In vivo phosphorus spectroscopy in human subjects on a clinical 3T MRI system

Alors que l’Imagerie par résonance magnétique (IRM) permet d’obtenir un large éventail de données anatomiques et fonctionnelles, les scanneurs cliniques sont généralement restreints à l’utilisation du proton pour leurs images et leurs applications spectroscopiques. Le phosphore jouant un rôle prépondérant dans le métabolisme énergétique, l’utilisation de cet atome en spectroscopie RM présente un énorme avantage dans l’observation du corps humain. Cela représente un certain nombre de déEis techniques à relever dus à la faible concentration de phosphore et sa fréquence de résonance différente. L’objectif de ce projet a été de développer la capacité à réaliser des expériences de spectroscopie phosphore sur un scanneur IRM clinique de 3 Tesla. Nous présentons ici les différentes étapes nécessaires à la conception et la validation d’une antenne IRM syntonisée à la fréquence du phosphore. Nous présentons aussi l’information relative à réalisation de fantômes utilisés dans les tests de validation et la calibration. Finalement, nous présentons les résultats préliminaires d’acquisitions spectroscopiques sur un muscle humain permettant d’identiEier les différents métabolites phosphorylés à haute énergie. Ces résultats s’inscrivent dans un projet de plus grande envergure où les impacts des changements du métabolisme énergétique sont étudiés en relation avec l’âge et les pathologies.Although magnetic resonance imaging (MRI) provides a wide array of anatomical and functional contrasts, clinical MRI systems are typically limited to imaging of tissue water and spectroscopy based on hydrogen atoms in more complex molecules. Given the important role of phosphate metabolism in virtually all biological processes, there has been considerable interest in the development of technology allowing detection and spectroscopy of magnetic resonance signals associated with phosphorus in the human body. This poses a number of technical challenges, due to the lower natural abundance and resonant frequency of phosphorus. The objective of this project was to develop and implement a basic in vivo phosphorus capability on a clinical 3 Tesla MRI scanner. We present here the various steps toward building and testing a working prototype of MR coil tuned to phosphorus frequency. We also provide information on building a set of test phantoms required for this project that can be used for signal calibration. Finally we show preliminary phosphorus spectra from muscle, in human subjects, demonstrating the main constituents in high-energy phosphate metabolism. These results pave the way for future applications in which the metabolic physiology of the human brain will be studied during aging and in disease

Hyperpolarized 13 C magnetic resonance spectroscopy detects toxin-induced neuroinflammation in mice.

Lipopolysaccharide (LPS) is a commonly used agent for induction of neuroinflammation in preclinical studies. Upon injection, LPS causes activation of microglia and astrocytes, whose metabolism alters to favor glycolysis. Assessing in vivo neuroinflammation and its modulation following therapy remains challenging, and new noninvasive methods allowing for longitudinal monitoring would be highly valuable. Hyperpolarized (HP) 13 C magnetic resonance spectroscopy (MRS) is a promising technique for assessing in vivo metabolism. In addition to applications in oncology, the most commonly used probe of [1-13 C] pyruvate has shown potential in assessing neuroinflammation-linked metabolism in mouse models of multiple sclerosis and traumatic brain injury. Here, we aimed to investigate LPS-induced neuroinflammatory changes using HP [1-13 C] pyruvate and HP 13 C urea. 2D chemical shift imaging following simultaneous intravenous injection of HP [1-13 C] pyruvate and HP 13 C urea was performed at baseline (day 0) and at days 3 and 7 post-intracranial injection of LPS (n = 6) or saline (n = 5). Immunofluorescence (IF) analyses were performed for Iba1 (resting and activated microglia/macrophages), GFAP (resting and reactive astrocytes) and CD68 (activated microglia/macrophages). A significant increase in HP [1-13 C] lactate production was observed at days 3 and 7 following injection, in the injected (ipsilateral) side of the LPS-treated mouse brain, but not in either the contralateral side or saline-injected animals. HP 13 C lactate/pyruvate ratio, without and with normalization to urea, was also significantly increased in the ipsilateral LPS-injected brain at 7 days compared with baseline. IF analyses showed a significant increase in CD68 and GFAP staining at 3 days, followed by increased numbers of Iba1 and GFAP positive cells at 7 days post-LPS injection. In conclusion, we can detect LPS-induced changes in the mouse brain using HP 13 C MRS, in alignment with increased numbers of microglia/macrophages and astrocytes. This study demonstrates that HP 13 C spectroscopy has substantial potential for providing noninvasive information on neuroinflammation

Methodological developement in X nuclei NMR for in vivo study of aging brain metabolism

