD). Our results indicate that under these experimental conditions, cultured macrophages engage in a M2 activation pattern and afford partial protection from OGD-induced neuronal loss through paracrine mechanisms. We also conclude that microglia is susceptible to hypoxia-induced cell death in vitro and in vivo

La sclérose en plaques est une maladie chronique. Elle se caractérise par une inflammation focale et diffuse, une démyélinisation, une souffrance axonale et une dégénérescence neuronale. L’apparition d’une incapacité progressive et irréversible en est la principale morbidité. Mieux comprendre les liens entre les différents mécanismes impliqués dans la SEP et la part de chacun dans la genèse du handicap est fondamental.L’inflammation dans la SEP est polymorphe. Une famille de produits de contraste, l’USPIO, permet le marquage des macrophages et leur suivi en IRM. Ainsi, 35 patients ont été inclus. Le rehaussement par l’USPIO était spatialement et temporellement différent de celui observé après injection de gadolinium. Les lésions caractérisées par une activité macrophagique induisent localement une déstructuration tissulaire plus importante sur l’IRM initiale et après 1 an.À cette inflammation aiguë s’ajoutent l’inflammation et la démyélinisation chroniques, qui entraînent une accumulation de sodium. La deuxième étude a permis de montrer cette accumulation en IRM chez 20 patients souffrant de la forme progressive, où prédominent l’inflammation et la démyélinisation chroniques.L'importance de l’accumulation corticale de sodium chez ces patients nous a conduit à entreprendre une troisième étude chez des patients souffrant de troubles cognitifs. Ainsi 58 patients rémittents ont été inclus dans cette étude. Une accumulation de sodium a été mise en évidence chez les patients présentant une atteinte cognitive. Cette accumulation touche le néocortex, en particulier préfrontal, cingulaire et le precuneus. De plus, cette accumulation survient avant l’atrophie. / Multiple sclerosis (MS) is a chronic disease leading to permanent disability. Demyelination, inflammation and diffuse axonal and neuronal loss are histological hallmarks of MS. Better understand the links between these different mechanisms and their role in the genesis of disability is fundamental. Inflammation in MS is polymorphic. A family of recent contrast agents, the USPIO, allows the labelling and monitoring of macrophages. 35 patients were included. Enhancement by USPIO was spatially and temporally different from gadolinium. Lesions characterized by macrophage activity locally induce a more significant tissue destructuration at baseline and at one year.Moreover, chronic inflammation and chronic demyelination lead to intra-neuronal sodium accumulation. Because of the physical properties of 23Na and technical improvements, sodium MRI imaging is to date possible. Thus in the second study, sodium accumulation was demonstrated in 20 patients at a progressive phase, particularly susceptible to chronic inflammation and demyelination. Then, the discovery of the importance of cortical sodium accumulation in this study led us to undertake a third study in patients prone to cognitive disorders, were cortical involvement seems to be more pronounced. 58 remitting patients were enrolled. Sodium accumulation was demonstrated in patients with cognitive impairment. This accumulation affects the neocortex (prefrontal cortices, cingulate and the precuneus). This accumulation occurs before atrophy.

