Imaging Brain Metabolism Using Hyperpolarized 13C Magnetic Resonance Spectroscopy
Authors: Lydia M. Le Page, Caroline Guglielmetti, Celine Taglang, Myriam M. Chaumeil
Journal: Trends in Neurosciences (2020)

Hyperpolarized 13C MRS is an emerging metabolic imaging technique that provides in vivo assessment of enzymatic fluxes after injection of hyperpolarized probes ( e. g., [1-13C]pyruvate).This technology provides valuable diagnostic and treatment monitoring information in the oncological and cardiovascular fields, where measurement of hyperpolarized pyruvate-to-lactate conversion has been translated to the clinic.The technique has recently been applied preclinically in several non-oncological neurological disorders, successfully detecting metabolic impairment.Valuable metabolic data assessing hyperpolarized pyruvate-to-lactate conversion have been acquired in animal models of multiple sclerosis, traumatic brain injury, neuroinflammation, cognitive impairment, and stroke.Clinical trials on healthy volunteers and traumatic brain injury patients are currently underway. Aberrant metabolism is a key factor in many neurological disorders. The ability to measure such metabolic impairment could lead to improved detection of disease progression, and development and monitoring of new therapeutic approaches. Hyperpolarized 13C magnetic resonance spectroscopy (MRS) is a developing imaging technique that enables non-invasive measurement of enzymatic activity in real time in living organisms. Primarily applied in the fields of cancer and cardiac disease so far, this metabolic imaging method has recently been used to investigate neurological disorders. In this review, we summarize the preclinical research developments in this emerging field, and discuss future prospects for this exciting technology, which has the potential to change the clinical paradigm for patients with neurological disorders. Aberrant metabolism is a key factor in many neurological disorders. The ability to measure such metabolic impairment could lead to improved detection of disease progression, and development and monitoring of new therapeutic approaches. Hyperpolarized 13C magnetic resonance spectroscopy (MRS) is a developing imaging technique that enables non-invasive measurement of enzymatic activity in real time in living organisms. Primarily applied in the fields of cancer and cardiac disease so far, this metabolic imaging method has recently been used to investigate neurological disorders. In this review, we summarize the preclinical research developments in this emerging field, and discuss future prospects for this exciting technology, which has the potential to change the clinical paradigm for patients with neurological disorders. Metabolic changes are increasingly being recognized as key players in neurological diseases, and may reveal exciting new avenues for treatment of these disorders. It is critical to develop new tools for monitoring brain metabolism through disease progression and in response to treatment, but such development remains complex due to our inability to easily sample brain tissue. New neuroimaging methods are beginning to address the crucial need for non-invasive assessment of brain metabolism. Hyperpolarized 13C MRS/magnetic resonance spectroscopic imaging (MRSI, see Glossary) is an emerging technology that enables in vivo acquisition of dynamic metabolic data, providing unprecedented insights into the in vivo metabolic status of the organ of interest. 1H and nonhyperpolarized, thermal 13C MRS have both been used to acquire clinical data on steady-state human brain metabolism [1.Albrecht D. S. et al.In vivo imaging of human neuroinflammation.ACS Chem. Neurosci. 2016; 7: 470-483Crossref PubMed Scopus (126) Google Scholar, 2.Cheshkov S. et al.Oxidation of [U-(13) C]glucose in the human brain at 7T under steady state conditions.Magn. Reson. Med. 2017; 78: 2065-2071Crossref PubMed Scopus (19) Google Scholar, 3.Boumezbeur F. et al.Altered brain mitochondrial metabolism in healthy aging as assessed by in vivo magnetic resonance spectroscopy.J. Cereb. Blood Flow Metab. 