Improved ¹H-[¹³C] and ¹H NMR methods for in vivo measurement of cerebral metabolism

Nuclear magnetic resonance (NMR) spectroscopy can be applied in vivo to measure static or dynamic biochemical information, e.g., concentrations of metabolites and metabolic fluxes, using various nuclei such as 1H, 13C, 31P and 15N. The work of this thesis involves both 1H and 13C nuclei and focuses on improving 1H NMR detection methods for measuring metabolites in vivo in rat brain. 13C NMR spectroscopy can be used to monitor the flow of 13C label from a 13C enriched substrate, such as glucose or acetate, into different NMR detectable metabolites, e.g., glutamate (Glu) and glutamine (Gln), for the quantitative study of cerebral metabolism in vivo. 1H-observed 13C-edited (1H-[13C]) NMR spectroscopy is an alternative to direct 13C NMR detection of 13C labeled metabolites, allowing a higher spatial and temporal resolution, albeit at a lower spectral resolution. In this context, a hybrid full signal intensity 1H-[13C] NMR sequence, combining a 13C editing block based on an inversion B1 insensitive spectral editing pulse (BISEP) with a spin-echo based localization (SPECIAL), was developed and implemented at ultra-high magnetic field (14.1 T) to benefit from increased sensitivity and spectral resolution at high B0. As a result, high quality 1H-[13C] NMR spectra were obtained, leading to an improved quantification of 13C labeled metabolites, which allowed the measurement of time courses of Glu C4, Gln C4, as well as, for the first time, of Glu C3 and of Gln C3, with high temporal resolution from a small acquisition volume. Although at high magnetic field spectral resolution and sensitivity are improved, spectral overlap is still present, e.g., the N-acetylaspartate (NAA) C6 is easily obscured by the intensive labeling of Glu C3 and Gln C3 (Glx C3), due to their complex coupling patterns. To improve the detection of unresolved 13C labeled metabolites, we developed an alternative 1H-[13C] NMR editing sequence termed RACED-STEAM (selective Resonances suppression by Adiabatic Carbon Editing and Decoupling single-voxel STimulated Echo Acquisition Mode), which can selectively suppress 1H resonances bound to a specific 13C to uncover resonances that are difficult to resolve in the 1H-[13C] NMR spectrum. Results showed the efficient suppression of Glx C3 and Glu C4, allowing the detection of NAA C6 and Gln C4 at 9.4 T. The application of this method to measure the time course of NAA C6 demonstrated that NAA C6 turnover can be measured at very low levels of isotopic enrichment in a small volume, within a time frame of a few hours. The proposed scheme could also be extended to lower magnetic fields provided that the 13C chemical shifts remain sufficiently resolved. Similarly, due to limited spectral resolution and sensitivity, editing techniques for the in vivo detection of metabolites using 1H NMR spectroscopy are still in development. Many in vivo 1H NMR editing sequences are usually performed at moderate to long echo times (TE). Furthermore, several 1H NMR spectroscopy studies are performed at long TE to avoid the confounding effect of macromolecular signals on metabolite quantification. In such cases, proton T2 relaxation times of metabolites have to be taken into account for proper quantification of metabolite concentrations. While T2 relaxation times of singlets have been characterized in several studies, due to the relative experimental simplicity, similar information is lacking from coupled spin resonances of cerebral metabolites. In this thesis, spectral simulations based on the density matrix formalism were initially performed to predict the response of spin systems to the pulse sequence used. T2 relaxation times of coupled spin resonances and singlet resonances of cerebral metabolites were then measured in rat brain in vivo at 9.4 T. Data analysis was performed using LCModel combined with simulated TE-specific spectra. The aforementioned spectral simulations were further used to optimize pulse sequence parameters for the detection of metabolites, such as glycine, whose resonance signal is overlapped with the more intense myo-inositol resonances. A favorable short TE (i.e., 20 ms) was sufficient to reduce the signal intensity of myo-inositol, allowing the detection of glycine in vivo in rat brain at 9.4 T. This work suggests that at high magnetic fields, glycine can be measured at a relatively short TE without additional editing efforts. Moreover, glycine is present at a particularly high concentration in the medulla oblongata (MO). Therefore, we further measured regional distribution of glycine in the hippocampus, cortex, striatum and MO of the rat brain, as well as the highly specific neurochemical profile of the MO. In conclusion, dedicated approaches for 1H NMR detection developed and validated in this thesis lead to improved dynamic measurement of 13C labeling time courses such as the time courses of 13C labeling of Glu C3 and Gln C3, as well as the spectral resolved NAA C6, the direct in vivo detection of glycine in rat brain at 9.4 T and the measurement of T2 relaxation times of numerous J-coupled metabolites for the first time.

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

Biophysical Basis of the Diffusion-Weighted Magnetic Resonance Signal in the Rat Brain

