Proton diffusion spectroscopy and modeling of brain metabolism at 14.1T

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.

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

Diffusion-weighted MRS of acetate in the rat brain

Masoumeh Dehghani Moghadam, Rolf Gruetter, Nicolas Kunz, Bernard Lanz, Hikari Ananda Infinity Yoshihara

Acetate has been proposed as an astrocyte-specific energy substrate for metabolic studies in the brain. The determination of the relative contribution of the intracellular and extracellular compartments to the acetate signal using diffusion-weighted magnetic resonance spectroscopy can provide an insight into the cellular environment and distribution volume of acetate in the brain. In the present study, localized (1) H nuclear magnetic resonance (NMR) spectroscopy employing a diffusion-weighted stimulated echo acquisition mode (STEAM) sequence at an ultra-high magnetic field (14.1 T) was used to investigate the diffusivity characteristics of acetate and N-acetylaspartate (NAA) in the rat brain in vivo during prolonged acetate infusion. The persistence of the acetate resonance in (1) H spectra acquired at very large diffusion weighting indicated restricted diffusion of acetate and was attributed to intracellular spaces. However, the significantly greater diffusion of acetate relative to NAA suggests that a substantial fraction of acetate is located in the extracellular space of the brain. Assuming an even distribution for acetate in intracellular and extracellular spaces, the diffusion properties of acetate yielded a smaller volume of distribution for acetate relative to water and glucose in the rat brain.

Wiley-Blackwell2017

In Vivo Magnetic Resonance Spectroscopy (MRS) of the Activated Human Brain at 7T

Benoît Schaller

The principal aim of this thesis was to investigate human brain energy metabolism during physiological activation using proton (1H) magnetic resonance spectroscopy (MRS) at 7 Tesla (T). High magnetic fields (≥ 7 T) provide several advantages in human brain imaging and spectroscopy. In particular, signal-to-noise ratio (SNR) and chemical shift dispersion increase at higher magnetic fields, allowing for improved accuracy and precision of metabolite quantification. To take profit of the benefits of MRS at high field, one needs to provide an advanced methodology including hardware, pulse sequences experimental setup and data analysis. Each of these aspects was thoroughly explored in this thesis. In the field of functional studies, functional MRS (fMRS) has recently been used as a research tool to investigate the neuroenergetic and metabolic basis of physiologic brain activation. It provides direct insight into brain metabolism by non-invasively determining concentrations of metabolites. This is critical for the basic understanding of overall brain function, and potentially of the pathogenesis of many neurodegenerative diseases. In general, an optimized MR system and accurate acquisition methodology are required for high sensitivity and reliable metabolite quantification. The metabolites of interest for fMRS studies (e.g. lactate, glucose) are present in low concentration and metabolite changes are small under activation. Hence, studies of metabolite changes, in particular lactate, have led to inconsistent reports in the literature over the last decades. To address these challenges, it was essential to develop a robust MR protocol for the quantitative measurement of metabolite changes. A fMRS study was performed to investigate metabolite changes during visual stimulation using the enhanced sensitivity of the SPin ECho full Intensity Acquired Localized (SPECIAL) sequence. Small but significant increases of lactate (19 ± 4 %, P < 0.05) and glutamate (4 ± 1 %, P < 0.001) were observed using a small number of subjects (n = 6). With the exception of glucose (12 ± 5 %, P < 0.001), no other significant metabolite concentration changes beyond experimental error were observed. Based on this successful fMRS study, we further investigated brain energy metabolism. A subsequent fMRS study was performed to determine metabolite changes occurring during motor activation in the human brain. This second study demonstrated that increases in lactate (17 ± 5 %, P < 0.001) and glutamate (2 ± 1 %, P < 0.005) during motor stimulation were small, but similar to those observed during visual stimulation. These metabolite changes were further analyzed, and they supported the hypothesis of an increase in the change of cerebral metabolic rate of oxygen, ∆CMRO2, that is transiently lower than that of glucose, ∆CMRGlc, during the first one to two minutes of stimulation. Finally, we hypothesized that the observed glutamate and lactate increases were a general manifestation of the blood-oxygenation level dependent effect. The accuracy and the reliability of metabolite quantification is particularly challenging at short echo times due to the presence of broad underlying resonances overlapping with those of metabolite. Such resonances arise from macromolecules, which are characterized by short T1 and T2 relaxation times. Two studies were performed at 3 T and 7 T to investigate the characteristics of the macromolecule signal. An important finding was that the mathematical approximation (spline baseline) was a sufficient estimation of the macromolecule contribution to 1H spectra at 3 T compared to the experimentally measured macromolecule signal for healthy subjects. As a caveat, small, but significant, differences in the quantification may need to be taken into account when comparing metabolite concentrations obtained with these two different approaches, such as for glutamate and for γ-amino-butyric, which were more reliably quantified when using the measured macromolecule baseline. In addition, a study on the tissue-specificity of the macromolecule signal in the healthy human occipital lobe at 7 T reported that a general average macromolecule signal ensured reliable metabolite quantification. In general, these studies helped to set up a robust and reliable methodology in order to investigate human brain metabolism with improved accuracy. Despite their great potential for MRS, high main magnetic fields also compose challenges. In particular, the wavelength of the proton spin resonance becomes comparable to the size of a human head (at 7 T, λ = 11 cm). The phase variation across the brain produces signal addition (constructive interference, central bright spot) and signal cancellation (destructive interference, signal drop). Transceive arrays have been developed to address this issue. In this study, we designed, tested, and built an eight-channel transceive array, which was used to acquire images of the human head at 7 T (297 MHz). The design aimed for a low mutual coupling between the microstrip elements (> -15 dB), a relatively large penetration depth in the loading sample, reduced radiation losses and load-invariant tuning and matching. Electromagnetic simulations were run using Microwave Studio (CST, Darmstadt, Germany) for design optimization. The model was further demonstrated with the construction of the transceive array. Finally, the resulting array coil was shown to be handling, stable and also suited for techniques in which the transmission signals from all channels with different amplitudes and phases are combined. To conclude, this thesis provided complementary and new metabolic information about cerebral energy metabolism during physiological activation. Furthermore, the macromolecule studies and the building of the transceive array allowed to improve the accuracy and sensitivity of NMR measurements.