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Type C hepatic encephalopathy (HE) is a neuropsychiatric disease caused by chronic liver disease. Management of type C HE remains an important challenge because treatment options are limited. Both the antibiotic rifaximin and probiotics have been reported to reduce the symptoms of HE, but longitudinal studies assessing their effects on brain metabolism are lacking and the molecular mechanisms underpinning their effects are not fully understood. Therefore, we evaluated in detail the effects of these different treatments on the neurometabolic changes associated with type C HE using a multimodal approach including ultra-high field in vivo 1H MRS. We analyzed longitudinally the effect of rifaximin alone or in combination with the probiotic Vivomixx on the brain metabolic profile in the hippocampus and cerebellum of bile duct ligated (BDL) rats, an established model of type C HE. Overall, while rifaximin alone appeared to induce no significant effect on the neurometabolic profile of BDL rats, its association with the probiotic resulted in more attenuated neurometabolic alterations in BDL rats followed longitudinally (i.e. a smaller increase in Gln and milder decrease in Glu and Cr levels). Given that both rifaximin and some probiotics are used in the treatment of HE, the implications of these findings may be clinically relevant.

This working group of the International Society of Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN) was commissioned to summarize and update current efforts in the development and characterization of animal models of hepatic encephalopathy (HE). As defined in humans, HE in animal models is based on the underlying degree and severity of liver pathology. Although hyperammonemia remains the key focus in the pathogenesis of HE, other factors associated with HE have been identified, together with recommended animal models, to help explore the pathogenesis and pathophysiological mechanisms of HE. While numerous methods to induce liver failure and disease exist, less have been characterized with neurological and neurobehavioural impairments. Moreover, there still remains a paucity of adequate animal models of Type C HE induced by alcohol, viruses and non-alcoholic fatty liver disease; the most common etiologies of chronic liver disease.

Glioblastoma (GBM) is the most aggressive brain tumor type in adults. GBM is heterogeneous, with a compact core lesion surrounded by an invasive tumor front. This front is highly relevant for tumor recurrence but is generally non-detectable using standard imaging techniques. Recent studies demonstrated distinct metabolic profiles of the invasive phenotype in GBM. Magnetic resonance (MR) of hyperpolarized 13C-labeled probes is a rapidly advancing field that provides real-time metabolic information. Here, we applied hyperpolarized 13C-glucose MR to mouse GBM models. Compared to controls, the amount of lactate produced from hyperpolarized glucose was higher in the compact GBM model, consistent with the accepted “Warburg effect”. However, the opposite response was observed in models reflecting the invasive zone, with less lactate produced than in controls, implying a reduction in aerobic glycolysis. These striking differences could be used to map the metabolic heterogeneity in GBM and to visualize the infiltrative front of GBM.

Creatine (Cr) is a nitrogenous organic acid and plays roles such as fast phosphate energy buffer to replenish ATP, osmolyte, antioxidant, neuromodulator, and as a compound with anabolic and ergogenic properties in muscle. Cr is taken from the diet or endogenously synthetized by the enzymes arginine:glycine amidinotransferase and guanidinoacetate methyltransferase, and specifically taken up by the transporter SLC6A8. Loss-of-function mutations in the genes encoding for the enzymes or the transporter cause creatine deficiency syndromes (CDS). CDS are characterized by brain Cr deficiency, intellectual disability with severe speech delay, behavioral troubles, epilepsy, and motor dysfunction. Among CDS, the X-linked Cr transporter deficiency (CTD) is the most prevalent with no efficient treatment so far. Different animal models of CTD show reduced brain Cr levels, cognitive deficiencies, and together they cover other traits similar to those of patients. However, motor function was poorly explored in CTD models, and some controversies in the phenotype exist in comparison with CTD patients. Our recently described Slc6a8^(Y389C) knock-in rat model of CTD showed mild impaired motor function, morphological alterations in cerebellum, reduced muscular mass, Cr deficiency, and increased guanidinoacetate content in muscle, although no consistent signs of muscle atrophy. Our results indicate that such motor dysfunction co-occurred with both nervous and muscle dysfunctions, suggesting that muscle strength and performance as well as neuronal connectivity might be affected by this Cr deficiency in muscle and brain., Journal of Inherited Metabolic Disease, 45 (2), ISSN:1573-2665, ISSN:0141-8955

Type C hepatic encephalopathy (HE) is a complex neuropsychiatric disorder occurring as a consequence of chronic liver disease. Alterations in energy metabolism have been suggested in type C HE, but $\textit{in vivo}$ studies on this matter remain sparse and have reported conflicting results. Here, we propose a novel preclinical $^{18}$F-FDG PET methodology to compute quantitative 3D maps of the regional cerebral metabolic rate of glucose (CMR$_{glc}$) from a labelling steady-state PET image of the brain and an image-derived input function. This quantitative approach shows its strength when comparing groups of animals with divergent physiology, such as HE animals. PET CMR$_{glc}$ maps were registered to an atlas and the mean CMR$_{glc}$ from the hippocampus and the cerebellum were associated to the corresponding localized $^{1}$H MR spectroscopy acquisitions. This study provides for the first time local and quantitative information on both brain glucose uptake and neurometabolic profile alterations in a rat model of type C HE. A 2-fold lower brain glucose uptake, concomitant with an increase in brain glutamine and a decrease in the main osmolytes was observed in the hippocampus and in the cerebellum. These novel findings are an important step towards new insights into energy metabolism in the pathophysiology of HE., 30 pages, 6 figures