Le but de ce travail de thèse a été de développer à MIRC en une capacité à observer deux aspects clefs du métabolisme cérébral chez le rongeur par spectroscopie RMN des noyaux X : le métabolisme mitochondrial du glucose à partir de l’observation du 13C et la synthèse d’ATP par observation du 31P. Ces développements s’inscrivent à la fois dans une recherche fondamentale pour améliorer notre compréhension du signal RMN et raffiner son analyse ainsi que du métabolisme cérébral chez les sujets sains. Ils s’inscrivent aussi dans une recherche translationnelle avec la possibilité d’évaluer certains aspects du métabolisme comme potentiels biomarqueurs de maladies neurodégénératives. Ces travaux ont pu être réalisés à très haut champ (11.7T) permettant d’obtenir un meilleur rapport signal à bruit. Dans un premier temps, nous présenterons le développement d’une séquence de transfert de saturation pour la mesure des flux de synthèse d’adénosine triphosphate (ATP) et de phosphocréatine (PCr). Cette séquence a été optimisée pour sélectionner avec un module de localisation ISIS le signal émis par le cerveau uniquement. Avec l’augmentation de la résolution spectrale à haut champ, cette séquence a pu être utilisée pour caractériser le phosphate inorganique extracellulaire, et prévenir un biais de quantification possible à plus bas champ. De plus, elle a permis l’observation de l’adaptation du métabolisme cérébral chez les rats transgéniques BACHD, modèles de la maladie de Huntington. Ces rats présentent une augmentation d’environ 10% de la concentration de PCr permettant de pallier à leur plus faible taux de synthèse d’ATP qui lui est diminué de moitié. Dans un second temps, la mise en place d’un pipeline automatisé d’analyse des données a permis d’explorer le modèle métabolique bicompartimental de la consommation de glucose observée en spectroscopie 13C, qui prend en compte le cycle de Krebs dans les neurones et les astrocytes. Deux corrections majeures ont été apportées au modèle traditionnel permettant d’expliquer les dynamiques à moyen et long termes. La première est la mise en évidence d’un pool de glutamate vésiculaire agissant comme tampon temporel au marquage du glutamate, la seconde est la présence d’une dilution 6 fois plus importante de pyruvate vers les astrocytes que vers les neurones. Ces résultats viennent renforcer les hypothèses entourant le couplage métabolique entre ces deux types cellulaires. Ces hypothèses ont pu être testées après l’optimisation d’une séquence d’acquisition du signal RMN des noyaux 13C par transfert de polarisation (DEPT), testée in vivo dans le cerveau du rat sain. Finalement, l’utilisation combinée de la spectroscopie 31P et 13C a été appliquée chez le rat sous intoxication chronique au 3-NP, une toxine inhibant le cycle de Krebs et utilisée comme modèle de la maladie de Huntington.The aim of this thesis was to develop at MIRCen new capabilities to observe two key aspects of energy metabolism in rodent brains using X nuclei NMR spectroscopy: glucose consumption with 13C spectroscopy and adenosine triphosphate (ATP) synthesis with 31P measurements. These developments will be used to both expand general understanding of brain metabolism in healthy subjects but also provide technical tools to search for biomarkers in translational projects of drug development applied to neurodegenerative diseases. This work was done at very high field (11.7T) where signal to noise could be maximized. In the first part, we present the optimization of saturation transfer sequence to measure ATP synthesis rate as well as phosphocreatine (PCr) synthesis rate. With ISIS module, the signal was localized to a voxel containing only the brain, eliminating outside source of signal. With the higher spectral resolution offered by high fields, a second, extracellular pool of Pi was characterized which could prevent possible biases in flux quantification of ATP synthesis. This sequence was also applied to measure metabolic adaptation of BACHD rat models (models of Huntington’s disease, HD) where it was found that the 10% increase in PCr concentration could palliate the ATP synthase activity that is halved in this model. In the second part, we present how deeper analysis of 13C data using automatic differential equation writing script was used to better understand the bicompartmental model of glucose degradation to glutamate and glutamine, which accounts for TCA cycle in neurons and astrocytes. Two major corrections were made to the traditional model, to fit mid- and long-term unexplained dynamics. Looking at glutamate and glutamine isotopomer labeling dynamics, the necessity of adding a vesicular glutamate temporal buffer was made evident. The distinction between astrocytic and neuronal pyruvate dilution also showed that astrocytes use up to 6 times more pyruvate than neurons showing intricate metabolic coupling between the two cell types. These results have then been tested in vivo after optimization of the ISIS-DEPT sequence to observe 13C labeling in the rat brain. Finally, experiments combining 31P and 13C spectroscopy were performed on rats chronically intoxicated with 3-NP, a toxin inhibiting TCA cycle which is used as a model of HD

Brief Communication

International audienceWith the increased spectral resolution made possible at high fields, a second, smaller inorganic phosphate resonance can be resolved on $^{31}$P magnetic resonance spectra in the rat brain. Saturation transfer was used to estimate de $novo$ adenosine triphosphate synthesis reaction rate. While the main inorganic phosphate pool is used by adenosine triphos-phate synthase, the second pool is inactive for this reaction. Accounting for this new pool may not only help us understand $^{31}$P magnetic resonance spectroscopy metabolic profiles better but also better quantify adenosine triphos-phate synthesis

Experimental strategies for in\ vivo 13^{13}C NMR spectroscopy

International audience$In\ vivo$ carbon-13 ($^{13}$C) MRS opens unique insights into the metabolism of intact organisms, and has led to major advancements in the understanding of cellular metabolism under normal and pathological conditions in various organs such as skeletal muscles, the heart, the liver and the brain. However, the technique comes at the expense of significant experimental difficulties. In this review we focus on the experimental aspects of non-hyperpolarized $^{13}$C MRS $in\ vivo$. Some of the enrichment strategies which have been proposed so far are described; the various MRS acquisition paradigms to measure $^{13}$C labeling are then presented. Finally, practical aspects of $^{13}$C spectral quantification are discussed

