In vivo magnetic resonance studies of glycogen and hyperpolarized lithium in the rat brain

Nuclear magnetic resonance (NMR) is used for a large array of applications, ranging from chemical characterization to oil drilling to medical imaging. In these fields NMR is used as an investigational tool, but new techniques and applications are continuously being developed as well. The latter is also the subject of this thesis. After an introduction to NMR theory, it focuses on the development of new methodologies for two separate areas of investigation: the metabolism of brain glycogen and hyperpolarization via dynamic nuclear polarization (DNP). Brain glycogen is the main storage form of glucose in the brain. However, because it is present in much lower concentrations in the brain than in other tissues, the significance of this storage and other roles are subject to debate. To better understand the roles of brain glycogen, it is important to quantify its metabolic parameters. The only currently available method to detect brain glycogen in vivo is 13C NMR spectroscopy. Incorporation of 13C-labeled glucose is necessary to allow glycogen measurement, but might be affected by changes in the turnover between glucose and glycogen. We therefore established a protocol to measure the glycogen absolute concentration in the rat brain by eliminating label turnover as a variable. The approach is based on establishing an increased, constant 13C isotopic enrichment (IE). 13C-glucose infusion is then performed at this IE of brain glycogen. As glycogen IE cannot be assessed in vivo, we validated that it can be inferred from the N-acetyl-aspartate (NAA) IE in vivo: after [1-13C]-glucose ingestion, glycogen IE was 2.2 ± 0.1 times that of NAA. Glycogen concentration measured in vivo by 13C NMR (5.8 ± 0.7 µmol/g) was in excellent agreement with post-mortem biochemistry (6.4 ± 0.6 µmol/g). A second glycogen NMR protocol was then implemented to measure concentration and turnover simultaneously. After reaching isotopic steady state for glycogen C1 using [1-13C]-glucose administration, [1,6-13C2]-glucose was infused such that isotopic steady state was maintained at the C1 position, but the C6 position reflected 13C label incorporation. To overcome the large chemical shift displacement error between the C1 and C6 resonances of glycogen, 2D gradient-based localization using the Fourier series window approach was implemented. The glycogen concentration of 5.1 ± 1.6 µmol/g measured from the C1 position was in excellent agreement with concomitant biochemical determinations, while glycogen turnover measured from the rate of label incorporation into the C6 position of glycogen in the α-chloralose anesthetized rat was 0.7 µmol/g/h. The contrast agent Omniscan was added to a formic acid-filled glass reference sphere in the abovementioned protocols in order to shorten its T1 relaxation time and to accelerate the calibration procedure it is used in; therefore it was established that Omniscan in pure 13C formic acid has a relaxivity of 2.9 mM-1s-1. The study was expanded, and the relaxivity of several commercially available gadolinium (Gd)-based contrast agents was determined for X-nuclei resonances with long intrinsic relaxation times. The relaxivity of Omniscan on the glutamate C1 and C5 in aqueous solution was ~ 0.5 mM-1s-1. These relaxivities allow the preparation of solutions with a predetermined short 13C T1. Another isotope with a long relaxation time is lithium-6 (6Li). Lithium is an ion that is widely used in psychotherapy, and 6Li has an intrinsic relaxation time on the order of several minutes, which was strongly affected by the contrast agents. Its relaxivity ranged from ~ 0.1 mM-1s-1 for Omniscan to 0.3 for Magnevist, whereas the relaxivity of Gd-DOTP was 11 mM-1s-1. Overall, these experiments suggested that the presence of submicromolar contrast agent concentrations should be detectable, provided sufficient sensitivity is available, such as that afforded by hyperpolarization. This submicromolar contrast agent concentration detection was demonstrated in a subsequent study: first it was established that lithium-6 can be readily hyperpolarized through DNP within 30 min while retaining a long polarization relaxation time on the order of a minute. The shortening of this relaxation time was used for the detection of 500 nM contrast agent in vitro. Hyperpolarized lithium-6 was then administered to the rat, where its signal retained a relaxation time on the order of 70 s in vivo. Localization experiments implied that the lithium signal originated from within the brain and that it was detectable up to 5 min after administration. Next, ~ 1.5 µM of contrast agent was injected in the rat, and its effect on previously injected hyperpolarized lithium-6 was successfully quantified. It was concluded that the detection of submicromolar contrast agent concentrations using hyperpolarized NMR nuclei such as 6Li may provide a novel avenue for molecular imaging. In a separate set of of experiments, hyperpolarization through DNP was applied to 15N-labeled choline, 13C-labeled bicarbonate and 13C-labeled acetate. All three metabolites were successfully polarized and were detected both in vitro and in vivo.

