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Carbon-13 NMR spectroscopy ((13)C MRS) offers the unique capability to measure brain metabolic rates in vivo. Hyperpolarized (13)C reduces the time required to assess brain metabolism from hours to minutes compared to conventional (13)C MRS. This study investigates metabolism of hyperpolarized [1-(13)C]pyruvate and [2-(13)C]pyruvate in the rat brain in vivo under various anesthetics: pentobarbital, isoflurane, α-chloralose, and morphine. The apparent metabolic rate from pyruvate to lactate modeled using time courses obtained after injection of hyperpolarized [1-(13)C]pyruvate was significantly greater for isoflurane than for all other anesthetic conditions, and significantly greater for morphine than for α-chloralose. The apparent metabolic rate from pyruvate to bicarbonate was significantly greater for morphine than for all other anesthetic conditions, and significantly lower for pentobarbital than for α-chloralose. Results show that relative TCA rates determined from hyperpolarized (13)C data are consistent with TCA cycle rates previously measured using conventional (13)C MRS under similar anesthetic conditions, and that using morphine for sedation greatly improves detection of downstream metabolic products compared to other anesthetics.

Two variants of a widely used two-compartment model were prepared for fitting the time course of [1,6-13C2]glucose metabolism in rat brain. Features common to most models were included, but in one model the enrichment of the substrates entering the glia and neuronal citric acid cycles was allowed to differ. Furthermore, the models included the capacity to analyze multiplets arising from 13C spin-spin coupling, known to improve parameter estimates in heart. Data analyzed were from a literature report providing time courses of [1,6-13C2]glucose metabolism. Four analyses were used, two comparing the effect of different pyruvate enrichment in glia and neurons, and two for determining the effect of multiplets present in the data. When fit independently, the enrichment in glial pyruvate was less than in neurons. In the absence of multiplets, fit quality and parameter values were typical of those in the literature, whereas the multiplet curves were not modeled well. This prompted the use of robust statistical analysis (the Kolmogorov-Smirnov test of goodness of fit) to determine whether individual curves were modeled appropriately. At least 50% of the curves in each experiment were considered poorly fit. It was concluded that the model does not include all metabolic features required to analyze the data.

To improve the ability to move from preclinical trials in mouse models of Huntington's disease (HD) to clinical trials in humans, biomarkers are needed that can track similar aspects of disease progression across species. Brain metabolites, detectable by magnetic resonance spectroscopy (MRS), have been suggested as potential biomarkers in HD. In this study, the R6/2 transgenic mouse model of HD was used to investigate the relative sensitivity of the metabolite profiling and the brain volumetry to anticipate the disease progression. Magnetic resonance imaging (MRI) and 1H MRS data were acquired at 9.4 T from the R6/2 mice and wild-type littermates at 4, 8, 12, and 15 weeks. Brain shrinkage was detectable in striatum, cortex, thalamus, and hypothalamus by 12 weeks. Metabolite changes in cortex paralleled and sometimes preceded those in striatum. The entire set of metabolite changes was compressed into principal components (PCs) using Partial Least Squares-Discriminant Analysis (PLS-DA) to increase the sensitivity for monitoring disease progression. In comparing the efficacy of volume and metabolite measurements, the cortical PC1 emerged as the most sensitive single biomarker, distinguishing R6/2 mice from littermates at all time points. Thus, neurochemical changes precede volume shrinkage and become potential biomarkers for HD mouse models.

Evaluating early changes in cerebral metabolism in hydrocephalus can help in the decision making and the timing of surgical intervention. This study was aimed at examining the tricarboxylic acid (TCA) cycle rate and (13)C label incorporation into neurotransmitter amino acids and other compounds 2 weeks after rats were subjected to kaolin-induced progressive hydrocephalus. In vivo and ex vivo magnetic resonance spectroscopy (MRS), combined with the infusion of [1,6-(13)C]glucose, was used to monitor the time courses of (13)C label incorporation into the different carbon positions of glutamate in the forebrains of rats with hydrocephalus as well as in those of controls. Metabolic rates were determined by fitting the measured data into a one-compartment metabolic model. The TCA cycle rate was 1.3 ± 0.2 μmoles/gram/minute in the controls and 0.8 ± 0.4 μmoles/gram/minute in the acute hydrocephalus group, the exchange rate between α-ketoglutarate and glutamate was 4.1 ± 2.5 μmoles/gram/minute in the controls and 2.7 ± 2.6 μmoles/gram/minute in the hydrocephalus group calculated from in vivo MRS. There were no statistically significant differences between these rates. Hydrocephalus caused a decrease in the amounts of glutamate, alanine and taurine. In addition, the concentration of the neuronal marker N-acetyl aspartate was decreased. (13)C Labelling of most amino acids derived from [1,6-(13)C]glucose was unchanged 2 weeks after hydrocephalus induction. The only indication of astrocyte impairment was the decreased (13)C enrichment in glutamine C-2. This study shows that hydrocephalus causes subtle but significant alterations in neuronal metabolism already early in the course of the disease. These sub-lethal changes, however, if maintained and if ongoing might explain the delayed and programmed neuronal damage as seen in chronic hydrocephalus.

