Cerebral glutamine metabolism under hyperammonemia determined in vivo by localized H-1 and N-15 NMR spectroscopy

Brain glutamine synthetase (GS) is an integral part of the glutamate-glutamine cycle and occurs in the glial compartment. In vivo Magnetic Resonance Spectroscopy (MRS) allows noninvasive measurements of the concentrations and synthesis rates of metabolites. N-15 MRS is an alternative approach to C-13 MRS. Incorporation of labeled N-15 from ammonia in cerebral glutamine allows to measure several metabolic reactions related to nitrogen metabolism, including the glutamate-glutamine cycle. To measure N-15 incorporation into the position 5N of glutamine and position 2N of glutamate and glutamine, we developed a novel N-15 pulse sequence to simultaneously detect, for the first time, [5-N-15] Gln and [2-N-15]Gln + Glu in vivo in the rat brain. In addition, we also measured for the first time in the same experiment localized H-1 spectra for a direct measurement of the net glutamine accumulation. Mathematical modeling of H-1 and N-15 MRS data allowed to reduce the number of assumptions and provided reliable determination of GS (0.30 +/- 0.050 mu mol/g per minute), apparent neurotransmission (0.26 +/- 0.030 mu mol/g per minute), glutamate dehydrogenase (0.029 +/- 0.002 mu mol/g per minute), and net glutamine accumulation (0.033 +/- 0.001 mu mol/g per minute). These results showed an increase of GS and net glutamine accumulation under hyperammonemia, supporting the concept of their implication in cerebral ammonia detoxification. Journal of Cerebral Blood Flow & Metabolism (2012) 32, 696-708; doi:10.1038/jcbfm.2011.173; published online 14 December 2011

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

Development and implementation of ¹H magnetic resonance techniques for an improved in vivo assessment of models of cerebral metabolic disorders

Mélanie Craveiro

Nuclear magnetic resonance (NMR) is a physical phenomenon that is widely used in the biomedical field due to its non-invasive and non-destructive properties, which make it an optimal tool for the in vivo investigation of living organs such as the brain. This thesis focused on the development of 1H magnetic resonance (MR) techniques at ultra-high magnetic field strength to improve the measurement and quantification of the 1H MR signal in the rodent brain, and to accurately assess the alterations of the metabolite and water concentrations in several cerebral metabolic disorders. In rodents, 1H MR spectroscopy (MRS) allows the assessment of the concentration of up to 20 metabolites that take part in many cerebral metabolic processes such as neurotransmission, cell energy metabolism, cell growth and osmosis. 1H MRS thus provides a unique tool to non-invasively investigate the cerebral metabolism in healthy and pathological conditions in vivo. However, it is essential for reliable quantification that systematic errors and overlap of the measured metabolite signals are minimized. To that aim, the impact of a potential additional short T2 relaxation time component, which might affect the glutamine quantification at long echo times, was assessed in this thesis. The J-difference editing technique MEGA-SPECIAL was optimized to obtain the unequivocal detection of the glutamine signal at moderate echo time. As a control, the glutamine concentration obtained with this method was then compared to short echo time 1H MR spectroscopy measurements. Since the two measurements of the glutamine concentration at short and moderate echo time did not result in significant differences, this study concluded that there is a low probability of an effect of a short T2 relaxation time component on the glutamine concentration measurement. Since a reliable quantification of 1H spectra partly relies on the accurate assessment of the overlapping macromolecule contribution to the metabolites, an optimized method for the post-processing of the measured macromolecule signal was developed to ensure an accurate assessment of its contribution. This method was applied to investigate potential regional differences in the mouse brain macromolecule signals that may affect metabolite quantification when not taken into account, as well as for the assessment of macromolecule alterations in a human-glioma mouse model. No regional macro- molecule variation was found to significantly affect the metabolite quantification in the healthy mouse brain, which supports the common use of a general macromolecule spec- trum for healthy rodent brain 1H spectra quantification. However, several alterations of the macromolecule spectrum, some of which were reported for the first time, were observed in glioma tissues, and their accurate assessment was shown to be necessary for reliable metabolite quantification. Localized 1H MR spectroscopy techniques that are designed to acquire the1H MR signal from a 3D volume can also be combined with MR imaging (MRI) to acquire information on the spatial distribution of the individual metabolites, which is often of interest when heterogeneous tissues are investigated (such as in pathological conditions). This MR spectroscopic imaging (MRSI) technique is however limited in its conventional implementation by the long measurement time that is necessary to acquire the spatial information. A fast MRSI technique, spectroscopic RARE (spRARE), was therefore implemented at high magnetic field strength in this thesis in order to benefit from the advantage of high magnetic fields in addition to the rapid spatial encoding scheme offered by spRARE. Several optimizations of the metabolite signal detection were implemented in spRARE, before the efficiency of the technique was validated at 9.4 T through a comparison of the acquired in vitro metabolite signals with numerical simulations using the density matrix formalism. The in vivo quantification of the water content is also of interest as it offers perspec- tives for investigating cerebral disease progression that is associated with brain water content alterations. In addition, when the water signal is used as an internal reference for metabolite concentration quantification in MRS, such knowledge can also contribute to a more reliable quantification. A method that is based on the multi-spin-echo MRI technique was therefore developed for the absolute quantification of the water content in the rodent brain, with the aim of assessing alterations of its content in pathological conditions. It was then applied on a bile-duct-ligation rat model of chronic hepatic encephalopathy (HE) to assess the brain water content changes associated with the disease as well as its metabolic alterations. The absence of significant brain water content changes detected in the investigated HE rat model, which was supported by post-mortem brain water content measurements using gravimetry and the dry/wet weight-ratio technique, potentially opens the way for investigating cerebral metabolic processes in chronic HE in absence of edema formation. Localized 1H MRS and conventional MRSI were also applied for the investigation of rodent models of brain disorders in order to assess the metabolic profile associated with those disorders. These studies for the first time allowed the assessment of GABA as a potential early Parkinson’s disease marker, while they also provided new insights into the role of the PPAR-β brain receptor in the early neuroprotective effects following ischemia.

