Measuring Glycolytic Activity with Hyperpolarized [2H7, U-13C6] D-Glucose in the Naive Mouse Brain under Different Anesthetic Conditions

Glucose is the primary fuel for the brain; its metabolism is linked with cerebral function. Different magnetic resonance spectroscopy (MRS) techniques are available to assess glucose metabolism, providing complementary information. Our first aim was to investigate the difference between hyperpolarized 13C-glucose MRS and non-hyperpolarized 2H-glucose MRS to interrogate cerebral glycolysis. Isoflurane anesthesia is commonly employed in preclinical MRS, but it affects cerebral hemodynamics and functional connectivity. A combination of low doses of isoflurane and medetomidine is routinely used in rodent fMRI and shows similar functional connectivity, as in awake animals. As glucose metabolism is tightly linked to neuronal activity, our second aim was to assess the impact of these two anesthetic conditions on the cerebral metabolism of glucose. Brain metabolism of hyperpolarized 13C-glucose and 2H-glucose was monitored in two groups of mice in a 9.4 T MRI system. We found that the very different duration and temporal resolution of the two techniques enable highlighting the different aspects in glucose metabolism. We demonstrate (by numerical simulations) that hyperpolarized 13C-glucose reports on de novo lactate synthesis and is sensitive to CMRGlc. We show that variations in cerebral glucose metabolism, under different anesthesia, are reflected differently in hyperpolarized and non-hyperpolarized X-nuclei glucose MRS.

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

In vivo cerebral metabolism of glioblastoma xenografts assessed with ¹H MRS, ¹³C MRS and ¹⁸F-FDG PET

Marta Lai

Magnetic resonance spectroscopy (MRS) is a powerful tool when studying metabolism in intact and living organs preserving cells in their natural microenvironment with minimal external interference. The unique insights offered by the observation of metabolic processes in vivo are available thanks to the non-invasiveness of the MR technique, due to the employment of non-ionizing radiations at room temperature. These conditions are ideal when studying cerebral metabolism in vivo, since the brain is a particularly complex organ where neurons and glial cells are highly interconnected and dependent from each other. Moreover, brain cells are protected by the blood-brain barrier, which regulates the delivery of nutrients and chemicals. The work of this thesis focused on the application of 1H and 13C MRS in vivo to study cerebral metabolism in mice. 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) was used in complement to the abovementioned MR techniques to determine the cerebral metabolic rate of glucose (CMRglc) or local glucose uptake under specific physiological and pathological conditions. At first, we validated the application of 13C MRS to small volumes in the mouse brain in combination to compartmental modeling for neuro-glial metabolism, as previously applied to humans and rats in larger volumes. The second part of the thesis focused on the translation of these techniques to the study of brain metabolism during glioblastoma invasion. A highly infiltrative mouse xenograft model of glioblastoma derived from human glioma spheres was employed in our study to determine the evolution over time of the neurochemical profile detected with 1H MRS at 14.1 Tesla. 13C-labeling experiments with [1,6-13C]glucose were performed at late stage after extensive glioma invasion in the whole brain. Metabolic fluxes were determined under these conditions reflecting energetic metabolism in neurons and glioma/glial cells. Globally, infiltrating cells did not show a highly glycolytic metabolism, as often observed in cancer cells and in glioma cells close to the tumor core, suggesting that environmental stimuli induced by a central necrosis, high cellular density and poor vascularization may be crucial to the occurrence of a metabolic switch. Finally, a novel patient-derived mouse xenograft model of glioblastoma was tested and characterized longitudinally with 1H MRS at 14.1 Tesla. Glioma cells were implanted intracranially after patient tumor resection and minimal manipulation in vitro in order to minimize a selective pressure and promote a close resemblance of the xenograft with the parental tumor. This study showed that patient-derived xenografts obtained from freshly injected glioma cells offer a reliable model to study glioma invasion and related treatments while raising intriguing questions on the mechanisms of adaptation of human cells to the brain of the host.

EPFL2017

Development and applications of hyperpolarized 13C and 1H MR spectroscopy of cerebral metabolism at ultra-high field

Emmanuelle Ines Flatt

This thesis is composed of four studies centered on investigating cerebral metabolism using magnetic resonance spectroscopy (MRS) of hyperpolarized and non-hyperpolarized compounds at ultra-high field. In the first two chapters, we studied longitudinally the effects of different treatments for hepatic encephalopathy (HE) on the neurometabolic changes associated with chronic HE using a rat model of type C HE and a multimodal approach including 1H MRS. The effects of the antibiotic rifaximin administered at different doses as well as its combination with probiotics were studied in the established model of bile duct ligated (BDL) rats. We showed for the first time the beneficial effects of the combined rifaximin and the probiotic Vivomixx® on the neurometabolic changes in vivo and longitudinally in a rat model of type C HE. The longitudinal changes of some brain metabolites concentration (glutamine, glutamate, creatine), as well as gut bifidobacteria concentration, were significantly less pronounced in the group of rats treated with rifaximin and probiotics compared to non-treated rats. We also showed that rifaximin treatment alone had limited efficacy. When administered at human dose in rats, its effects appeared only at the early stages of the disease, whereas at a higher dose some neurometabolic changes associated with HE were attenuated but the general condition of the rats was worse. In the third study, we used dissolution dynamic nuclear polarization (DNP) to hyperpolarize [2H7, U-13C6]-D-glucose, which enabled us to monitor real-time glycolysis in the healthy mouse brain. Given that glucose metabolism is tightly linked to neuronal activity, we first investigated how the change in anesthesia impacts the cerebral metabolism of hyperpolarized glucose. Anesthetics are known to influence brain activity, and in this study we showed for the first time that switching from isoflurane anesthesia to a combination of lower doses of isoflurane and medetomidine had a high impact on cerebral glucose uptake and glycolytic flow, as reflected by the increased labelling of downstream [1-13C] lactate. The second part of this study aimed at evaluating the feasibility of quantifying cerebral glucose metabolism kinetics by evaluating two different mathematical models, where kinetic rate constants were determined by fitting the models on 13C curves by non-linear regression using the Levenberg-Marquardt algorithm. We showed that a 3-compartment model is more stable and reliable than a 4-compartment model. Finally, since lithium salts are widely used for treating bipolar disorder but its mechanism of action is still not clearly understood, we evaluated the potential of hyperpolarized 6Li injected at pharmacological doses to assess its real-time bio-distribution and pharmacokinetic in the rat brain. We demonstrated first that hyperpolarized 6Li can be detected at pharmacological concentration in the rat head with high signal-to-noise ratio (SNR). We also showed that the transport of lithium through the intact blood-brain barrier brain is a limiting factor and demonstrated a striking difference between apparent 6Li T1 values in a brain with normal or disrupted blood-brain barrier.

EPFL2021