Methods for Molecular Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy using Hyperpolarized Nuclei

Riccardo Balzan

Since the introduction 10 years ago of the dissolution method, Dynamic Nuclear Polarization (DNP) became a widely applied and powerful technique to enhance nuclear magnetic resonance (NMR) signals of low naturally abundant, insensitive nuclear spins for analytical chemistry and biomedical research. The aim of DNP is to obtain a very high degree of polarization on the nuclei of interest in cryogenic conditions (i.e. 1 K ), a hundred or more times higher than the thermal polarization. Then, trough a fast dissolution process the molecules hosting the nuclear spins are mixed in a room temperature solution and, since their polarization is conserved if their relaxation times are long enough, they can be used for in vitro or in vivo experiments with a SNR enhanced by 104 times or more. Although the DNP technique was established about 60 years ago, the hardware and cryogenic equipment needed to perform DNP and the subsequent dissolution, still are a technological challenge. In this thesis we will cover different DNP aspects, ranging from hardware installation, software development, solid state measurement, in vitro and in vivo experiments and a possible application of DNP to MRI studies of granular materials. A new DNP cryostat was developed and tested to determine its cryogenic performances and then optimized for fast cooldown, helium holdtime and overall minimal operational consumption. We will show the characteristics of the cryostat and the performances on standard operations like cooling and DNP at 1 K . Another important aspect the development tackled was automation. The management of the system was condensed in a single electronic box capabile to handle the interface with all the cryostat instruments. This box is driven by a single common USB port trough a custom made software interface we developed. Although a few manual operations are still needed, we achieved a high degree of automation. The solid state polarization enhancement ε is defined as the ratio between the DNP enhanced and the thermal polarization signals. It can be determined by measuring the DNP enhanced signal after the system stabilizes in the polarized state (i.e. after about 5 buildup time) and thermal polarization signal after the spins have fully relaxed to equilibrium (i.e. after about 5T1). As a first application of the new system, we present a method that exploits the behavior of steady states magnetization produced with trains of evenly spaced pulses, at constant flip angle. This method allows to precisely determine the thermally polarized signal of a sample with known T1 and a given flip angle in a small fraction (as low as half of a T1 or less depending on the requested accuracy) of the time needed in the usual "wait 5 T1 and pulse" scheme. The main drawback of the dissolution DNP is that the large polarization achieved will ineluctably relax towards its thermal equilibrium value in a few times T1, that for typical 13C labeled molecules is shorter than a minute. On the other hand some nuclei as 6Li and quaternary 15N show a very long T1 on the order of few minutes in biological conditions, allowing measurements as long as several minutes. 6Li is very sensitive to contrast agents such as Gd-doped contrast agents, O2 and de-oxygenated hemoglobin. We studied the effect of the oxygenation of human and rat blood on the 6Li T1, showing clear changes between oxygenated and de-oxygenated samples. Thus we showed that 6Li could be used to map blood oxygenation levels in vitro, and suggest possible in vivo applications, even in brain due to its capability to easily pass the blood brain barrier. 15N labeled choline has a very long 15N T1 and can be used to probe rate of transport or metabolism in brain. Although it has a long T1, the spectral dispersion between the precursor, choline, and its metabolites is very small. The spectral dispersion is much larger for 1 H spins and thus being able to partially transfer repeatedly the 15N to 1H before detection would greatly increase the spectral separation between metabolites while still being able to measure the time course of the metabolic process, which is not the case with a full transfer like with INEPT. We demonstrate that a Hartmann–Hahn polarization transfer can be used to partially transfer the hyperpolarization of 15N to surrounding 1H before detection on 1H. This method can then be applied to in vivo DNP experiments. Even with a very high signal it is very difficult to quantify metabolites that are very close in the NMR spectrum, compared to the actual in vivo linewidth. To improve the spectral resolution it is worth performing the experiments in high magnetic fields. We show the first in vivo rat muscle DNP measurement of acetate at 14.1 T , to resolve e.g. the glutamate peak from acetate substrate. Finally we discuss a segmentation algorithm used to precisely reconstruct granular samples composed of up to 104 spheres. We used a standard Gradient Echo imaging sequence on a Siemens 7 T MRI human scanner to obtain high resolution images of a phantom composed of a cylinder filled with Cu2+ doped water and plastic spheres. Then the 3D images where segmented trough a thresholded Hugh transform method and the beads centers determined. The datasets were studied to extract structural and geometrical information.

