Late post‐natal neurometabolic development in healthy male rats using 1 H and 31 P Magnetic Resonance Spectroscopy

Brain metabolism evolves rapidly during early post‐natal development in the rat. While changes in amino acids, energy metabolites, antioxidants or metabolites involved in phospholipid metabolism have been reported in the early stages, neurometabolic changes during the later post‐natal period are less well characterized. Therefore, we aimed to assess the neurometabolic changes in male Wistar rats between post‐natal days 29 and 77 (p29‐p77) using longitudinal magnetic resonance spectroscopy (MRS) in vivo at 9.4 Tesla. 1H MRS was performed in the hippocampus between p29‐p77 at one‐week intervals (n=7) and in the cerebellum between p35‐p77 at two‐week intervals (n=7) using the SPECIAL sequence at ultra‐short echo‐time. NOE enhanced and 1H decoupled 31P MR spectra were acquired at p35, p48 and p63 (n=7) in a larger voxel covering cortex, hippocampus and part of the striatum. The hippocampus showed a decrease in taurine concentration and an increase in glutamate (with more pronounced changes until p49), seemingly a continuation of their well described changes in the early post‐natal period. A constant increase in myo‐inositol‐ and choline‐containing compounds in the hippocampus (in particular glycero‐phosphocholine as shown by 31P MRS) was measured throughout the observation period, probably related to membrane metabolism and myelination. The cerebellum showed only a significant increase in myo‐inositol between p35‐p77. In conclusion, the present study showed important changes in brain metabolites in both the hippocampus and cerebellum in the later post‐natal period (p29/p35‐p77) of male rats, something previously unreported. Based on these novel data, changes in some neurometabolites beyond p28‐35, conventionally accepted as the cut off for adulthood, should be taken into account in both experimental design and data interpretation in this animal model.

Brain metabolism during Chronic Hepatic Encephalopathy studied by in vivo 1H and 31P MRS

Veronika Rackayová

Magnetic resonance spectroscopy (MRS) is a powerful tool increasingly used in biomedical research and also in clinical practice. This thesis focused on the improvements of in vivo 31P MRS and 31P magnetization transfer methods at high magnetic field (9.4T). Then, in vivo 31P MRS was combined with 1H MRS to longitudinally study neurometabolic changes during chronic hepatic encephalopathy (CHE) in adult and developing brain using a well-recognized animal model of cholestatic chronic liver disease and HE type C â bile duct ligated (BDL) rats. CHE is neuro-psychiatric disorder caused by chronic liver disease (CLD), with not well-understood molecular mechanisms, inducing important and sometimes lethal brain changes. In a next step, potential protective treatments (probiotics and high creatine diet) were tested. To the best of our knowledge such a detailed approach was not yet published. Firstly, we showed by in vivo 1H MRS in hippocampus that adult BDL rats suffered from increase in brain glutamine, decrease in metabolites involved in osmoregulation including creatine, decrease in neurotransmitters and ascorbate and we were able to show the importance of early metabolic changes compared to late changes thanks to longitudinal measurements. 31P MRS study in larger brain volume found only mild perturbation of energy metabolism such as non-significant trend in ATP decrease in adult BDL rats. Secondly, studies in young BDL rats at two developmental stages by 1H MRS in the hippocampus revealed similar changes that the one observed in adult brain during CHE. However almost all the neurometabolic changes were more profound or appeared earlier in the evolution of the disease in young BDL rats. A new surface 1H-31P double-tuned coil was built and several optimizations and development were done to improve the spectral resolution, SNR and localization, including implementation of 1H-31P NOE enhancement and 1H decoupling in static 31P MRS, optimization of localization method and also development of an optimal protocol for 31P magnetization transfer. 31P MRS study in p21 BDL rats was performed with an improved 31P MRS protocol and revealed a decreased creatine kinase rate constant 2 weeks after BDL surgery and significant changes in metabolites involved in high-phosphate metabolism, in phospholipid metabolism and also cellular redox state 6 weeks after BDL. In the last part of this thesis, some therapeutic approaches were tested. A probiotic treatment delayed some neurometabolic changes present in CHE in adult BDL rats and improved motor activity. Based on previously shown devastating effects of decreased brain creatine during brain development, we proposed the high creatine diet as a possible treatment for young BDL rats. We demonstrated that oral administration of creatine partially restored brain creatine levels and also had a positive effect on other neurometabolic changes present in CHE. In conclusion, the work done in this thesis revealed for the first time a large spectrum of neurometabolic alterations present in adult and young BDL rats suffering from CLD and CHE, together with the time evolution of these changes during the disease progression, using in vivo longitudinal 1H MRS, improved 31P MRS and 31P magnetization transfer protocols developed herein. In addition, we brought some insight on the positive effects of two innovative treatments on the progression of CHE in adult and young BDL rats.

EPFL2018

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

Lijing Xin

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.

EPFL2009

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.

EPFL2013