Recent studies with a conditional mouse model of spinocerebellar ataxia type 1 (SCA1) suggest that neuronal dysfunction is reversible and neurodegeneration preventable with early interventions. Success of such interventions will depend on early detection of neuronal and glial abnormalities before cell loss and availability of objective methods to monitor progressive neurodegeneration. Cerebellar concentrations ofN-acetylaspartate (NAA),myo-inositol, and glutamate as measured by magnetic resonance spectroscopy (MRS) correlate with ataxia scores of patients with SCA1, indicating their potential as reliable biomarkers of neurodegeneration. Here we investigated whether neurochemical levels are altered by early, presymptomatic disease and whether they gauge disease progression in a mouse model of SCA1. Cerebellar neurochemical profiles of transgenic mice that overexpress the mutant human ataxin-1 (theSCA1[82Q] line) were measured longitudinally up to 1 year by MRS at 9.4 T and compared to those of transgenic mice that overexpress the normal human ataxin-1 (theSCA1[30Q] line) and wild-type controls. Multiple neurochemicals distinguished theSCA1[82Q] mice from controls with no overlap at all ages. Six neurochemicals were significantly different inSCA1[82Q] mice at 6 weeks, before major pathological and neurological changes. Alterations in NAA,myo-inositol, and glutamate progressively worsened and were significantly correlated (p< 0.0001) with disease progression as assessed by histology (molecular layer thickness and an overall severity score). Therefore, the neurochemicals that correlate with clinical status in patients reflected progressive pathology in the mouse model. These data demonstrate that presymptomatic and progressive neurodegeneration in SCA1 can be noninvasively monitored using MRS.

Acetate, a glial-specific substrate, is an attractive alternative to glucose for the study of neuronal-glial interactions. The present study investigates the kinetics of acetate uptake and utilization in the rat brain in vivo during infusion of [2-13C]acetate using NMR spectroscopy. When plasma acetate concentration was increased, the rate of brain acetate utilization (CMR(ace)) increased progressively and reached close to saturation for plasma acetate concentration2-3 mM, whereas brain acetate concentration continued to increase. The Michaelis-Menten constant for brain acetate utilization (K(M)(util) = 0.01 +/- 0.14 mM) was much smaller than for acetate transport through the blood-brain barrier (BBB) (K(M)(t) = 4.18 +/- 0.83 mM). The maximum transport capacity of acetate through the BBB (V(max)(t) = 0.96 +/- 0.18 micromol/g/min) was nearly twofold higher than the maximum rate of brain acetate utilization (V(max)(util) = 0.50 +/- 0.08 micromol/g/min). We conclude that, under our experimental conditions, brain acetate utilization is saturated when plasma acetate concentrations increase above 2-3 mM. At such high plasma acetate concentration, the rate-limiting step for glial acetate metabolism is not the BBB, but occurs after entry of acetate into the brain.

In this work the feasibility of measuring neuronal-glial metabolism in rat brain in vivo using co-infusion of [1,6-(13)C(2)]glucose and [1,2-(13)C(2)]acetate was investigated. Time courses of (13)C spectra were measured in vivo while infusing both (13)C-labeled substrates simultaneously. Individual (13)C isotopomers (singlets and multiplets observed in (13)C spectra) were quantified automatically using LCModel. The distinct (13)C spectral pattern observed in glutamate and glutamine directly reflected the fact that glucose was metabolized primarily in the neuronal compartment and acetate in the glial compartment. Time courses of concentration of singly and multiply-labeled isotopomers of glutamate and glutamine were obtained with a temporal resolution of 11 min. Although dynamic metabolic modeling of these (13)C isotopomer data will require further work and is not reported here, we expect that these new data will allow more precise determination of metabolic rates as is currently possible when using either glucose or acetate as the sole (13)C-labeled substrate.