2010; 30: 211-221Crossref PubMed Scopus (184) Google Scholar]. However, these methods are not used widely in the clinic and, in particular, thermal 13C MRS requires long scan times and infusions of 13C-labeled substrate. Hyperpolarized 13C MRS brings new information to the field of metabolic imaging. First published in 2003 [4.Ardenkjaer-Larsen J. H. et al.Increase in signal-to-noise ratio of > 10,000 times in liquid-state NMR.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10158-10163Crossref PubMed Scopus (2222) Google Scholar], this method requires the hyperpolarization and rapid liquid dissolution of a 13C-labeled compound, typically pyruvate, a process typically carried out in an automated fashion using a hyperpolarizer. Hyperpolarization of a compound causes it to become more than 10 000-fold more visible by magnetic resonance imaging (MRI), due to the increased signal:noise ratio. Figure 1 (Key Figure) summarizes the typical steps of a hyperpolarized 13C MRS/I preclinical experiment. Given that the hyperpolarized state lasts only for a short time once in solution (referred to as the relaxation time, or T1), injection into the biological system of interest (cells, animal model, or human) must be rapid (10–15 s in animals) and coincide with the time of data acquisition. Typical MR hardware is then used to obtain 13C MR spectra (slab, 2D or 3D, single time point or dynamic), where the resonances of the injected substrate and its downstream metabolic products can be seen. Following injection of hyperpolarized [1-13C]pyruvate, the most commonly used probe, the resonances of hyperpolarized [1-13C]lactate, hyperpolarized [1-13C]alanine, and, in some cases, hyperpolarized 13C bicarbonate, are observed in the living brain, reflecting enzymatic conversion via lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and pyruvate dehydrogenase (PDH), respectively. Many new compounds for injection are under development, and are discussed later. MR spectra showing area(s) under the curve(s), corresponding metabolic ratios of product:substrate ( e. g., lactate:pyruvate), or kinetic pseudo-rate constants for substrate to product conversion ( e. g., kPL) can then be reported from hyperpolarized data. By using preclinical models, hyperpolarized 13C metabolic data can be validated either through correlation analyses with 'ground truth' ex vivo biochemical analyses, or through comparison to more widely applied clinical imaging techniques to assess the added value of the method. Clinically, hyperpolarized 13C MRS/I can provide access to otherwise inaccessible metabolic information, given the rarity of human brain tissue biopsy sampling. So far, hyperpolarized 13C MRS/I has been primarily applied in the fields of cancer and, to a lesser extent, cardiovascular disease, with clinical studies underway in both areas. More recently, exciting data have been acquired in preclinical models of neurological disorders and neuroinflammation, in which metabolic changes have been observed with disease progression and following treatment. This new investigative direction of neurological disorders may improve our understanding of in vivo brain metabolism not only preclinically, but also in patient populations, since hyperpolarized 13C MRS/I has already been translated to the clinic. In this review, we summarize the current applications of hyperpolarized 13C MRS/I in the non-oncological neurological field, consider current challenges, and discuss prospects for future work. The conversion of pyruvate to lactate sits at a key intersection in energetic metabolism pathways, with LDH being present in most cell types across the body. This enzymatic step is affected in a multitude of diseases and, consequently, often modulated in response to treatment. As a result, an ability to non-invasively visualize pyruvate to lactate conversion opens the door to possible applications in disease monitoring and assessment of therapeutic response. Understanding hyperpolarized 13C MRS/I data from the healthy brain at different ages is essential if one is to investigate disease development and treatment response. The adult brain has been investigated by hyperpolarized 13C MRS/I in several preclinical proof-of-concept studies. The feasibility of using both hyperpolarized [1-13C]pyruvate to study brain metabolism using a variety of MR systems and radiofrequency (RF) coils has been confirmed, with conversion of hyperpolarized [1-13C]pyruvate to hyperpolarized [1-13C]lactate and hyperpolarized 13C bicarbonate detected in mouse and rat brain via imaging at 1 or 3 Tesla [5.Miloushev V. Z. et al.Hyperpolarized (13)C pyruvate mouse brain metabolism with absorptive-mode EPSI at 1T.J. Magn. Reson. 