Type C hepatic encephalopathy (HE) is a complex neuropsychiatric disorder occurring as a consequence of chronic liver disease. Alterations in energy metabolism have been suggested in type C HE, but in vivo studies on this matter remain sparse and have reported conflicting results. Here, we propose a novel preclinical

The invasive behavior of glioblastoma, the most aggressive primary brain tumor, is considered highly relevant for tumor recurrence. However, the invasion zone is difficult to visualize by Magnetic Resonance Imaging (MRI) and is protected by the blood brain barrier, posing a particular challenge for treatment. We report biological features of invasive growth accompanying tumor progression and invasion based on associated metabolic and transcriptomic changes observed in patient derived orthotopic xenografts (PDOX) in the mouse and the corresponding patients’ tumors. The evolution of metabolic changes, followed in vivo longitudinally by 1H-Magnetic Resonance Spectroscopy (MRS) at ultra-high field, reflected growth and the invasive properties of the human glioblastoma transplanted into the brains of mice (PDOX). Comparison of MRS derived metabolite signatures, reflecting temporal changes of tumor development and invasion in PDOX, revealed high similarity to spatial metabolite signatures of combined multi-voxel MRS analyses sampled across different areas of the patients’ tumors. Pathway analyses of the transcriptome associated with the metabolite profiles of the PDOX, identified molecular signatures of invasion, comprising extracellular matrix degradation and reorganization, growth factor binding, and vascular remodeling. Specific analysis of expression signatures from the invaded mouse brain, revealed extent of invasion dependent induction of immune response, recapitulating respective signatures observed in glioblastoma. Integrating metabolic profiles and gene expression of highly invasive PDOX provided insights into progression and invasion associated mechanisms of extracellular matrix remodeling that is essential for cell-cell communication and regulation of cellular processes. Structural changes and biochemical properties of the extracellular matrix are of importance for the biological behavior of tumors and may be druggable. Ultra-high field MRS reveals to be suitable for in vivo monitoring of progression in the non-enhancing infiltration zone of glioblastoma.

Purpose Reliable detection and fitting of macromolecules (MM) are crucial for accurate quantification of brain short‐echo time (TE) 1H‐MR spectra. An experimentally acquired single MM spectrum is commonly used. Higher spectral resolution at ultra‐high field (UHF) led to increased interest in using a parametrized MM spectrum together with flexible spline baselines to address unpredicted spectroscopic components. Herein, we aimed to: (1) implement an advanced methodological approach for post‐processing, fitting, and parametrization of 9.4T rat brain MM spectra; (2) assess the concomitant impact of the LCModel baseline and MM model (ie, single vs parametrized); and (3) estimate the apparent T2 relaxation times for seven MM components. Methods A single inversion recovery sequence combined with advanced AMARES prior knowledge was used to eliminate the metabolite residuals, fit, and parametrize 10 MM components directly from 9.4T rat brain in vivo 1H‐MR spectra at different TEs. Monte Carlo simulations were also used to assess the concomitant influence of parametrized MM and DKNTMN parameter in LCModel. Results A very stiff baseline (DKNTMN ≥ 1 ppm) in combination with a single MM spectrum led to deviations in metabolite concentrations. For some metabolites the parametrized MM showed deviations from the ground truth for all DKNTMN values. Adding prior knowledge on parametrized MM improved MM and metabolite quantification. The apparent T2 ranged between 12 and 24 ms for seven MM peaks. Conclusion Moderate flexibility in the spline baseline was required for reliable quantification of real/experimental spectra based on in vivo and Monte Carlo data. Prior knowledge on parametrized MM improved MM and metabolite quantification.

Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdhki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdhki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdhki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdhki/ki rats showed imbalance of intra-and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdhki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new ther-apeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery., (c) 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Creatine is an organic compound used as fast phosphate energy buffer to recycle ATP, important in tissues with high energy demand such as muscle or brain. Creatine is taken from the diet or endogenously synthetized by the enzymes AGAT and GAMT, and specifically taken up by the transporter SLC6A8. Deficit in the endogenous synthesis or in the transport leads to Cerebral Creatine Deficiency Syndromes (CCDS). CCDS are characterized by brain creatine deficiency, intellectual disability with severe speech delay, behavioral troubles such as attention deficits and/or autistic features, and epilepsy. Among CCDS, the X-linked creatine transporter deficiency (CTD) is the most prevalent with no efficient treatment so far. Different mouse models of CTD were generated by doing long deletions in the Slc6a8 gene showing reduced brain creatine and cognitive deficiencies or impaired motor function. We present a new knock-in (KI) rat model of CTD holding an identical point mutation found in patients with reported lack of transporter activity. KI males showed brain creatine deficiency, increased urinary creatine/creatinine ratio, cognitive deficits and autistic-like traits. The Slc6a8 Y389C KI rat fairly enriches the spectrum of CTD models and provides new data about the pathology, being the first animal model of CTD carrying a point mutation.