Hibernating mammals use reduced metabolism, hypothermia, and stored fat to survive up to 5 or 6 mo without feeding. We found serum levels of the fat-derived ketone, d-β-hydroxybutyrate (BHB), are highest during deep torpor and exist in a reciprocal relationship with glucose throughout the hibernation season in the thirteen-lined ground squirrel ( Spermophilus tridecemlineatus). Ketone transporter monocarboxylic acid transporter 1 (MCT1) is upregulated at the blood-brain barrier, as animals enter hibernation. Uptake and metabolism of 13C-labeled BHB and glucose were measured by high-resolution NMR in both brain and heart at several different body temperatures ranging from 7 to 38°C. We show that BHB and glucose enter the heart and brain under conditions of depressed body temperature and heart rate but that their utilization as a fuel is highly selective. During arousal from torpor, glucose enters the brain over a wide range of body temperatures, but metabolism is minimal, as only low levels of labeled metabolites are detected. This is in contrast to BHB, which not only enters the brain but is also metabolized via the tricarboxylic acid (TCA) cycle. A similar situation is seen in the heart as both glucose and BHB are transported into the organ, but only 13C from BHB enters the TCA cycle. This finding suggests that fuel selection is controlled at the level of individual metabolic pathways and that seasonally induced adaptive mechanisms give rise to the strategic utilization of BHB during hibernation.

Commercial solutions for pentobarbital anesthesia typically contain water, ethanol, and propylene glycol (PG). The last two are characterized by resonances that can affect the determination of metabolite concentrations from 1H spectra. The purpose of the present study was to measure the concentration of metabolites in the rat brain in vivo under pentobarbital anesthesia using 1H MRS. Resonances of PG, but not ethanol, were observed in the rat brain. Chemical shifts and J-coupling constants for PG were measured at 37°C and pH 7.1 and used for spectral simulation. Inclusion of the simulated PG spectrum in the basis set for LCModel analysis enabled accurate fitting of in vivo spectra. This work demonstrates that concentration of brain metabolites can be reliably measured using 1H spectroscopy under pentobarbital anesthesia. The chemical shifts and J-coupling values reported here can be used to simulate the spectrum of PG at any field strength, with various pulse sequences. Magn Reson Med, 2008. © 2008 Wiley-Liss, Inc.

The brain of a hibernating mammal withstands physiological extremes that would result in cerebral damage and death in a non-hibernating species such as humans. To examine the possibility that this neuroprotection results from alterations in cerebral metabolism, we used in vivo(1)H NMR spectroscopy at high field (9.4 T) to measure the concentration of 18 metabolites (neurochemical profile) in the brain of 13-lined ground squirrels (Spermophilus tridecemlineatus) before, during, and after hibernation. Resolved in vivo(1)H NMR spectra were obtained even at low temperature in torpid hibernators ( approximately 7 degrees C). The phosphocreatine-to-creatine ratio was increased during torpor (+143%) indicating energy storage, and remained increased to a lesser extent during interbout arousal (IBA) (+83%). The total gamma-aminobutyric acid concentration was increased during torpor (+135%) and quickly returned to baseline during IBA. Glutamine (Gln) was decreased (-54%) during torpor but quickly returned to normal levels during IBA and after terminal arousal in the spring. Glutamate (Glu) was also decreased during torpor (-17%), but remained decreased during IBA (-20% compared with fall), and returned to normal level in the spring. Our observation that Glu and Gln levels are depressed in the brain of hibernators suggests that the balance between anaplerosis and loss of Glu and Gln (because of glutamatergic neurotransmission or other mechanisms) is altered in hibernation.

In vivo 13C NMR spectroscopy has the unique capability to measure metabolic fluxes noninvasively in the brain. Quantitative measurements of metabolic fluxes require analysis of the 13C labeling time courses obtained experimentally with a metabolic model. The present work reviews the ingredients necessary for a dynamic metabolic modeling study, with particular emphasis on practical issues. © 2006 Elsevier Inc. All rights reserved.