As applications of nanotechnologies for life and health sciences get booming, magnetic nanovectors undergo a considerable development. Such composite structures made from polymer spheres encapsulating magnetic nanoparticles or from a nanoparticular magnetic core surrounded by an organic coverage exhibit a combination of physical, chemical and magnetic properties very appropriate for diagnostic by imaging such as Magnetic Resonance Imaging (MRI), or for therapy: targeted pharmaceutical vectorization, therapeutic hyperthermia... When such vectors exhibit a nanometric size, intravenous injection and easy spread in the body of the patients are allowed, while effects related to the specific surface area are increased. The present doctoral work was concerned by two important families of magnetic nanovectors: - nanospheres of biocompatible polymer having loaded a radioactivable compound for tumoral therapy and having encapsulated magnetite nanoparticles for diagnostic by MRI: a system for thera-diagnostic is thus obtained.- contrast agents for MRI of vascular or cerebral inflammation, consisting of a nanometric iron oxide (maghemite or magnetite) core i.e. ultrasmall superparamagnetic iron oxide - USPIO - surrounded by an organic coverage for targeting the affected region. These USPIO were used to study inflammation in the atherotic plaque of the aorta in a murine model.Most of the time, such nanovectors are administered to the patients in liquid suspensions by intravenous injection. It is thus crucial to characterize both the collective behaviour and the individual structure of the vectors in liquid suspension. On the other hand their interactions with the targeted regions in the body have to be understood. For this purposes, a multiscale approach of the structure and properties of such nanovectors has been developed, with structural studies carried out through innovative developments based on electron microscopies down to subnanometric resolution and correlated with physical properties. To achieve characterization of nanovectors in liquid media we have developed the application of Wet-STEM, a new mode in transmission of environmental scanning electron microscopy (ESEM), to image the internal structure of the magnetic nanoparticles in liquid suspension and image calculations by Monte Carlo simulations have shown that a nanometric resolution could be theoretically achieved. By the same technique, stability or tendency to flocculation in suspensions can be evidenced with respect to the collective behavior of different nanovectors.In a second step we have investigated the interactions of the nanoparticles with targeted regions. The biodistribution and biotransformation of the USPIO contrast agents in the tissular and cellular environments were investigated at increasing spatial resolution using different techniques. The biodistribution of a MRI contrast agent grafted with a fluorophore, in ex vivo samples from atherotic aorta and spleen were revealed by biphoton microscopy with a resolution of a few hundred nanometers, down to macrophage scale. Then preparation of ex vivo samples for transmission electron microscopy (TEM) was adapted from standard protocols especially with respect to staining after inclusion in resin. This way, the first high resolution HR(TEM) images and electron diffraction patterns of crystallized USPIO contrast agents in the aorta or the spleen of an atheromatous mouse were obtained. Combining such structural studies with measurement (using a SQUID setup) of magnetic properties, a longitudinal follow-up of USPIO nanoparticles injected in mice for MRI of the atherotic plaque has been completed for USPIO particles embedded in the aorta and the spleen: the results were interpreted in terms of agglomeration of the particles with a decreasing size depending on time after injection and found consistent with a model of in vitro degradation in acidic environment proposed to mimick the lysosomal metabolism.

Staphylococcus aureus est une des espèces bactériennes les plus fréquemment responsable des cas d’endophtalmie et d’arthrite aiguës chez l’homme. Nous avons étudié la pharmacodynamie oculaire du linézolide et de la daptomycine, ainsi que leur toxicité, dans un modèle d’endophtalmie expérimentale chez le lapin. Il est apparu que seule une dose très élevée delinézolide administrée par voie intravitréenne (30mg/0,1mL) présentait une efficacité bactérioclinique. Aucune altération de la fonction visuelle n’a été mise en évidence. La dose intravitréenne efficace de daptomycine (1mg/0,1mL) était quant à elle responsable d’une altération significative de l’intégrité fonctionnelle de la rétine. Enfin, la pharmacodynamie de ces deux antibiotiques dans le compartiment oculaire présentait des différences significatives avec les autres sites tissulaires d’infection étudiés à ce jour. Dans la deuxième partie de nos travaux nous nous sommes intéressés à une nouvelle technique d’IRM, associée à des particules d’USPIOs (Ultrasmall SuperParamagnetic Iron Oxide). Nous avons montré que cette techniqued’imagerie permettait d’une part la mise en évidence de l’infiltration des macrophages dans la synoviale infectée, et, d’autre part, la visualisation de la guérison de l’articulation, contrairement à l’imagerie conventionnelle à base de chélates de gadolinium. Cette technique innovante offre donc une nouvelle dimension à l’imagerie ostéo-articulaire grâce à une mise en évidence in vivo plus spécifique des phénomènes pathologiques. / Staphylococcus aureus (S. aureus) is a frequent cause of acute endophthalmitis and arthritis in humans. In the first part, we investigated the ocular pharmacodynamics and safety of two recently approved antistaphylcoccal antibiotics, linezolid and daptomycin. Only a very high intravitreal dose of linezolid (30 mg / 0.1 mL) showed a bactericidal and clinical efficacy. Suchintraocular concentrations appeared to be safe for the retinal function, and linezolide could be considered as a promising therapeutic alternative. The effective intravitreal dose of daptomycin (1 mg / 0.1 mL) was responsible for a significant impairment of the functional integrity of the retina. Finally, the ocular pharmacodynamics of these two antibiotics showed special features in comparison with the one of other types of tissue infection. In a second part, we evaluated a newimaging method in experimental infectious arthritis, by using MRI enhanced by ultrasmall superparamagnetic iron oxide (USPIO). We first showed this method can depict the macrophage infiltration in infected synovium, and secondly it can demonstrate resolution of joint infection, in contrast to conventional MRI performed by gadolinium chelates. This in vivo non invasive imaging method therefore presents a new dimension in musculoskeletal imaging by accurately helping monitor bacterial joint infection.