2017; 275: 120-126Crossref PubMed Scopus (7) Google Scholar, 6.Hurd R. E. et al.Cerebral dynamics and metabolism of hyperpolarized [1-(13)C]pyruvate using time-resolved MR spectroscopic imaging.J. Cereb. Blood Flow Metab. 2010; 30: 1734-1741Crossref PubMed Scopus (46) Google Scholar, 7.Mayer D. et al.Dynamic and high-resolution metabolic imaging of hyperpolarized [1-13C]-pyruvate in the rat brain using a high-performance gradient insert.Magn. Reson. Med. 2011; 65: 1228-1233Crossref PubMed Scopus (40) Google Scholar]. Hyperpolarized [1-13C]alanine has also been detected, although this metabolite is likely to arise from the soft tissue surrounding the healthy brain [5.Miloushev V. Z. et al.Hyperpolarized (13)C pyruvate mouse brain metabolism with absorptive-mode EPSI at 1T.J. Magn. Reson. 2017; 275: 120-126Crossref PubMed Scopus (7) Google Scholar]. Before moving to the clinic, with the first clinical manuscript reported in 2013, hyperpolarized 13C MRS/I was also shown to be feasible in the nonhuman healthy primate brain using a clinical 3 Tesla MR system and the clinical SpinLab hyperpolarizer [8.Park I. et al.Dynamic hyperpolarized carbon-13 MR metabolic imaging of nonhuman primate brain.Magn. Reson. Med. 2014; 71: 19-25Crossref PubMed Scopus (26) Google Scholar]. Interestingly, values for hyperpolarized 13C lactate:pyruvate were found to be similar to those seen in rodent studies. To our knowledge, only one preclinical study to date has used hyperpolarized 13C MRS/I to investigate brain development. This longitudinal study in mice measured cerebral conversion of hyperpolarized [1-13C]pyruvate to hyperpolarized [1-13C]lactate at multiple timepoints between postnatal days 18 and 60, and observed that this conversion decreased with age [9.Chen Y. et al.Pyruvate to lactate metabolic changes during neurodevelopment measured dynamically using hyperpolarized 13C imaging in juvenile murine brain.Dev. Neurosci. 2016; 38: 34-40Crossref PubMed Scopus (13) Google Scholar]. However, total brain volume as measured by conventional MRI remained largely unchanged, demonstrating the added value of metabolic imaging [9.Chen Y. et al.Pyruvate to lactate metabolic changes during neurodevelopment measured dynamically using hyperpolarized 13C imaging in juvenile murine brain.Dev. Neurosci. 2016; 38: 34-40Crossref PubMed Scopus (13) Google Scholar]. In the healthy aging brain, hyperpolarized 13C MRS/I research remains limited; there is also much opportunity for understanding age-linked neurological disorders, such as Alzheimer's disease (AD) [10.An Y. et al.Evidence for brain glucose dysregulation in Alzheimer's disease.Alzheimers Dement. 2018; 14: 318-329Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar]. As an initial step into the aging field, hyperpolarized 13C MRS has been used to study healthy older mice. Administration of dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, was shown to decrease cerebral hyperpolarized [1-13C]pyruvate to hyperpolarized [1-13C]lactate conversion. An impairment in spatial learning was observed with DCA treatment over time, assessed using a Morris water maze [11.Harris R. A. et al.Aerobic glycolysis is required for spatial memory acquisition but not memory retrieval in mice.eNeuro. 