Glial cells are thought to supply energy for neurotransmission by increasing nonoxidative glycolysis; however, oxidative metabolism in glia may also contribute to increased brain activity. To study glial contribution to cerebral energy metabolism in the unanesthetized state, we measured neuronal and glial metabolic fluxes in the awake rat brain by using a double isotopic-labeling technique and a two-compartment mathematical model of neurotransmitter metabolism. Rats (n= 23) were infused simultaneously with14C-bicarbonate and [1-13C]glucose for up to 1 hr. The14C and13C labeling of glutamate, glutamine, and aspartate was measured at five time points in tissue extracts using scintillation counting and13C nuclear magnetic resonance of the chromatographically separated amino acids. The isotopic13C enrichment of glutamate and glutamine was different, suggesting significant rates of glial metabolism compared with the glutamate-glutamine cycle. Modeling the13C-labeling time courses alone and with14C confirmed significant glial TCA cycle activity (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(V_{\mathrm{PDH}}^{(\mathrm{g})},{\sim}0.5\) \end{document}μmol · gm-1· min-1) relative to the glutamate-glutamine cycle (VNT) (∼0.5-0.6 μmol · gm-1· min-1). The glial TCA cycle rate was ∼30% of total TCA cycle activity. A high pyruvate carboxylase rate (VPC, ∼0.14-0.18 μmol · gm-1· min-1) contributed to the glial TCA cycle flux. This anaplerotic rate in the awake rat brain was severalfold higher than under deep pentobarbital anesthesia, measured previously in our laboratory using the same13C-labeling technique. We postulate that the high rate of anaplerosis in awake brain is linked to brain activity by maintaining glial glutamine concentrations during increased neurotransmission.

Quantification of (31)P NMR spectra is commonly performed using line-fitting techniques with prior knowledge. Currently available time- and frequency-domain analysis software includes AMARES (in jMRUI) and CFIT respectively. Another popular frequency-domain approach is LCModel, which has been successfully used to fit both (1)H and (13)C in vivo NMR spectra. To the best of our knowledge LCModel has not been used to fit (31)P spectra. This study demonstrates the feasibility of using LCModel to quantify in vivo (31)P MR spectra, provided that adequate prior knowledge and LCModel control parameters are used. Both single-voxel and MRSI data are presented, and similar results are obtained with LCModel and with AMARES. This provides a new method for automated, operator-independent analysis of (31)P NMR spectra.

A new scheme is proposed to edit the 3.0 ppm GABA resonance without macromolecule (MM) contamination. Like previous difference spectroscopy approaches, the new scheme manipulates J-modulation of this signal using a selective editing pulse. The elimination of undesirable MM contribution at 3.0 ppm is obtained by applying this pulse symmetrically about the J-coupled MM resonance, at 1.7 ppm, in the two steps of the editing scheme. The effectiveness of the method is demonstrated in vitro, using lysine to mimic MM, and in vivo. As compared to the most commonly used editing scheme, which necessitates the acquisition and processing of two distinct difference spectroscopy experiments, the new scheme offers a reduction in experimental time (-33%) and an increase in accuracy. Magn Reson Med 45:517-520, 2001.

To investigate the effect of Carr-Purcell (CP) pulse trains on transverse relaxation times, T2, of tissue water and metabolites (both noncoupled and J-coupled spins) in the rat brain at 9.4 Tesla (T) using LASER, CP-LASER, and T2ρ-LASER sequences.Proton NMR spectra were measured in rat brain in vivo at 9.4T. Spectra were acquired at multiple echo times ranging from 18 to 402 ms. All spectra were analyzed using LCModel with simulated basis sets. Signals of metabolites as a function of echo time were fitted using a mono-exponential function to determine their T2 relaxation times.Measured T2 s for tissue water and all metabolites were significantly longer with CP-LASER and T2ρ-LASER compared with LASER. The T2 increased by a factor of ∼ 1.3 for noncoupled and weakly coupled spins (e.g., N-acetylaspartate and total creatine) and by a factor of ∼ 2 (e.g., glutamine and taurine) to ∼ 4 (e.g., glutamate and myo-inositol) for strongly coupled spins.Application of a CP pulse train results in a larger increase in T2 relaxation times for strongly coupled spins than for noncoupled (singlet) and weakly coupled spins. This needs to be taken into account when correcting for T2 relaxation in CP-like sequences such as LASER.

Impairment of energy metabolism is a key feature of Huntington disease (HD). Recently, we reported longitudinal neurochemical changes in R6/2 mice measured by in-vivo proton magnetic resonance spectroscopy (1H MRS; Zacharoff et al, 2012 ). Here, we present similar 1H MRS measurements at an early stage in the milder Q111 mouse model. In addition, we measured the concentration of ATP and inorganic phosphate (Pi), key energy metabolites not accessible with 1H MRS, using 31P MRS both in Q111 and in R6/2 mice. Significant changes in striatal creatine and phosphocreatine were observed in Q111 mice at 6 weeks relative to control, and these changes were largely reversed at 13 weeks. No significant change was detected in ATP concentration, in either HD mouse, compared with control. Calculated values of [ADP], phosphorylation potential, relative rate of ATP synthase ( v/Vmax(ATP)), and relative rate of creatine kinase ( v/Vmax(CK)) were calculated from the measured data. ADP concentration and v/Vmax(ATP) were increased in Q111 mice at 6 weeks, and returned close to normal at 13 weeks. In contrast, these parameters were normal in R6/2 mice. These results suggest that early changes in brain energy metabolism are followed by compensatory shifts to maintain energetic homeostasis from early ages through manifest disease.