Chronic liver disease leads to neuropsychiatric complications called hepatic encephalopathy (HE). Current treatments have some limitations in their efficacy and tolerability, emphasizing the need for alternative therapies. Modulation of gut bacterial flora using probiotics is emerging as a therapeutic alternative. However, knowledge about how probiotics influence brain metabolite changes during HE is missing. In the present study, we combined the advantages of ultra-high field in vivo 1H MRS with behavioural tests to analyse whether a long-term treatment with a multistrain probiotic mixture (VIVOMIXX) in a rat model of type C HE had a positive effect on behaviour and neurometabolic changes. We showed that the prophylactic administration of this probiotic formulation led to an increase in gut Bifidobacteria and attenuated changes in locomotor activity and neurometabolic profile in a rat model of type C HE. Both the performance in behavioural tests and the neurometabolic profile of BDL + probiotic rats were improved compared to the BDL group at week 8 post-BDL. They displayed a significantly lesser increase in brain Gln, a milder decrease in brain mIns and a smaller decrease in neurotransmitter Glu than untreated animals. The clinical implications of these findings are potentially far-reaching given that probiotics are generally safe and well-tolerated by patients.

Glioblastoma (GBM) is the most aggressive brain tumor type in adults. GBM is heterogeneous, with a compact core lesion surrounded by an invasive tumor front. This front is highly relevant for tumor recurrence but is generally non-detectable using standard imaging techniques. Recent studies demonstrated distinct metabolic profiles of the invasive phenotype in GBM. Magnetic resonance (MR) of hyperpolarized 13C-labeled probes is a rapidly advancing field that provides real-time metabolic information. Here, we applied hyperpolarized 13C-glucose MR to mouse GBM models. Compared to controls, the amount of lactate produced from hyperpolarized glucose was higher in the compact GBM model, consistent with the accepted “Warburg effect”. However, the opposite response was observed in models reflecting the invasive zone, with less lactate produced than in controls, implying a reduction in aerobic glycolysis. These striking differences could be used to map the metabolic heterogeneity in GBM and to visualize the infiltrative front of GBM.

Magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) allow the chemical analysis of physiological processes in vivo and provide powerful tools in the life sciences and for clinical diagnostics. Excellent homogeneity of the static B

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.

Creatine (Cr) is an important organic compound acting as intracellular high-energy phosphate shuttle and in energy storage. While located in most cells where it plays its main roles in energy metabolism and cytoprotection, Cr is highly concentrated in muscle and brain tissues, in which Cr also appears to act in osmoregulation and neurotransmission. This review discusses the basis of Cr metabolism, synthesis and transport within brain cells. The importance of Cr in brain function and the consequences of its impaired metabolism in primary and secondary Cr deficiencies are also discussed. Cr and phosphocreatine (PCr) in living systems can be well characterized using in vivo magnetic resonance spectroscopy (MRS). This review describes how (1)H MRS allows the measurement of Cr and PCr, and how (31)P MRS makes it possible to estimate the creatine kinase (CK) rate constant and so detect dynamic changes in the Cr/PCr/CK system. Absolute quantification by MRS using creatine as internal reference is also discussed. The use of in vivo MRS to study brain Cr in a non-invasive way is presented, as well as its use in clinical and preclinical studies, including diagnosis and treatment follow-up in patients.

In vivo Magnetic Resonance Spectroscopy is a useful tool to characterize brain biochemistry as well as its alteration in a large number of major central nervous system diseases. The present review will focus on the study of the glutamate-glutamine cycle, an important biochemical pathway in excitatory neurotransmission, analyzed using in vivo MRS of different accessible nuclei: 1H, 13C, 15N and 31P. The different methodological aspects of data acquisition, processing and absolute quantification of the MRS data for each nucleus will be presented, as well as the description of the mathematical modeling approach to interpret the MRS measurements in terms of biochemical kinetics. The unique advantages of MRS, especially its non-invasive nature enabling longitudinal monitoring of brain disease progression and/or effect of treatment is illustrated in the particular context of hyperammonemic disorders with a specific focus on animal models. We review the current possibilities given by in vivo MRS to investigate some of the molecular mechanisms involved in hyperammonemic disorders and to give a better understanding of the process of development of hepatic encephalopathy, a severe neuropsychiatric disorder that frequently accompanies liver disease.

Hepatic encephalopathy (HE) is a major complication of liver failure/disease which frequently develops during the progression of end-stage liver disease. This metabolic neuropsychiatric syndrome involves a spectrum of symptoms, including cognition impairment, attention deficits and motor dysfunction which eventually can progress to coma and death. Pathologically, HE is characterized by swelling of the astrocytes which consequently leads to brain edema, a common feature found in patients with acute liver failure (ALF) as well as in cirrhotic patients suffering from HE. The pathogenic factors involved in the onset of astrocyte swelling and brain edema in HE are unresolved. However, the role of astrocyte swelling/brain edema in the development of HE remains ambiguous and therefore measuring brain edema as an endpoint to evaluate HE is questioned. The following review will determine the effect of astrocyte swelling and brain edema on neurological function, discuss the various possible techniques to measure brain edema and lastly to propose a number of neurobehavioral tests to evaluate HE.