2019; 6 (ENEURO.0389-18.2019)Crossref PubMed Scopus (29) Google Scholar]. Immunometabolism refers to the metabolic alterations that occur following activation of immune cells, generally in response to pathogens or noxious stimuli. Although immune cell activation is central to many brain diseases, clinically translatable methods that enable the specific imaging of cells from the immune system and immune subtypes are still lacking. On activation, innate immune cells undergo metabolic reprogramming that gives rise to pro- or anti-inflammatory subsets [12.Galvan-Pena S. O'Neill L. A. Metabolic reprograming in macrophage polarization.Front. Immunol. 2014; 5: 420PubMed Google Scholar]. Therefore, hyperpolarized 13C MRS/I is a promising technique for non-invasively detecting and monitoring inflammatory status. Classically activated proinflammatory macrophages, generally associated with tissue damage, have been shown to increase their glucose uptake, glycolysis, and conversion of pyruvate into lactate [12.Galvan-Pena S. O'Neill L. A. Metabolic reprograming in macrophage polarization.Front. Immunol. 2014; 5: 420PubMed Google Scholar]. In vitro studies conducted in mouse macrophage cells using hyperpolarized [1-13C]pyruvate showed that, after activation using the toxin lipopolysaccharide (LPS), hyperpolarized 13C lactate:pyruvate was significantly increased compared with nontreated macrophages [13.Sriram R. et al.Molecular detection of inflammation in cell models using hyperpolarized (13)C-pyruvate.Theranostics. 2018; 8: 3400-3407Crossref PubMed Scopus (0) Google Scholar]. This increased ratio was explained by an increase in LDH activity and gene transcription, increased cofactor NADH, and decreased expression of monocarboxylate transporters 1 and 4 (MCT1/4), resulting in an increased intracellular lactate concentration (see also Table S1 in the supplemental information online). Furthermore, hyperpolarized [1-13C]pyruvate was able to detect the effect of a nonsteroidal anti-inflammatory drug affecting LDH affinity. In line with these findings, a single intracranial injection of LPS in the mouse brain resulted in increased hyperpolarized [1-13C]lactate and hyperpolarized 13C lactate:pyruvate at the site of injection [14.Le Page L. M. et al.Hyperpolarized (13) C magnetic resonance spectroscopy detects toxin-induced neuroinflammation in mice.NMR Biomed. 2019; 32e4164Crossref PubMed Scopus (15) Google Scholar]. These changes were in alignment with increased numbers of microglia/macrophages and astrocytes. Activation of lymphocyte T cells and the resultant altered metabolism [15.Zhu J. Paul W. E. CD4 T cells: fates, functions, and faults.Blood. 2008; 112: 1557-1569Crossref PubMed Scopus (1170) Google Scholar] have also been detected using hyperpolarized [1-13C]pyruvate; activated CD4+ T cells showed a threefold higher hyperpolarized 13C lactate:pyruvate compared with nonactivated cells [16.Can E. et al.Noninvasive rapid detection of metabolic adaptation in activated human T lymphocytes by hyperpolarized (13)C magnetic resonance.Sci. Rep. 2020; 10: 200Crossref PubMed Scopus (7) Google Scholar]. Energetic imbalance and abnormal energy metabolism have been implicated in most neurodegenerative disorders [17.Camandola S. Mattson M. P. Brain metabolism in health, aging, and neurodegeneration.EMBO J. 2017; 36: 1474-1492Crossref PubMed Scopus (315) Google Scholar,18.Procaccini C. et al.Role of metabolism in neurodegenerative disorders.Metabolism. 2016; 65: 1376-1390Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar]. There are multiple causes of alterations in brain metabolism that may arise from direct insults to the brain tissue itself and/or as a result of systemic modifications occurring in peripheral organs. So far, only a small number of preclinical studies have used hyperpolarized 13C MRS/I approaches to assess metabolic impairment in the diseased brain outside the oncological field, and these are limited to hyperpolarized [1-13C]pyruvate. In models of traumatic brain injury (TBI), hyperpolarized 13C lactate:pyruvate increased at the injury site within hours and remained elevated for several weeks following impact [19.Guglielmetti C. et al.In vivo metabolic imaging of traumatic brain injury.Sci. Rep. 2017; 7: 17525Crossref PubMed Scopus (25) Google Scholar,20.DeVience S. J. et al.Metabolic imaging of energy metabolism in traumatic brain injury using hyperpolarized [1-(13)C]pyruvate.Sci. Rep. 