Huntington disease (HD) is a severe neurodegenerative disorder for which there is a critical need of disease modifying drugs. Clinical onset typically occurs between the age of 30 and 50, and the disease evolves over 15 to 20 years with worsening psychiatric, motor, and cognitive symptoms (Novak and Tabrizi, 2010).

Determination of oxidative metabolism in the brain using in vivo ¹³C NMR spectroscopy (¹³C MRS) typically requires repeated blood sampling throughout the study to measure blood glucose concentration and fractional enrichment (input function). However, drawing blood from small animals, such as young rats, placed deep inside the magnet is technically difficult due to their small total blood volume. In the present study, a custom-built animal holder enabled temporary removal of the animal from the magnet for blood collection, followed by accurate repositioning in the exact presampling position without degradation of B₀ shimming. ¹³C label incorporation into glutamate C4 and C3 positions during a 120 min [1,6-¹³C₂] glucose infusion was determined in 28-day-old rats (n = 4) under α-chloralose sedation using localized, direct-detected in vivo ¹³C MRS at 9.4T. The tricarboxylic acid cycle activity rate (V(TCA)) determined using a one-compartment metabolic modeling was 0.67 ± 0.13 μmol/g/min, a value comparable to previous ex vivo studies. This methodology opens the avenue for in vivo measurements of brain metabolic rates using ¹³C MRS in small animals.

The low sensitivity of 13C spectroscopy can be enhanced using dynamic nuclear polarization. Detection of hyperpolarized [1-(13)C]pyruvate and its metabolic products has been reported in kidney, liver, and muscle. In this work, the feasibility of measuring 13C signals of hyperpolarized 13C metabolic products in the rat brain in vivo following the injection of hyperpolarized [1-(13)C]pyruvate and [2-(13)C]pyruvate is investigated. Injection of [2-(13)C]pyruvate led to the detection of [2-(13)C]lactate, but no other downstream metabolites such as TCA cycle intermediates were detected. Injection of [1-(13)C]pyruvate enabled the detection of both [1-(13)C]lactate and [13C]bicarbonate. A metabolic model was used to fit the hyperpolarized 13C time courses obtained during infusion of [1-(13)C]pyruvate and to determine the values of VPDH and VLDH.

To quantitatively investigate the effects of pentobarbital anesthesia on brain activity, brain metabolite concentrations and cerebral metabolic rate of glucose, in vivo proton MR spectra, and electroencephalography were measured in the rat brain with various doses of pentobarbital. The results show that (1) the resonances attributed to propylene glycol, a solvent in pentobarbital injection solution, can be robustly detected and quantified in the brain; (2) the concentration of most brain metabolites remained constant under the isoelectric state (silent electroencephalography) with a high dose of pentobarbital compared to mild isoflurane anesthesia condition, except for a reduction of 61% in the brain glucose level, which was associated with a 37% decrease in cerebral metabolic rate of glucose, suggesting a significant amount of “housekeeping” energy for maintaining brain cellular integrity under the isoelectric state; and (3) electroencephalography and cerebral metabolic activities were tightly coupled to the pentobarbital anesthesia depth and they can be indirectly quantified by the propylene glycol resonance signal at 1.13 ppm. This study indicates that in vivo proton MR spectroscopy can be used to measure changes in cerebral metabolite concentrations and cerebral metabolic rate of glucose under varied pentobarbital anesthesia states; moreover, the propylene glycol signal provides a sensitive biomarker for quantitatively monitoring these changes and anesthesia depth noninvasively.

Unraveling the energy metabolism and the hemodynamic outcomes of excitatory and inhibitory neuronal activity is critical not only for our basic understanding of overall brain function, but also for the understanding of many brain disorders. Methodologies of magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) are powerful tools for the noninvasive investigation of brain metabolism and physiology. However, the temporal and spatial resolution of in vivo MRS and MRI is not suitable to provide direct evidence for hypotheses that involve metabolic compartmentalization between different cell types, or to untangle the complex neuronal microcircuitry, which results in changes of electrical activity. This review aims at describing how the current models of brain metabolism, mainly built on the basis of in vitro evidence, relate to experimental findings recently obtained in vivo by 1H MRS, 13C MRS, and MRI. The hypotheses related to the role of different metabolic substrates, the metabolic neuron—glia interactions, along with the available theoretical predictions of the energy budget of neurotransmission will be discussed. In addition, the cellular and network mechanisms that characterize different types of increased and suppressed neuronal activity will be considered within the sensitivity-constraints of MRS and MRI.