Hyperammonemia can be caused by various acquired or inherited disorders such as urea cycle defects. The brain is much more susceptible to the deleterious effects of ammonium in childhood than in adulthood. Hyperammonemia provokes irreversible damage to the developing central nervous system: cortical atrophy, ventricular enlargement and demyelination lead to cognitive impairment, seizures and cerebral palsy. The mechanisms leading to these severe brain lesions are still not well understood, but recent studies show that ammonium exposure alters several amino acid pathways and neurotransmitter systems, cerebral energy metabolism, nitric oxide synthesis, oxidative stress and signal transduction pathways. All in all, at the cellular level, these are associated with alterations in neuronal differentiation and patterns of cell death. Recent advances in imaging techniques are increasing our understanding of these processes through detailed in vivo longitudinal analysis of neurobiochemical changes associated with hyperammonemia. Further, several potential neuroprotective strategies have been put forward recently, including the use of NMDA receptor antagonists, nitric oxide inhibitors, creatine, acetyl-L-carnitine, CNTF or inhibitors of MAPKs and glutamine synthetase. Magnetic resonance imaging and spectroscopy will ultimately be a powerful tool to measure the effects of these neuroprotective approaches.

Glioblastoma are notorious for their highly invasive growth, diffusely infiltrating adjacent brain structures that precludes complete resection, and is a major obstacle for cure. To characterize this "invisible" tumor part, we designed a high resolution multimodal imaging approach assessing in vivo the metabolism of invasively growing glioma xenografts in the mouse brain. Animals were subjected longitudinally to magnetic resonance imaging (MRI) and

In vivo (13)C magnetic resonance spectroscopy (MRS) enables the investigation of cerebral metabolic compartmentation while, e.g. infusing (13)C-labeled glucose. Metabolic flux analysis of (13)C turnover previously yielded quantitative information of glutamate and glutamine metabolism in humans and rats, while the application to in vivo mouse brain remains exceedingly challenging. In the present study, (13)C direct detection at 14.1 T provided highly resolved in vivo spectra of the mouse brain while infusing [1,6-(13)C(2)]glucose for up to 5 h. (13)C incorporation to glutamate and glutamine C4, C3, and C2 and aspartate C3 were detected dynamically and fitted to a two-compartment model: flux estimation of neuron-glial metabolism included tricarboxylic acid cycle (TCA) flux in astrocytes (V(g) = 0.16 ± 0.03 µmol/g/min) and neurons (V(TCA)(n )= 0.56 ± 0.03 µmol/g/min), pyruvate carboxylase activity (V(PC) = 0.041 ± 0.003 µmol/g/min) and neurotransmission rate (V(NT) = 0.084 ± 0.008 µmol/g/min), resulting in a cerebral metabolic rate of glucose (CMR(glc)) of 0.38 ± 0.02 µmol/g/min, in excellent agreement with that determined with concomitant (18)F-fluorodeoxyglucose positron emission tomography ((18)FDG PET).We conclude that modeling of neuron-glial metabolism in vivo is accessible in the mouse brain from (13)C direct detection with an unprecedented spatial resolution under [1,6-(13)C(2)]glucose infusion.

Keywords: CIBM-AIT Reference EPFL-ARTICLE-229510doi:10.1016/j.ab.2017.05.014View record in Web of Science Record created on 2017-07-10, modified on 2017-07-18

Quantification of short-echo time proton magnetic resonance spectroscopy results in >18 metabolite concentrations (neurochemical profile). Their quantification accuracy depends on the assessment of the contribution of macromolecule (MM) resonances, previously experimentally achieved by exploiting the several fold difference in T(1). To minimize effects of heterogeneities in metabolites T(1), the aim of the study was to assess MM signal contributions by combining inversion recovery (IR) and diffusion-weighted proton spectroscopy at high-magnetic field (14.1 T) and short echo time (= 8 msec) in the rat brain. IR combined with diffusion weighting experiments (with delta/Delta = 1.5/200 msec and b-value = 11.8 msec/mum(2)) showed that the metabolite nulled spectrum (inversion time = 740 msec) was affected by residuals attributed to creatine, inositol, taurine, choline, N-acetylaspartate as well as glutamine and glutamate. While the metabolite residuals were significantly attenuated by 50%, the MM signals were almost not affected (< 8%). The combination of metabolite-nulled IR spectra with diffusion weighting allows a specific characterization of MM resonances with minimal metabolite signal contributions and is expected to lead to a more precise quantification of the neurochemical profile.

In (1)H magnetic resonance spectroscopy, macromolecule signals underlay metabolite signals, and knowing their contribution is necessary for reliable metabolite quantification. When macromolecule signals are measured using an inversion-recovery pulse sequence, special care needs to be taken to correctly remove residual metabolite signals to obtain a pure macromolecule spectrum. Furthermore, since a single spectrum is commonly used for quantification in multiple experiments, the impact of potential macromolecule signal variability, because of regional differences or pathologies, on metabolite quantification has to be assessed. In this study, we introduced a novel method to post-process measured macromolecule signals that offers a flexible and robust way of removing residual metabolite signals. This method was applied to investigate regional differences in the mouse brain macromolecule signals that may affect metabolite quantification when not taken into account. However, since no significant differences in metabolite quantification were detected, it was concluded that a single macromolecule spectrum can be generally used for the quantification of healthy mouse brain spectra. Alternatively, the study of a mouse model of human glioma showed several alterations of the macromolecule spectrum, including, but not limited to, increased mobile lipid signals, which had to be taken into account to avoid significant metabolite quantification errors.