EPFL2013

Ultra high magnetic field for glial contribution into brain metabolism studied by MR spectroscopy and CEST methods for molecular imaging of glycogen

Elise Marie Catherine Vinckenbosch

Magnetic resonance at ultra-high field increases signal and spectral dispersion. In this thesis, I use those characteristics to investigate three different subjects in 13C spectroscopy, hyperpolarized methods and chemical exchange saturation transfer (CEST) used for molecular imaging. In the brain, metabolism is driven by glia-neuron interactions and is composed of at least two compartments with distinct TCA cycle kinetics. The first study examines cerebral metabolic adaptation after an induced glial TCA-cycle impairment using 1H and 13C spectroscopy. We treated rats with fluoroacetate, which specifically blocks glial aconitase and measured changes in neurochemical profile at euglycemic and hyperglycemic condi-tions by in vivo 1H MR spectroscopy. In addition, we evaluated the brain-compartmentalized metabolic adap-tation by following 13C-incorporation into brain amino acids by modern dynamic 13C MRS methods during infu-sion of [1,6-13C2]glucose. By following [1,6-13C2]glucose incorporation, we successfully observed an augmen-tation of pyruvate carboxylase and glutamine synthetase fluxes and a 20% decrease of glial TCA cycle flux. Neuronal metabolism was also affected. In the second study, we focused on hyperpolarized methods to increase acetate 13C polarization allowing us to follow the kinetics of its metabolic products in the TCA cycle. Indeed, infusion of hyperpolarized [1-13C]acetate, an astrocyte-specific precursor, and the in vivo detection of 2-oxo[5-13C]glutarate (2OG), a TCA intermediate, enables the direct analysis of glial Krebs cycle activity. We examined the effect of hyperpolar-ized acetate concentration on its cerebral metabolism and calculate the production rate of 2OG. A healthy group was compared to rodents treated with fluoroacetate. The conversion rates of acetate to 2OG were calculated and were found to depend on the substrate dose. We successfully estimated 2OG production rates. In partially inhibited TCA cycle conditions, the production rate of 2OG was reduced by a quarter. This level of reduction is agrees with the percentage of inhibition calculated in the first study. Finally, we investigated glycogen imaging in skeletal muscle using the CEST strategy. It has been demonstrat-ed in perfused liver that glycogen can be detected indirectly through the water signal using CEST. Glycogen detection is, due to its resonance close to water, strongly sensitive to spectral dispersion and improved by a higher static magnetic field. In contrast to liver, which contains ~300 mmol/g of glycogen and enjoys good glycogen specificity, myocellular content is also rich in creatine, which produces a spectral signal on the same order of amplitude as its glycogen content of ~80-30 mmol/g. In this study, we allowed glycogen to be de-tectable in vivo and distinguishable from the neighboring creatine at 14.1T. We propose to use creatine as an internal reference for muscular glycoCEST analysis. Scanning muscle after physical exercise, resulted in a 50% reduction of glycoCEST compared to muscle at resting state. This result supportis the specificity of the CEST method and is consistent with literature. This last study demonstrates for the first time that glycoCEST imag-ing is feasible in vivo.

EPFL2018

Mathematical Modeling of Brain Energy Metabolism, Measured with PET and MRS in Rodents