In in vivo (1)H spectroscopy, the signal at 1.32 ppm is usually assigned to lactate. This resonance position is shared with threonine at physiological pH. The similarity of spectral patterns of lactate and threonine renders the separate measurement of either threonine or lactate without and even with editing technically challenging. In this study, the threonine signal was detected using a single-shot multiple-bond editing technique and quantified in vivo in both rat and human brains. A threonine concentration was estimated at 0.8 +/- 0.3 mM (mean +/- SD, n = 6) in the rat brain and at approximately 0.33 mM in the human brain.

In NMR spectroscopy, volume selection can be advantageously achieved using adiabatic π pulses, which enable high bandwidth and B 1 insensitivity. In order to avoid the generation of non-linear phase profiles and the subsequent signal loss caused by incoherent averaging, adiabatic π pulses are usually used in pairs for volume selection in each spatial dimension. Alternatively, when performing spectroscopic imaging (SI), a high enough spatial resolution results in negligible phase dispersion within each pixel. This allows using only one pulse per selected spatial dimension, resulting in a reduced echo-time and reduced power deposition. In this work, the feasibility of such an approach is explored theoretically and numerically, allowing the derivation of explicit conditions to obtain SI images without artifact. Adequate spatial and spectral post-processing procedures are described to compensate for the effect of non-linear phase profiles. These developments are applied to SI in the rat brain at 9.4 T, using a new adiabatic sequence named Pseudo-LASER.

All 13C NMR studies of brain glycogen to date relied on observing the incorporation of 13C label into glycogen, and thus interpretation was potentially affected by changes in 13C label turnover rates. The goal of this study was to quantify total brain glycogen concentration under conditions of hypoglycemia or normoglycemia using biochemical methods. Rats were sacrificed using a focused microwave fixation device. The results showed that metabolism of brain glycogen was Glc- and insulin-sensitive and that insulin-induced hypoglycemia promoted a gradual glycogenolysis. Moreover, we show that there are very mild effects of isoflurane and α-chloralose anesthesia on brain glycogen concentration. Altogether these results show that total brain glycogen serves as a substantial source of glucosyl units during insulin-induced moderate hypoglycemia and therefore may be neuroprotective. Finally we also conclude that previous interpretation of 13C NMR spectroscopy data accurately reflected the changes in total brain glycogen content. © 2006 Elsevier Ltd. All rights reserved.

Most in vivo 13C NMR spectroscopy studies in the brain have been performed using 1H decoupling during acquisition. Decoupling imposes significant constraints on the experimental setup (particularly for human studies at high magnetic field) in order to stay within safety limits for power deposition. We show here that incorporation of the 13C label from 13C-labeled glucose into brain amino acids can be monitored accurately using localized 13C NMR spectroscopy without the application of 1H decoupling. Using LCModel quantification with prior knowledge of one-bond and multiple-bond JCH coupling constants, the uncertainty on metabolites concentrations was only 35% to 91% higher (depending on the carbon resonance of interest) in undecoupled spectra compared to decoupled spectra in the rat brain at 9.4 Tesla. Although less sensitive, 13C NMR without decoupling dramatically reduces experimental constraints on coil setup and pulse sequence design required to keep power deposition within safety guidelines. This opens the prospect of safely measuring 13C NMR spectra in humans at varied brain locations (not only the occipital lobe) and at very high magnetic fields above 4 Tesla. Magn Reson Med, 2006. © 2005 Wiley-Liss, Inc.

Currently no definitive biomarker of Alzheimer's disease (AD) is available, and this impedes both clinical diagnosis in humans and drug discovery in transgenic animal models. Proton magnetic resonance spectroscopy ( 1 H MRS) provides a noninvasive way to investigate in vivo neurochemical abnormalities. Each observable metabolite can potentially provide information about unique in vivo pathological processes at the molecular or cellular level. In this study, the age-dependent 1 H MRS profile of transgenic AD mice was compared to that of wild-type mice. Twenty-seven APP-PS1 mice (which coexpress mutated human presenilin 1 and amyloid-β precursor protein) and 30 wild-type mice age 66-904 days were examined, some repeatedly. A reduction in the levels of N -acetylaspartate and glutamate, compared with total creatine levels, was found in APP-PS1 mice with advancing age. The most striking finding was a dramatic increase in the concentration of myo -inositol with age in APP-PS1 mice, which was not observed in wild-type mice. The age-dependent neurochemical changes observed in APP-PS1 mice agree with results obtained from in vivo human MRS studies. Among the different transgenic mouse models of AD that have been studied with 1 H MRS, APP-PS1 mice seem to best match the neurochemical profile exhibited in human AD. 1 H MRS could serve as a sensitive in vivo surrogate indicator of therapeutic efficacy in trials of agents designed to reduce neurotoxicity due to microglial activation. Because of its noninvasive and repeatable nature, MRS in transgenic models of AD could substantially accelerate drug discovery for this disease.