MPI region of interest (ROI) analysis and quantification of iron in different volumes

MPI directly detects superparamagnetic iron oxides (SPIOs), which should enable precise, accurate and linear quantification. However, selecting a region of interest (ROI) has strong effects on MPI quantification results. Ideally, ROI selection should be simple, user-independent, and widely applicable. In this work, we describe and compare four MPI ROI selection methods and assesses their performance in vitro and in vivo. To explore the effect of ROI selection, ten ferucarbotran phantoms were imaged, each contained the same amount of iron but varied in volume. Three users tested the accuracy of the ROI methods for quantification of these samples. Lastly, quantification of ferucarbotran-labeled stem cells in vivo was demonstrated with the four ROI methods. We demonstrate that each ROI method has strengths. We conclude there is an important trade-off between ROI size and the accuracy of iron quantification, therefore the choice of ROI selection method for each study must be carefully informed

Hyperpolarized 13 C magnetic resonance spectroscopy detects toxin-induced neuroinflammation in mice.

Lipopolysaccharide (LPS) is a commonly used agent for induction of neuroinflammation in preclinical studies. Upon injection, LPS causes activation of microglia and astrocytes, whose metabolism alters to favor glycolysis. Assessing in vivo neuroinflammation and its modulation following therapy remains challenging, and new noninvasive methods allowing for longitudinal monitoring would be highly valuable. Hyperpolarized (HP) 13 C magnetic resonance spectroscopy (MRS) is a promising technique for assessing in vivo metabolism. In addition to applications in oncology, the most commonly used probe of [1-13 C] pyruvate has shown potential in assessing neuroinflammation-linked metabolism in mouse models of multiple sclerosis and traumatic brain injury. Here, we aimed to investigate LPS-induced neuroinflammatory changes using HP [1-13 C] pyruvate and HP 13 C urea. 2D chemical shift imaging following simultaneous intravenous injection of HP [1-13 C] pyruvate and HP 13 C urea was performed at baseline (day 0) and at days 3 and 7 post-intracranial injection of LPS (n = 6) or saline (n = 5). Immunofluorescence (IF) analyses were performed for Iba1 (resting and activated microglia/macrophages), GFAP (resting and reactive astrocytes) and CD68 (activated microglia/macrophages). A significant increase in HP [1-13 C] lactate production was observed at days 3 and 7 following injection, in the injected (ipsilateral) side of the LPS-treated mouse brain, but not in either the contralateral side or saline-injected animals. HP 13 C lactate/pyruvate ratio, without and with normalization to urea, was also significantly increased in the ipsilateral LPS-injected brain at 7 days compared with baseline. IF analyses showed a significant increase in CD68 and GFAP staining at 3 days, followed by increased numbers of Iba1 and GFAP positive cells at 7 days post-LPS injection. In conclusion, we can detect LPS-induced changes in the mouse brain using HP 13 C MRS, in alignment with increased numbers of microglia/macrophages and astrocytes. This study demonstrates that HP 13 C spectroscopy has substantial potential for providing noninvasive information on neuroinflammation

Using 31P-MRI of hydroxyapatite for bone attenuation correction in PET-MRI: proof of concept in the rodent brain

Abstract Background The correction of γ-photon attenuation in PET-MRI remains a critical issue, especially for bone attenuation. This problem is of great importance for brain studies due to the density of the skull. Current techniques for skull attenuation correction (AC) provide indirect estimates of cortical bone density, leading to inaccurate estimates of brain activity. The purpose of this study was to develop an alternate method for bone attenuation correction based on NMR. The proposed approach relies on the detection of hydroxyapatite crystals by zero echo time (ZTE) MRI of 31P, providing individual and quantitative assessment of bone density. This work presents a proof of concept of this approach. The first step of the method is a calibration experiment to determine the conversion relationship between the 31P signal and the linear attenuation coefficient μ. Then 31P-ZTE was performed in vivo in rodent to estimate the μ-map of the skull. 18F-FDG PET data were acquired in the same animal and reconstructed with three different AC methods: 31P-based AC, AC neglecting the bone and the gold standard, CT-based AC, used to comparison for the other two methods. Results The calibration experiment provided a conversion factor of 31P signal into μ. In vivo 31P-ZTE made it possible to acquire 3D images of the rat skull. Brain PET images showed underestimation of 18F activity in peripheral regions close to the skull when AC neglected the bone (as compared with CT-based AC). The use of 31P-derived μ-map for AC leads to increased peripheral activity, and therefore a global overestimation of brain 18F activity. Conclusions In vivo 31P-ZTE MRI of hydroxyapatite provides μ-map of the skull, which can be used for attenuation correction of 18F-FDG PET images. This study is limited by several intrinsic biases associated with the size of the rat brain, which are unlikely to affect human data on a clinical PET-MRI system