Glutamine has multiple roles in brain metabolism and its concentration can be altered in various pathological conditions. An accurate knowledge of its concentration is therefore highly desirable to monitor and study several brain disorders in vivo. However, in recent years, several MRS studies have reported conflicting glutamine concentrations in the human brain. A recent hypothesis for explaining these discrepancies is that a short T2 component of the glutamine signal may impact on its quantification at long echo times. The present study therefore aimed to investigate the impact of acquisition parameters on the quantified glutamine concentration using two different acquisition techniques, SPECIAL at ultra-short echo time and MEGA-SPECIAL at moderate echo time. For this purpose, MEGA-SPECIAL was optimized for the first time for glutamine detection. Based on the very good agreement of the glutamine concentration obtained between the two measurements, it was concluded that no impact of a short T2 component of the glutamine signal was detected.

To determine the single spin-echo T 2 relaxation times of uncoupled and J-coupled metabolites in rat brain in vivo at 14.1 T and to compare these results with those previously obtained at 9.4 T. Measurements were performed on five rats at 14.1 T using the SPECIAL sequence and TE-specific basis-sets for LCModel analysis. The T 2 of singlets ranged from 98 to 148 ms and T 2 of J-coupled metabolites ranged from 72 ms (glutamate) to 97 ms (myo-inositol). When comparing the T 2s of the metabolites measured at 14.1 T with those previously measured at 9.4 T, a decreasing trend was found (p

Hepatic encephalopathy (HE) is a common and severe neuropsychiatric complication present in acute and chronic liver disease. The unique advantages of high field (1)H MRS provide a method for assessing pathogenic mechanism, diagnosis and monitoring of HE, as well as for treatment assessment or recovery after liver transplantation, in a reproducible and reliable non-invasive way. The purpose of the present review is to present some new features of in vivo proton Magnetic Resonance Spectroscopy ((1)H MRS) at high magnetic fields combined with some basic requirements for reliable metabolic profiling. Finally, in vivo applications of (1)H MRS in different HE animal models are presented.

The recent developments in high magnetic field 13C magnetic resonance spectroscopy with improved localization and shimming techniques have led to important gains in sensitivity and spectral resolution of 13C in vivo spectra in the rodent brain, enabling the separation of several 13C isotopomers of glutamate and glutamine. In this context, the assumptions used in spectral quantification might have a significant impact on the determination of the 13C concentrations and the related metabolic fluxes. In this study, the time domain spectral quantification algorithm AMARES (advanced method for accurate, robust and efficient spectral fitting) was applied to 13 C magnetic resonance spectroscopy spectra acquired in the rat brain at 9.4 T, following infusion of [1,6-(13)C2 ] glucose. Using both Monte Carlo simulations and in vivo data, the goal of this work was: (1) to validate the quantification of in vivo 13C isotopomers using AMARES; (2) to assess the impact of the prior knowledge on the quantification of in vivo 13C isotopomers using AMARES; (3) to compare AMARES and LCModel (linear combination of model spectra) for the quantification of in vivo 13C spectra. AMARES led to accurate and reliable 13C spectral quantification similar to those obtained using LCModel, when the frequency shifts, J-coupling constants and phase patterns of the different 13C isotopomers were included as prior knowledge in the analysis.

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. 15N MRS is an alternative approach to 13C MRS. Incorporation of labeled 15N from ammonia in cerebral glutamine allows to measure several metabolic reactions related to nitrogen metabolism, including the glutamate—glutamine cycle. To measure 15N incorporation into the position 5N of glutamine and position 2N of glutamate and glutamine, we developed a novel 15N pulse sequence to simultaneously detect, for the first time, [5-15N]Gln and [2-15N]Gln + Glu in vivo in the rat brain. In addition, we also measured for the first time in the same experiment localized 1H spectra for a direct measurement of the net glutamine accumulation. Mathematical modeling of 1H and 15N MRS data allowed to reduce the number of assumptions and provided reliable determination of GS (0.30 ± 0.050 μmol/g per minute), apparent neurotransmission (0.26 ± 0.030 μmol/g per minute), glutamate dehydrogenase (0.029 ± 0.002 μmol/g per minute), and net glutamine accumulation (0.033 ± 0.001 μmol/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.