An efficient shim system and an optimized localization sequence were used to measure in vivo 1H NMR spectra from cerebral cortex, hippocampus, striatum, and cerebellum of C57BL/6 mice at 9.4 T. The combination of automatic first- and second-order shimming (FASTMAP) with strong custom-designed second-order shim coils (shim strength up to 0.04 mT/cm2) was crucial to achieve high spectral resolution (water line width of 11-14 Hz). Requirements for second-order shim strengths to compensate field inhomogeneities in the mouse brain at 9.4 T were assessed. The achieved spectral quality (resolution, S/N, water suppression, localization performance) allowed reliable quantification of 16 brain metabolites (LCModel analysis) from 5-10-microL brain volumes. Significant regional differences (up to 2-fold, P0.05) were found for all quantified metabolites but Asp, Glc, and Gln. In contrast, 1H NMR spectra measured from the striatum of C57BL/6, CBA, and CBA/BL6 mice revealed only small (13%, P0.05) interstrain differences in Gln, Glu, Ins, Lac, NAAG, and PE. It is concluded that 1H NMR spectroscopy at 9.4 T can provide precise biochemical information from distinct regions of the mouse brain noninvasively that can be used for monitoring of disease progression and treatment as well as phenotyping in transgenic mice models.

We detected glutamate C4 and C3 labeling in the monkey brain during an infusion of [U- 1 3 C 6 ]glucose, using a simple 1 H PRESS sequence without 1 3 C editing or decoupling. Point-resolved spectroscopy (PRESS) spectra revealed decreases in 1 2 C-bonded protons, and increases in 1 3 C-bonded protons of glutamate. To take full advantage of the simultaneous detection of 1 2 C- and 1 3 C-bonded protons, we implemented a quantitation procedure to property measure both glutamate C4 and C3 enrichments. This procedure relies on LCModel analysis with a basis set to account for simultaneous signal changes of protons bound to 1 2 C and 1 3 C. Signal changes were mainly attributed to 1 2 C- and 1 3 C-bonded protons of glutamate. As a result, we were able to measure the tricarboxylic acid (TCA) cycle flux in a 3.9 cm 3 voxel centered in the monkey brain on a whole-body 3 Tesla system (V T C A = 0.55 ′ 0.04 μmol.g - 1 .min - 1 , N = 4). This work demonstrates that oxidative metabolism can be quantified in deep structures of the brain on clinical MRI systems, without the need for a 1 3 C radiofrequency (RF) channel.

The LCModel method was adapted to analyze localized in vivo (13)C NMR spectra obtained from the rat brain in vivo at 9.4 T. Prior knowledge of chemical-shifts, J-coupling constants and J-evolution was included in the analysis. Up to 50 different isotopomer signals corresponding to 10 metabolites were quantified simultaneously in 400 microl volumes in the rat brain in vivo during infusion of [1,6-(13)C(2)]glucose. The analysis remained accurate even at low signal-to-noise ratio of the order of 3:1. The relative distribution of isotopomers in glutamate, glutamine and aspartate determined in vivo in 22 min was in excellent agreement with that measured in brain extracts. Quantitation of time series of (13)C spectra yielded time courses of total (13)C label incorporation into up to 16 carbon positions, as well as time courses of individual isotopomer signals, with a temporal resolution as low as 5 min (dynamic isotopomer analysis). The possibility of measuring in vivo a wealth of information that was hitherto accessible only in extracts is likely to expand the scope of metabolic studies in the intact brain.

Localized (13)C NMR spectra were obtained from the rat brain in vivo over a broad spectral range (15-100 ppm) with minimal chemical-shift displacement error (10%) using semi-adiabatic distortionless enhancement by polarization transfer (DEPT) combined with (1)H localization. A new gradient dephasing scheme was employed to eliminate unwanted coherences generated by DEPT when using surface coils with highly inhomogeneous B(1) fields. Excellent sensitivity was evident from the simultaneous detection of natural abundance signals for N-acetylaspartate, myo-inositol, and glutamate in the rat brain in vivo at 9.4 T. After infusion of (13)C-labeled glucose, up to 18 (13)C resonances were simultaneously measured in the rat brain, including glutamate C2, C3, C4, glutamine C2, C3, C4, aspartate C2, C3, glucose C1, C6, N-acetyl-aspartate C2, C3, C6, as well as GABA C2, lactate C3, and alanine C3. (13)C-(13)C multiplets corresponding to multiply labeled compounds were clearly observed, suggesting that extensive isotopomer analysis is possible in vivo. This unprecedented amount of information will be useful for metabolic modeling studies aimed at understanding brain energy metabolism and neurotransmission in the rodent brain.