2017; 7: 1907Crossref PubMed Scopus (42) Google Scholar]. Furthermore, hyperpolarized 13C bicarbonate:lactate was decreased at early time points post injury [20.DeVience S. J. et al.Metabolic imaging of energy metabolism in traumatic brain injury using hyperpolarized [1-(13)C]pyruvate.Sci. Rep. 2017; 7: 1907Crossref PubMed Scopus (42) Google Scholar]. When investigated, these changes were found to be linked to decreased PDH activity with no changes in LDH activity. The cellular sources driving these metabolic changes remain unclear and most likely involve multiple cell types, such as dying neurons and inflammatory cells. Furthermore, interpretation of the results is challenging due to physical alterations of the brain, including blood–brain barrier (BBB) disruption, bleeding, and edema. However, interestingly, at subacute timepoints, microglial cells were shown to likely be a major contributor to the increased hyperpolarized 13C lactate:pyruvate, via use of a diet that specifically depletes the microglial population [19.Guglielmetti C. et al.In vivo metabolic imaging of traumatic brain injury.Sci. Rep. 2017; 7: 17525Crossref PubMed Scopus (25) Google Scholar]. Further evidence that proinflammatory macrophages can be responsible for the observed increased hyperpolarized 13C lactate:pyruvate was provided from two mouse models of multiple sclerosis (MS) [21.Guglielmetti C. et al.Hyperpolarized (13)C MR metabolic imaging can detect neuroinflammation in vivo in a multiple sclerosis murine model.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E6982-E6991Crossref PubMed Scopus (55) Google Scholar,22.Guglielmetti C. et al.Metabolic imaging of brain inflammation using hyperpolarized 13C MRSI of pyruvate and urea in a mouse model of multiple sclerosis.in: Proceedings of the International Society for Magnetic Resonance in Medicine Annual Meeting 2019. Abstr 0723. International Society for Magnetic Resonance in Medicine, 2019Google Scholar]. MS is an autoimmune disease that leads to destruction of myelin sheaths and results in motor and cognitive deficits [23.Reich D. S. et al.Multiple sclerosis.N. Engl. J. Med. 2018; 378: 169-180Crossref PubMed Scopus (1100) Google Scholar]. Increased hyperpolarized 13C lactate:pyruvate was seen in brain lesions, where high levels of macrophages were also detected. These changes were linked to macrophages upregulating the enzyme PDK1, leading to inhibition of PDH activity and increased flux towards lactate production. This increase was not detected in transgenic mice that do not elicit an immune response due to their lack of the fractalkine receptor (CX3CR1) [24.Jung S. et al.Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.Mol. Cell. Biol. 2000; 20: 4106-4114Crossref PubMed Scopus (1865) Google Scholar]. Hyperpolarized [1-13C]pyruvate was successfully used to identify metabolic alterations in a model of ischemic stroke induced by intracranial injections of the vasoconstrictor endothelin-1 [25.Xu Y. et al.Hyperpolarized (13)C magnetic resonance imaging can detect metabolic changes characteristic of penumbra in ischemic stroke.Tomography. 2017; 3: 67-73Crossref PubMed Scopus (17) Google Scholar]. Hyperpolarized [1-13C]pyruvate and hyperpolarized [1-13C]lactate, but not hyperpolarized 13C lactate:pyruvate, were increased in the stroke region (penumbra) compared with the contralateral hemisphere. Although no ex vivo validations were performed, these findings indicate an increased delivery of hyperpolarized [1-13C]pyruvate. This increased delivery is possibly linked to increased blood flow and/or perfusion or uptake across the BBB, as well as an increased conversion into lactate, which the authors suggest indicates increased LDH enzymatic activity. Increases in hyperpolarized 13C lactate:pyruvate were described in the brains of mice fed a high-fat diet for a 24-week period, and were correlated with decreased PDH activity, but not LDH activity, in the cortex and striatum [26.Choi Y. S. et al.Hyperpolarized [1-13C] pyruvate MR spectroscopy detect altered glycolysis in the brain of a cognitively impaired mouse model fed high-fat diet.Mol. Brain. 