The development of new diagnostic criteria for Alzheimer's disease (AD) requires new in vivo markers reflecting early pathological changes in the brain of patients. Magnetic resonance (MR) spectroscopy has been shown to provide useful information about the biochemical changes occurring in AD brain in vivo. The development of numerous transgenic mouse models of AD has facilitated the evaluation of early biomarkers, allowing researchers to perform longitudinal studies starting before the onset of the pathology. In addition, the recent development of high-field animal scanners enables the measurement of brain metabolites that cannot be reliably quantified at lower magnetic fields. In this report, we studied a new transgenic mouse model of AD, the 5xFAD model, by in vivo proton and phosphorus MR spectroscopy. This model, which is characterized by an early-onset and a robust amyloid pathology, developed changes in the neurochemical profile, which are typical in the human disease, i.e., an increase in myo-inositol and a decrease in N-acetylaspartate concentrations, as early as in the 40th week of age. In addition, a significant decrease in the γ-aminobutyrate concentration was observed in transgenic mice at this age compared to controls. The pseudo-first-order rate constant of the creatine kinase reaction as well as relative concentrations of phosphorus-containing metabolites were not changed significantly in the 36 and 72-week old transgenic mice. Overall, these results suggest that mitochondrial activity in the 5 × FAD mice is not substantially affected but that the model is relevant for studying early biomarkers of AD.

Manganese (Mn2+)-enhanced magnetic resonance imaging studies of the neuronal pathways of the hypothalamus showed that information about the regulation of food intake and energy balance circulate through specific hypothalamic nuclei. The dehydration-induced anorexia (DIA) model demonstrated to be appropriate for studying the hypothalamus with Mn2+-enhanced magnetic resonance imaging. Manganese is involved in the normal functioning of a variety of physiological processes and is associated with enzymes contributing to neurotransmitter synthesis and metabolism. It also induces psychiatric and motor disturbances. The molecular mechanisms by which Mn2+ produces alterations of the hypothalamic physiological processes are not well understood. 1H-magnetic resonance spectroscopy measurements of the rodent hypothalamus are challenging due to the distant location of the hypothalamus resulting in limited measurement sensitivity. The present study proposed to investigate the effects of Mn2+ on the neurochemical profile of the hypothalamus in normal, DIA, and overnight fasted female rats at 14.1 T. Results provide evidence that γ-aminobutyric acid has an essential role in the maintenance of energy homeostasis in the hypothalamus but is not condition specific. On the contrary, glutamine, glutamate, and taurine appear to respond more accurately to Mn2+ exposure. An increase in glutamine levels could also be a characteristic response of the hypothalamus to DIA.

The increase of total choline in tumors has become an important biomarker in cancer diagnosis. Choline and choline metabolites can be measured in vivo and in vitro using multinuclear MRS. Recent in vivo(13)C MRS studies using labeled substrates enhanced via dynamic nuclear polarization demonstrated the tremendous potential of hyperpolarization for real-time metabolic studies. The present study demonstrates the feasibility of detecting hyperpolarized (15)N labeled choline in vivo in a rat head at 9.4 T. We furthermore report the in vitro (172 +/- 16 s) and in vivo (126 +/- 15 s) longitudinal relaxation times. We conclude that with appropriate infusion protocols it is feasible to detect hyperpolarized (15)N labeled choline in live animals.

Tumor-initiating cells with stem cell properties are believed to sustain the growth of gliomas, but proposed markers such as CD133 cannot be used to identify these cells with sufficient specificity. We report an alternative isolation method purely based on phenotypic qualities of glioma-initiating cells (GICs), avoiding the use of molecular markers. We exploited intrinsic autofluorescence properties and a distinctive morphology to isolate a subpopulation of cells (FL1(+)) from human glioma or glioma cultures. FL1(+) cells are capable of self-renewal in vitro, tumorigenesis in vivo and preferentially express stem cell genes. The FL1(+) phenotype did not correlate with the expression of proposed GIC markers. Our data propose an alternative approach to investigate tumor-initiating potential in gliomas and to advance the development of new therapies and diagnostics.

Knowledge of T-1 relaxation times can be important for accurate relative and absolute quantification of brain metabolites, for sensitivity optimizations, for characterizing molecular dynamics, and for studying changes induced by various pathological conditions. H-1 T-1 relaxation times of a series of brain metabolites, including J-coupled ones, were determined using a progressive saturation (PS) technique that was validated with an adiabatic inversion-recovery (IR) method. The H-1 T-1 relaxation times of 16 functional groups of the neurochemical profile were measured at 14.1T and 9.4T. Overall, the T-1 relaxation times found at 14.1T were, within the experimental error, identical to those at 9.4T. The T(1)s of some coupled spin resonances of the neurochemical profile were measured for the first time (e.g., those of gamma-aminobutyrate [GABA], aspartate [Asp], alanine [Ala], phosphoethanolamine [PE], glutathione [GSH], N-acety-laspartylglutamate [NAAG], and glutamine [Gin]). Our results suggest that T-1 does not increase substantially beyond 9.4T. Furthermore, the similarity of T, among the metabolites (similar to 1.5 s) suggests that T-1 relaxation time corrections for metabolite quantification are likely to be similar when using rapid pulsing conditions. We therefore conclude that the putative T-1 increase of metabolites has a minimal impact on sensitivity when increasing B-0 beyond 9.4T. Magn Reson Med 62:862-867, 2009. (C) 2009 Wiley-Liss, Inc.