Nicolas Kunz

Nuclear magnetic resonance (NMR) can be used in-vivo in a vast array of applications, such as anatomical imaging (magnetic resonance imaging, MRI), localized chemical composition characterization (magnetic resonance spectroscopy, MRS), cellular structure assessment (diffusion tensor imaging, DTI) and cerebral activity mapping (functional imaging, fMRI) for the most important one. This thesis focused on the development of diffusion NMR spectroscopy and imaging methods at ultra-high magnetic field, with the aim of a better characterization of the diffusion mechanism in-vivo. DTI measures the water molecule displacement due to the thermal agitation in the sample. In cellular tissue, the molecules are restrained in compartments delimited by the cell membranes, which mean that DTI can provide information on the cerebral cellular microstructure. DTI is thus widely used to investigate cerebral disorders such as brain ischemia, trauma, and tumors, as well as the structural changes occurring during brain development and normal aging. However, the presence of water molecules in both the intra- and extra-cellular compartments may bias the assessed structural information. On the other hand, the metabolites, measured by 1H-MRS, are mostly localized in the intracellular compartment, which may provide a more precise insight of the cellular structure. With the availability of high magnetic field systems, 1H-MRS allows the measurement of 15 – 20 metabolites in-vivo in localized region in the order of &icro;l in the rodent brain. These metabolites are involved in cell energy management, neurotransmission, neuro-protection, neuronal development and membrane growth, and thus provide a rather complete status of the brain neurochemical profile. In this context, a diffusion tensor spectroscopy pulse sequence (DT-MRS) was implemented and used to investigate the metabolite diffusion properties in specific brain regions in-vivo at ultra-high magnetic field 14.1 T to benefit from the increased sensitivity and spectral resolution at high B0. The diffusion tensor of 5 metabolites were reconstructed in the corpus-callosum, CC, (NAA, Tau, Glu, Ins and Mac) and 8 in the cortex, Cx, (with in addition Cr, PCr, GABA and GSH). Metabolites apparent diffusion coefficients were significantly higher in the CC (0.115 – 0.153 µm2/ms) than in the Cx (0.087 – 0.107 µm2/ms) and so was the fractional anisotropy (0.51 – 0.62 and 0.34 – 0.51 respectively). In addition, the metabolite preferential diffusion directions showed an excellent agreement with the known rat brain structure. The aforementioned DW-MRS pulse sequence was then combined with an inversion recovery module, allowing direct assessment of the macromolecule (MM) signal, which overlaps with metabolites resonances. An inaccurate estimation of the MM baseline may result in a significant bias during the quantification process. At ultra-high magnetic field, the MM spectral shape becomes more complex, and therefore, the previously reported experimental techniques would require post-processing to remove residual metabolite signals. In this study, several metabolite contaminations remaining in the metabolite nulled spectrum were successfully identified and attributed to creatine, myo-inositol, taurine, choline, N-acetylaspartate, glutamine and glutamate. The macromolecule baseline assessed by DW-MRS resulted in a twofold reduction of the metabolite residuals with minimal effect on the macromolecule signal (i.e. < 8% signal reduction). Although at high magnetic field spectral resolution and sensitivity are improved, metabolite diffusion measurements remain very time consuming and challenging due to the low metabolite concentrations in-vivo (1 – 15 mM) compared to water (44 M). Therefore, a fast and robust EPI-based DTI pulse sequence was developed, implemented and validated to investigate the cerebral architecture in-vivo at 14.1 T through water diffusion. Combined with the use of a purpose-built quadrature surface coil and adiabatic RF-pulses, it resulted in images with a full coverage of the rat brain and a total acquisition time of one hour. The rapid signal loss arising from the use of a surface coil for the MRI acquisitions, which was previously in this thesis overcome experimentally, was also addressed in collaboration with the Signal Processing Core of CIBM, by a direct correction applied on reconstructed magnitude images. Based on a low-pass frequency mode; the developed method showed better accuracy in enhancing image intensity compared to standard approaches. In view of a future application of the presented diffusion NMR techniques, investigating structural changes during brain development, a study was performed to characterize different intra-uterine growth retardation (IUGR) models by 1H-MRS. Dexamethasone-treated animals (100 µg/kg/day induced from the day 15 of gestation) showed more severe alterations in cerebral energy metabolism and neurotransmitter action at postnatal day 7 and 21 than the reference IUGR model, caloric restriction (30% food intake of ad libitum fed dams from day 1 of gestation). In a continuation of the IUGR study, the exposure to the estrogen-like molecule (i.e. bisphenol-A, BPA), was investigated. Here, 1H-MRS showed a significant increase of the Glu/Asp ratio in the hippocampus of the BPA-treated group at 20 days old, indicating a possible deviation in the malate-aspartate shuttle. Finally, the diverse NMR techniques used and developed during this thesis have required the implementation of several data processing tools, which were centralized in a framework written in Matlab (Mathworks, Inc). Two principal modules were build: 1. to reconstruct DTI experiments data and 2. to facilitate and standardize the 1H-MRS spectra quantification by LCModel. The proposed NMR data processing framework was used to process most of the data presented in this thesis and has demonstrated good performance. In addition, this framework was tested and used by five external users to analyze for data-analysis in ongoing studies.

EPFL2010

Proton T-2 relaxation time of J-coupled cerebral metabolites in rat brain at 9.4T

Giulio Gambarota, Rolf Gruetter, Lijing Xin

Knowledge of proton T-2 relaxation time of metabolites is essential for proper quantitation of metabolite concentrations in localized proton spectroscopy, especially at moderate to long TEs. Although the T-2 relaxation time of singlets, such as that of creatine and N-acetylaspartate, has been characterized in several studies, similar information is lacking from coupled spin resonances of cerebral metabolites. In this study, the T-2 relaxation time of coupled spin resonances and singlet resonances of cerebral metabolites was measured in rat brain in vivo at 9.4T. Spectra were acquired at 11 TEs using the SPin ECho, full Intensity Acquired Localized (SPECIAL) spectroscopy method. Data analysis was performed in the frequency domain with the LCModel software using simulated TE-specific basis sets. The T-2 relaxation times in compounds showing singlet resonances were 113 +/- 3 ms (total creatine), 178 +/- 29 ms (total choline) and 202 +/- 12 ms (N-acetylaspartate). The T-2 values of J-coupled metabolites ranged from 89 +/- 8 ms (glutamate) to 148 +/- 14 ms (myo-inositol). Copyright (C) 2007 John Wiley & Sons, Ltd.