2018; 11: 74Crossref PubMed Scopus (8) Google Scholar]. Importantly, this study was the first to report a significant correlation between the hyperpolarized 13C lactate:pyruvate and cognitive deficits, evaluated in this case via a Morris Water Maze behavioral assessment. Interestingly, in a follow-up study of diabetic mice fed a high-fat diet for a 24-week period, hyperpolarized 13C lactate:total 13C was increased in the hippocampus [27.Choi Y. S. et al.Hyperpolarized [1-13C]lactate flux increased in the hippocampal region in diabetic mice.Mol. Brain. 2019; 12: 88Crossref PubMed Scopus (9) Google Scholar]. Ex vivo analyses of tissue revealed increased lactate levels that were not explained by modulations of LDH, but were correlated with the phosphorylated ATP citrate lyase protein, highlighting a metabolic adaptation in the presence of limited glucose availability. Following acute liver failure, 13C MRS/I of hyperpolarized [1-13C]pyruvate revealed increased brain hyperpolarized 13C lactate:pyruvate and hyperpolarized 13C alanine:pyruvate before the appearance of clinical symptoms, and further increases of these ratios with disease progression [28.Chavarria L. et al.Real-time assessment of (1)(3)C metabolism reveals an early lactate increase in the brain of rats with acute liver failure.NMR Biomed. 2015; 28: 17-23PubMed Google Scholar]. In this case, it is not yet determined whether [1-13C]alanine is being visualized in either the brain or the surrounding tissue. Although lacking validation through molecular ex vivo analyses of tissue, these findings shed light on the pathogenesis of cerebral edema in acute liver injury, and suggest that lactate produced from anaerobic metabolism is a crucial factor in the development of brain edema. The broad applications of hyperpolarized [1-13C]pyruvate have been discussed earlier, but there are many additional novel hyperpolarized probes in development. Such probes could provide valuable information on the changing metabolism in disease and enable early assessment of treatment response. While many metabolic pathways are of potential interest in neurological disorders, not all metabolites can be hyperpolarized. For success as a hyperpolarized probe, the metabolite of interest should have the following physical properties: (i) can be labeled with 13C at the position of interest (which is often costly); (ii) either be a liquid at room temperature, or dissolve in a safe solvent at high concentration (at least 1M); and (iii) form a glass when frozen quickly. From an MR perspective, both substrate and products of interest need to have distinct MR frequencies, and relaxation times long enough to be visualized during a MR experiment. Finally, from a biological perspective, the metabolite of interest should be rapidly transported to the organ of interest (and if necessary through the cell membrane), be metabolized rapidly, and be physiologically safe in the 1–200-mM range. Despite these steep requirements, many hyperpolarized probes in early development have so far been demonstrated feasible for application in healthy animals (Figure 2). Hyperpolarized [2-13C]pyruvate differs from the widely used [1-13C]pyruvate only by the position of the 13C label; protocols for its use are identical. However, from a metabolic perspective, hyperpolarized [2-13C]pyruvate enables investigation of tricarboxylic acid (TCA) cycle metabolism, because the 13C label in the C2 position results in the MR-visible products of not only [2-13C]lactate, but also [5-13C]glutamate and [1-13C]citrate [29.Park J. M. et al.Measuring mitochondrial metabolism in rat brain in vivo using MR spectroscopy of hyperpolarized [2-13C]pyruvate.NMR Biomed. 2013; 26: 1197-2203Crossref PubMed Scopus (47) Google Scholar]. Detection of metabolites besides [2-13C]lactate is not a given, and may be dependent on multiple biological and technical factors [30.Marjańska M. et al.In vivo 13C spectroscopy in the rat brain using hyperpolarized [1-13C]pyruvate and [2-13C]pyruvate.J. Magn. Reson. 