Ultra-short echo-time proton single voxel spectra of rat brain were obtained on a 14.1T 26 cm horizontal bore system. At this field, the fitted linewidth in the brain tissue of adult rats was about 11 Hz. New, separated resonances ascribed to phosphocholine, glycerophosphocholine and N-acetylaspartate were detected for the first time in vivo in the spectral range of 4.2-4.4 ppm. Moreover, improved separation of the resonances of lactate, alanine, gamma-aminobutyrate, glutamate and glutathione was observed. Metabolite concentrations were estimated by fitting in vivo spectra to a linear combination of simulated spectra of individual metabolites and a measured spectrum of macromolecules (LCModel). The calculated concentrations of metabolites were generally in excellent agreement with those obtained at 9.4T. These initial results further indicated that increasing magnetic field strength to 14.1T enhanced spectral resolution in (1)H NMR spectroscopy. This implies that the quantification of the neurochemical profile in rodent brain can be achieved with improved accuracy and precision.

By quantification of brain metabolites, localized brain proton MRS can non-invasively provide biochemical information from distinct regions of the brain. Quantification of short-TE signals is usually based on a metabolite basis set. The basis set can be obtained by two approaches: (1) by measuring the signals of metabolites in aqueous solution; (2) by quantum-mechanically simulating the theoretical metabolite signals. The purpose of this study was to compare the effect of these two approaches on metabolite concentration estimates. Metabolite concentrations were quantified with the QUEST method, using both approaches. A comparison was performed with the aid of Monte Carlo studies, by using signals simulated from both basis sets. The best results were obtained when the basis set used for the fit was the same as that used to simulate the Monte Carlo signals. This comparison was also performed using in vivo short-TE signals acquired at 7 T from the central region of rat brains. The concentration estimates, with confidence intervals, obtained using both basis sets were in good agreement with values from the literature. The in vivo study showed that, in general, the differences between the estimates obtained with the two basis sets were not statistically significant or scientifically important. Consequently, a simulated basis set can be used in place of a measured basis set.

Localized proton Magnetic Resonance Spectroscopy brain signals acquired at short echo-time contain contributions from metabolites, water and a ;background' which mainly originates from macromolecules and lipids. The purpose of the present study was to compare the influence of the background-accommodation strategy on the metabolite concentration estimates. Two strategies were investigated to accommodate the background, 1) the measured background signal was incorporated in the metabolite basis-set; and 2) the background signal was estimated and subtracted from the in vivo signal using Subtract-QUEST. The influence of the background-accommodation strategy was addressed with the aid of Monte Carlo and in vivo studies. For the considered signals of this study, the concentration estimates obtained using the first approach were below those obtained with Subtract-QUEST. Indeed, the presence of residual contribution of metabolite signals with short longitudinal relaxation times (T1) in the measured background led to an underestimation of metabolite concentration estimates. Conversely, the observed underestimation of the background contribution using Subtract-QUEST led to an overestimation of the metabolite estimates.

C57BL/6 mice are the most widely used strain of laboratory mice. Using in vivo proton Magnetic Resonance Spectroscopy (1H MRS), we have repeatedly observed an abnormal neurochemical profile in the brains of both wild-type and genetically modified mice derived from the C57BL/6J strain, consisting of a several fold increase in cerebral glutamine and two fold decrease in myo-inositol. This strikingly abnormal neurochemical “phenotype” resembles that observed in chronic liver disease or portosystemic shunting and appeared to be independent of transgene, origin or chow and was not associated with liver failure. As many as 25% of animals displayed the abnormal neurochemical profile, questioning the reliability of this model for neurobiology. We conducted an independent study to determine if this neurochemical profile was associated with portosystemic shunting. Our results showed that 100% of the mice with high brain glutamine displayed portosystemic shunting by concomitant portal angiography while all mice with normal brain glutamine did not. Since portosystemic shunting is known to cause alterations in gene expression in many organs including the brain, we conclude that portosystemic shunting may be the most significant problem associated with C57BL/6J inbreeding both for its effect on the central nervous system and for its systemic repercussions.

Type C hepatic encephalopathy (type C HE) is increasingly suspected in children with chronic liver disease (CLD), and believed to underlie long-term neurocognitive difficulties. The molecular underpinnings of type C HE in both adults and children are incompletely understood. In the present study we combined the experimental advantages of in vivo high field 1H magnetic resonance spectroscopy with immunohistochemistry to follow longitudinally over 8 weeks the neurometabolic changes in the hippocampus of animals having undergone bile duct ligation as pups. Rats who develop CLD early in life displayed pronounced neurometabolic changes in the hippocampus characterized by a progressive increase in glutamine concentration which correlated with plasma ammonia levels and a rapid decrease in brain myo-inositol. Other neurometabolic findings included a decrease in other organic osmolytes (taurine, choline-containing compounds and creatine), ascorbate and glutamate. At the cellular level, we observed an increase in glial fibrillary acidic protein (GFAP) and aquaporin 4 (AQP4) expression in the hippocampus at 4 weeks post bile duct ligation (BDL), together with astrocytic morphological alterations. These findings differ from observations in the brain of adult rats following BDL, and are in keeping with the commonly accepted theory of age-dependent vulnerability.