Inhibition of succinate dehydrogenase (SDH) by the mitochondrial toxin 3-nitropropionic acid (3-NP) has gained acceptance as an animal model of Huntington's disease. In this study 13C NMR spectroscopy was used to measure the tricarboxylic acid (TCA) cycle rate in the rat brain after 3-NP treatment. The time course of both glutamate C4 and C3 13C labelling was monitored in vivo during an infusion of [1-13C]glucose. Data were fitted by a mathematical model to yield the TCA cycle rate (Vtca) and the exchange rate between alpha-ketoglutarate and glutamate (Vx). 3-NP treatment induced a 18% decrease in Vtca from 0.71 +/- 0.02 micro mol/g/min in the control group to 0.58 +/- 0.02 micro mol/g/min in the 3-NP group (p0.001). Vx increased from 0.88 +/- 0.08 micro mol/g/min in the control group to 1.33 +/- 0.24 micro mol/g/min in the 3-NP group (p0.07). Fitting the C4 glutamate time course alone under the assumption that Vx is much higher than Vtca yielded Vtca=0.43 micro mol/g/min in both groups. These results suggest that both Vtca and Vx are altered during 3-NP treatment, and that both glutamate C4 and C3 labelling time courses are necessary to obtain a reliable measurement of Vtca.

A new scheme is proposed to edit separately glutamate C3 and C4 resonances of 1H bound to 13C, in order to resolve these two signals which overlap at intermediate magnetic fields (1.5 T-3 T), commonly available for human brain studies. The two edited spectra are obtained by combining the individual acquisitions from a four-scan measurement in two different ways. The four acquisitions correspond to the two steps of the classical POCE scheme combined with another two-scan module, where the relative phases of the C3 and C41H resonances are manipulated using zero quantum and double quantum coherence pathways. This new technique exhibits the same sensitivity as POCE and allows the 13C labeling of C3 and C4 glutamate from [1-13C]glucose to be monitored separately in the rat brain at 3 T. Magn Reson Med 44:395–400, 2000. © 2000 Wiley-Liss, Inc.

A novel single-shot spectral editing technique for in vivo proton NMR is proposed to recover resonances of low-concentration metabolites obscured by very strong resonances. With this new method, editing is performed by transferring transverse magnetization to J-coupled spins from selected coupling partners using a homonuclear Hartmann-Hahn polarization transfer with adiabatic pulses. The current implementation uses 1D-TOCSY with single-voxel localization based on LASER to recover the H1 proton of beta-glucose at 4.63 ppm from under water and the lactate methyl resonances from beneath a strong lipid signal. The method can be extended to further spin systems where conventional editing methods are difficult to perform.

Localized (13)C NMR spectroscopy provides a new investigative tool for studying cerebral metabolism. The application of (13)C NMR spectroscopy to living intact humans and animals presents the investigator with a number of unique challenges. This review provides in the first part a tutorial insight into the ingredients required for achieving a successful implementation of localized (13)C NMR spectroscopy. The difficulties in establishing (13)C NMR are the need for decoupling of the one-bond (13)C-(1)H heteronuclear J coupling, the large chemical shift range, the low sensitivity and the need for localization of the signals. The methodological consequences of these technical problems are discussed, particularly with respect to (a) RF front-end considerations, (b) localization methods, (c) the low sensitivity, and (d) quantification methods. Lastly, some achievements of in vivo localized (13)C NMR spectroscopy of the brain are reviewed, such as: (a) the measurement of brain glutamine synthesis and the feasibility of quantifying glutamatergic action in the brain; (b) the demonstration of significant anaplerotic fluxes in the brain; (c) the demonstration of a highly regulated malate-aspartate shuttle in brain energy metabolism and isotope flux; (d) quantification of neuronal and glial energy metabolism; and (e) brain glycogen metabolism in hypoglycemia in rats and humans. We conclude that the unique and novel insights provided by (13)C NMR spectroscopy have opened many new research areas that are likely to improve the understanding of brain carbohydrate metabolism in health and disease.