2010; 206: 210-218Crossref PubMed Scopus (76) Google Scholar]. Data acquired in the rat brain following injection of hyperpolarized [1-13C]ethyl pyruvate, a lipophilic probe used for its ability to more easily cross the BBB, showed a comparable average lactate level to that observed following hyperpolarized [1-13C]pyruvate injection [31.Hurd R. E. et al.Metabolic imaging in the anesthetized rat brain using hyperpolarized [1-13C] pyruvate and [1-13C] ethyl pyruvate.Magn. Reson. 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Lê T.P. et al.Evaluating the potential of hyperpolarised [1-13C] L-lactate as a neuroprotectant metabolic biosensor for stroke.Sci. Rep. 2020; 10: 5507Crossref PubMed Scopus (15) Google Scholar]. Interestingly, therapeutic strategies have identified lactate as a possible neuroprotective agent to limit cerebral damage after stroke and, thus, hyperpolarized [1-13C]lactate could be an effective theranostic agent, although use of hyperpolarized [1-13C]lactate may be BBB limited [34.Takado Y. et al.Hyperpolarized (13)C magnetic resonance spectroscopy reveals the rate-limiting role of the blood-brain barrier in the cerebral uptake and metabolism of l-lactate in vivo.ACS Chem. Neurosci. 2018; 9: 2554-2562Crossref PubMed Scopus (19) Google Scholar]. Widening the scope further, hyperpolarized 2-keto [1-13C]isocaproate (KIC) conversion to hyperpolarized [1-13C]leucine by branched-chain amino acid aminotransferase (BCAT) has been imaged in the healthy rat brain [35.Butt S. A. et al.Imaging cerebral 2-ketoisocaproate metabolism with hyperpolarized 13C magnetic resonance spectroscopic imaging.J. Cereb. Blood Flow Metab. 2012; 32: 1508-1514Crossref PubMed Scopus (31) Google Scholar], which could be informative with regards to the metabolism of glutamate, a key metabolite in astroglia, microglia, and neurons. Hyperpolarized [1-13C]glutamate and hyperpolarized [1-13C]acetate could provide valuable in vivo information. However, they currently face the same challenge: the resonance frequency of the product is close to that of the injected substrate, making data analysis complex. If this could be overcome, both probes could be usefully applied to the study of brain metabolism. Hyperpolarized [1-13C]glutamate has been shown to provide information on glutamate to glutamine conversion in the healthy rat brain, although it required BBB opening [36.Mazuel L. et al.Intracerebral synthesis of glutamine from hyperpolarized glutamate.Magn. Reson. Med. 2017; 78: 1296-1305Crossref PubMed Scopus (8) Google Scholar]. This probe is potentially useful for studying chronic excitotoxicity, a symptom associated with many neurodegenerative disorders [37.Lewerenz J. Maher P. Chronic glutamate toxicity in neurodegenerative diseases - what is the evidence?.Front. Neurosci. 2015; 9: 469Crossref PubMed Scopus (431) Google Scholar], but probe administration may have adverse effects [38.Hawkins R. A. The blood-brain barrier and glutamate.Am. J. Clin. Nutr. 2009; 90: 867S-874SCrossref PubMed Scopus (211) Google Scholar,39.Hawkins R. A. Vina J. R. How glutamate is managed by the blood-brain barrier.Biology (Basel). 2016; 5E37Crossref PubMed Scopus (49) Google Scholar]. Brain cells, such as astrocytes, also metabolize acetate as fuel, and reactive astrocytes are major players in multiple neurodegenerative disorders. Thus, it is promising that injection of hyperpolarized [1-13C]acetate in vivo resulted in observation of the TCA cycle intermediate α-ketoglutarate in the rat brain [40.Mishkovsky M. et al.In vivo detection of brain Krebs cycle intermediate by hyperpolarized magnetic resonance.J. Cereb. Blood Flow Metab. 2012; 32: 2108-2113Crossref PubMed Scopus (62) Google Scholar]. Oxidative stress is a key player in neuroinflammatory disorders and, thus, a non-invasive in vivo measure of it in the brain would be valuable. Hyperpolarized [1-13C]dehydroascorbic acid (DHA) has been shown to cross the BBB and be converted to hyperpolarized [1-13C]vitamin C alongside oxidation of glutathione (GSH). As such, the conversion of hyperpolarized [1-13C]DHA to hyperpolarized [1-13C]vitamin C could be used as a marker of oxidative stress [41.Keshari K. R. et al.Hyperpolarized 13C dehydr