Tumor-initiating cells with stem cell properties are believed to sustain the growth of gliomas, but proposed markers such as CD133 cannot be used to identify these cells with sufficient specificity. We report an alternative isolation method purely based on phenotypic qualities of glioma-initiating cells (GICs), avoiding the use of molecular markers. We exploited intrinsic autofluorescence properties and a distinctive morphology to isolate a subpopulation of cells (FL1(+)) from human glioma or glioma cultures. FL1(+) cells are capable of self-renewal in vitro, tumorigenesis in vivo and preferentially express stem cell genes. The FL1(+) phenotype did not correlate with the expression of proposed GIC markers. Our data propose an alternative approach to investigate tumor-initiating potential in gliomas and to advance the development of new therapies and diagnostics.

Background & Aims: The sequence of events in hepatic encephalopathy (HE) remains unclear. Using the advantages of in vivo 1H-MRS (9.4T) we aimed to analyse the time-course of disease in an established model of type C HE by analysing the longitudinal changes in a large number of brain metabolites together with biochemical, histological and behavioural assessment. We hypothesized that neurometabolic changes are detectable very early, and that these early changes will offer insight into the primary events underpinning HE. Methods: Wistar rats underwent bile-duct ligation (BDL) and were studied before BDL and at post-operative weeks 2, 4, 6 and 8 (n = 26). In vivo short echo-time H-1-MRS (9.4T) of the hippocampus was performed in a longitudinal manner, as were biochemical (plasma), histological and behavioural tests. Results: Plasma ammonium increased early after BDL and remained high during the study. Brain glutamine increased (+47%) as early as 2-4 weeks post-BDL while creatine (-8%) and ascorbate (-12%) decreased. Brain glutamine and ascorbate correlated closely with rising plasma ammonium, while brain creatine correlated with brain glutamine. The increases in brain glutamine and plasma ammonium were correlated, while plasma ammonium correlated negatively with distance moved. Changes in astrocyte morphology were observed at 4 weeks. These early changes were further accentuated at 6-8 weeks post-BDL, concurrently with the known decreases in brain organic osmolytes. Conclusion: Using a multimodal, in vivo and longitudinal approach we have shown that neurometabolic changes are already noticeable 2 weeks after BDL. These early changes are suggestive of osmotic/oxidative stress and are likely the premise of some later changes. Early decreases in cerebral creatine and ascorbate are novel findings offering new avenues to explore neuroprotective strategies for HE treatment. Lay summary: The sequence of events in chronic hepatic encephalopathy (HE) remains unclear, therefore using the advantages of in vivo proton magnetic resonance spectroscopy at 9.4T we aimed to test the hypothesis that neurometabolic changes are detectable very early in an established model of type C HE, offering insight into the primary events underpinning HE, before advanced liver disease confounds the findings. These early, previously unreported neurometabolic changes occurred as early as 2 to 4 weeks after bile-duct ligation, namely an increase in plasma ammonium and brain glutamine, a decrease in brain creatine and ascorbate together with behavioural and astrocyte morphology changes, and continued to progress throughout the 8-week course of the disease. (C) 2019 European Association for the Study of the Liver. Published by Elsevier B.V.

In vivo (1)H MR spectroscopy allows the non invasive characterization of brain metabolites and it has been used for studying brain metabolic changes in a wide range of neurodegenerative diseases. The prion diseases form a group of fatal neurodegenerative diseases, also described as transmissible spongiform encephalopathies. The mechanism by which prions elicit brain damage remains unclear and therefore different transgenic mouse models of prion disease were created. We performed an in vivo longitudinal (1)H MR spectroscopy study at 14.1 T with the aim to measure the neurochemical profile of Prnp -/- and PrPΔ32-121 mice in the hippocampus and cerebellum. Using high-field MR spectroscopy we were able to analyze in details the in vivo brain metabolites in Prnp -/- and PrPΔ32-121 mice. An increase of myo-inositol, glutamate and lactate concentrations with a decrease of N-acetylaspartate concentrations were observed providing additional information to the previous measurements.

In the last decade, evidence has emerged indicating that the growth of a vast majority of tumors including gliomas is sustained by a subpopulation of cancer cells with stem cell properties called cancer initiating cells. These cells are able to initiate and propagate tumors and constitute only a fraction of all tumor cells. In the present study, we showed that intracerebral injection of cultured glioma-initiating cells into nude mice produced fast growing tumors showing necrosis and gadolinium enhancement in MR images, whereas gliomas produced by injecting freshly purified glioma-initiating cells grew slowly and showed no necrosis and very little gadolinium enhancement. Using proton localized spectroscopy at 14.1 Tesla, decreasing trends of N-acetylaspartate, glutamate and glucose concentrations and an increasing trend of glycine concentration were observed near the injection site after injecting cultured glioma-initiating cells. In contrast to the spectra of tumors grown from fresh cells, those from cultured cells showed intense peaks of lipids, increased absolute concentrations of glycine and choline-containing compounds, and decreased concentrations of glutamine, taurine and total creatine, when compared with a contralateral non-tumor-bearing brain tissue. A decrease in concentrations of N-acetylaspartate and -aminobutyrate was found in both tumor phenotypes after solid tumor formation. Further investigation is needed to determine the cause of the dissimilarities between the tumors grown from cultured glioma-initiating cells and those from freshly purified glioma-initiating cells, both derived from human glioblastomas. Copyright (C) 2011 John Wiley & Sons, Ltd.