EPFL2013

Proton diffusion spectroscopy and modeling of brain metabolism at 14.1T

Masoumeh Dehghani Moghadam

As a field at the CIBM, Nuclear magnetic resonance (NMR)spectroscopy can be applied non-invasively to explore the metabolic fate of energy fuel substrates, as well as the rate at which they are consumed, using 13C and 1H nuclei. The work of this thesis encompasses both nuclei, and focuses on (1) improving the quantification and modeling of glucose-derived metabolites; and (2) characterizing diffusion-related parameters of the purportedly glial-specific energy substrate, acetate. Both aim to quantitatively explore cerebral energy metabolism, at ultra-high magnetic field, in vivo, in the healthy rat. 13C NMR spectroscopy, as a tool, enables measuring the progressive incorporation of 13C-glucose into brain glucose and then NMR detectable amino acids (glutamate and glutamine); this relies on the infusion of the 13C-labeled energy substrate. The experimentally obtained 13C labelling curves are analyzed using suitable mathematical models to provide an estimation of cerebral metabolic rates. Here, a dynamic model of time-courses of 13C multiplets arising from isotopomers was considered. So beyond the two-compartment neuronal-glial model, we took into account additional data on the dynamics of 13C isotopomers, available from the fine structure multiplets in 13C spectra of glutamate and glutamine, measured under prolonged [1-6,13C] glucose infusion. We concluded that the dynamic analyses of 13C multiplet time courses of glutamate and glutamine resulted in a higher precision for estimating the absolute values of most cerebral metabolic rates. Acetate metabolism is challenging because dynamic metabolic modeling requires prior knowledge of the transport and uptake kinetics of infused acetate. We sought this information by determining the apparent concentration and distribution volume (V_d) of cerebral acetate between the intracellular and the extracellular compartments. Experimentally, the diffusion characteristics of cerebral acetate were measured, relative to that of N-Acetylaspartate (NAA, known to be mainly intracellular) using diffusion-weighted 1H NMR spectroscopy at 14.1T, under prolonged acetate infusion. The detection of an acetate and NAA signal at large diffusion weighting provided direct experimental evidence of intracellular cerebral acetate and NAA, although a substantial fraction of acetate was extracellular. To estimate the apparent concentration of in vivo brain acetate, T1 and T2 relaxation times of acetate were measured. The longer T1 relaxation and shorter T2 relaxation times of acetate compared with NAA provided evidence of its small molecular size, and possibly different chemical environment. Our experimentally determined value of V_dled to cerebral metabolic rates of acetate (CMR acetate) of the same order reported for the glial Krebâs cycle rate, an indication that estimates of CMR acetate are highly dependent on V_d. Finally, in order to pursue metabolic mapping of cerebral acetate uptake in the rat, in vivo, at 14.1 T, the design and construction of a combined transmit-birdcage coil and receive-quadrature pair surface coil was considered. Its performance was compared to a single birdcage coil in the transmit/receive mode. So far, the preliminary results of the 2-coil configuration are promising: homogenous excitation and a gain in sensitivity up to a distance of 5 mm are achievable. Improvements are ongoing for NMR spectroscopic and imaging applications at 14.1 T.

EPFL2016