Bernard Lanz

Positron emission tomography (PET) and nuclear magnetic resonance spectroscopy (MRS) are two biomedical measurement techniques developed in the end of the XXth century, which drastically improved the amount of accessible information available in vivo. PET became popular through the most widely used tracer, fluorodeoxyglucose (FDG), which enables the measurement of the local glucose utilization and is nowadays routinely applied in clinical practice. Nuclear magnetic resonance is used in a large array of applications such as in analytical chemistry or chemical structure determination and is essentially known for its versatile medical imaging capabilities, grouped under the name of magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). In vivo MRS measures concentrations of metabolites by interacting with various nuclei such as 1H, 13C or 15N, with the major asset that MRS enables the identification of the chemical position at which the detected nuclei is located in the measured molecule. When coupled with labeled substrate infusions, dynamic MRS gives the opportunity to probe specific biochemical reactions directly in vivo, which opens the way to a wide range of metabolic studies. However, the correct interpretation of the elaborate dynamic labeling data acquired either with MRS or PET tracer experiments requires the application of adapted metabolic models to derive quantitative metabolic rates characterizing the biochemical processes under study. The work of this thesis involves both PET and MRS studies of brain energy metabolism in rodents and focuses on the development of adapted metabolic models to derive reliable metabolic fluxes characterizing various brain metabolic processes, such as glucose consumption, glial and neuronal oxidative metabolism and neurotransmission. FDG-PET enables the measurement of the cerebral metabolic rate of glucose (CMRGlc) using a three-compartment metabolic model for FDG transport across the blood-brain barrier and FDG phosphorylation to FDG-6P. Although this method is well established, a main drawback of CMRGlc measurement with FDG-PET is the necessity to characterize the available FDG concentration in the plasma over the experiment duration, the so-called arterial input function. This constraint is a strong limitation for preclinical studies in rodents, due to their low blood volume. We developed therefore a new method to extract the FDG input function directly from the PET images in rats and mice, using the time-activity curve of a voxel located in the inferior vena cava. The method was validated by comparison with a dedicated external blood counter and manual blood sampling. Using this method, a CMRGlc of ~0.22 μmol/g/min was determined in the mouse cortex. Glial oxidative metabolism can be specifically assessed using glial specific substrates, in particular [1-11C] acetate. However, no existing metabolic model described the 11C tissue activity curve in terms of neuroglial energy metabolism. In order to extract quantitative metabolic fluxes characterizing the glial TCA cycle rate and glutamate/glutamine cycling, we adapted the neuroglial two-compartment modeling approach previously used in 13C MRS cerebral metabolic studies to interpret positron emission data following 11C-acetate injection in the rat. The precision and accuracy of the estimated metabolic parameters was tested and a composite glial metabolic flux Vgtg=0.136 ± 0.042 μmol/g/min and a neurotransmission flux Vnt=0.170 ± 0.103 μmol/g/min were obtained. This approach enabled a direct comparison of the metabolic fluxes measured using 11C PET with the values determined with 13C MRS. In the field of 13C MRS, we took advantage of the higher sensitivity and spectral resolution available at high magnetic field (14.1T) to measure glial and neuronal oxidative metabolism, glutamatergic neurotransmission and pyruvate carboxylation in more details and with higher precision using both [1,6-13C2] glucose and [2-13C] acetate infusions in rats. In the acetate study, the glial specific uptake of acetate enabled a quantification of glial oxidative metabolism with unrivalled precision. We could estimate for the first time separately the transmitochondrial flux Vx in the glial and neuronal compartment, a biochemical pathway which has been subject of strong controversy. The glial and neuronal Vx were determined with high precision, as calculated through Monte Carlo simulation, and their value was on the same order of magnitude than the respective glial and neuronal TCA cycle flux. The model was extended to estimate the fraction of glutamate located in the glial compartment. A glial glutamate concentration of 0.6 ± 0.1 μmol/g was found. The values of the different metabolic fluxes characterizing the neuroglial system (Vtcag=0.27, Vxg=0.17, VPC=0.087, VNT=0.15, Vtcan=0.37 and Vxn=0.46 μmol/g/min) were in very good agreement with the values found using [1,6-13C2] glucose infusion. In addition, the effect of the MRS temporal resolution, experiment duration and signal- to-noise ratio on the precision of the derived metabolic fluxes was analyzed. Previous simulation studies showed that two-compartment modeling of neuroglial metabolism using 13C glucose infusion could lead to unreliable parameter estimations. However, using polarization transfer 13C MRS at 14.1T, the carbon positions C4, C3 and C2 of glutamate and glutamine as well as the positions C3 and C2 of aspartate could be measured with high SNR. The C2 positions were used in two-compartment modeling for the first time, enabling a precise measurement of the apparent glutamatergic neurotransmission and glial metabolism, including pyruvate carboxylation, as confirmed by Monte Carlo simulation. The glial dilution at the level of acetyl-CoA, which is related to the metabolism of other substrates than glucose, could also be characterized. Brain metabolism under hyperammonemic conditions was probed with 15N MRS under labeled ammonia infusion and modeled using a modified neuroglial two- compartment model describing the dynamics of [5-15N] glutamine, [2-15N] glutamate and [2-15N] glutamine. Due to detoxification processes, the total glutamine concentration, measured with 1H MRS, was steadily increasing. This non-steady- state metabolic condition resulting from the hyperammonemic stress was taken into account in the model, resulting in the determination of the apparent neurotransmission (0.26 ± 0.030 μmol/g/min), the glutamate dehydrogenase (0.029 ± 0.002 μmol/g/min) and net glutamine accumulation (0.033 ± 0.001 μmol/g/min). Finally, compartmental modeling was applied to 13C labeling studies in the awake rat, fed during 5, 24 or 48 h with a [1-13C]-labeled glucose solution, to determine brain glycogen and NAA turnover times (τGlyc=5.3 ± 3.2 h and τNAA=15.6 ± 6.5 h), using 13C spectra of brain extracts. A group of rats followed a mild brain activation protocol over 5 hours, which resulted in a decreased glycogen turnover time (τGlyc =2.9 ± 1.2 h).

EPFL2012