Imaging Brain Metabolism Using Hyperpolarized 13C Magnetic Resonance Spectroscopy

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 13 C 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. 1 H and nonhyperpolarized, thermal 13 C MRS have both been used to acquire clinical data on steadystate human brain metabolism [1][2][3]. However, these methods are not used widely in the clinic and, in particular, thermal 13 C MRS requires long scan times and infusions of 13 C-labeled substrate. Hyperpolarized 13 C MRS brings new information to the field of metabolic imaging. First published in 2003 [4], this method requires the hyperpolarization and rapid liquid dissolution of a 13 C-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 13 C 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 T 1 ), 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 13 C MR spectra (slab, 2D or 3D, single time point or dynamic), where the resonances of the injected substrate and its downstream metabolic Hyperpolarized [1- 13

C]Pyruvate Provides Non-invasive Metabolic Preclinical Information in the Healthy and Diseased Brain
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 13 C 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 13 C MRS/I in several preclinical proof-of-concept studies. The feasibility of using both hyperpolarized [1-13 C]pyruvate to study brain metabolism using a variety of MR systems and radiofrequency (RF) coils has been confirmed, with conversion of hyperpolarized [1-13 C]pyruvate to hyperpolarized [1-13 C]lactate and hyperpolarized 13 C bicarbonate detected in mouse and rat brain via imaging at 1 or 3 Tesla [5][6][7]. Hyperpolarized [1-13 C]alanine has also been detected, although this metabolite is likely to arise from the soft tissue surrounding the healthy brain [5].
Before moving to the clinic, with the first clinical manuscript reported in 2013, hyperpolarized 13 C 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]. Interestingly, values for hyperpolarized 13 C 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 13 C MRS/I to investigate brain development. This longitudinal study in mice measured cerebral conversion of Glossary Magnetic resonance imaging (MRI): typically used for visualizing and distinguishing between areas of varying physical properties of water molecules (via exploitation of the high MR visibility of protons). MRI is widely used in the clinic and can provide information on, for example, brain structure and presence of lesions, such as tumors or edema. Magnetic resonance spectroscopic imaging (MRSI): spatially localized spectra can be produced from multiple parts of the brain at once, to provide spatial information on steady-state metabolite levels. Magnetic resonance spectroscopy (MRS): enables visualization of steadystate levels of brain metabolites. Data are in the form of spectra. Proton spectroscopy enables detection of several metabolites with a sensitivity in the millimolar range (e.g., choline, N-acetyl aspartate, or creatine). Carbon spectroscopy is also viable but, due to the low natural abundance of the MR-detectable 13 C isotope, long acquisition times or infusion of 13 Clabelled compounds are necessary.
hyperpolarized [1-13 C]pyruvate to hyperpolarized [1-13 C]lactate at multiple timepoints between postnatal days 18 and 60, and observed that this conversion decreased with age [9]. However, total brain volume as measured by conventional MRI remained largely unchanged, demonstrating the added value of metabolic imaging [9].
In the healthy aging brain, hyperpolarized 13 C MRS/I research remains limited; there is also much opportunity for understanding age-linked neurological disorders, such as Alzheimer's disease (AD) [10]. As an initial step into the aging field, hyperpolarized 13 C 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 [1-13 C]pyruvate to hyperpolarized [1-13 C]lactate conversion. An impairment in spatial learning was observed with DCA treatment over time, assessed using a Morris water maze [11].  is placed into a hyperpolarizer for~1 h, where the sample sits at low temperature (T=1-2 K) while being irradiated with microwaves (F=~94 GHz).
Step 3: when hyperpolarization is complete, the sample is rapidly dissolved in a neutralizing buffer (within~10 s), which produces a pH 7, 37°C solution of [1-13 C]pyruvate in the range of 5-100 mM.
Step 4: this buffered solution is then immediately injected into the experimental subject placed inside a MR imaging (MRI) system, over a short period of time (~15 s). In the illustration, the injection is into a rodent model, via a tail-vein catheter. Acquisition of MR data (slab, 2D or 3D, dynamic or single timepoint acquisition) is timed with the injection of the hyperpolarized solution. Zoomed-in brain inset: In the rodent brain (zoomed-in brain inset), conversion of hyperpolarized [1-13 C]pyruvate to hyperpolarized [1-13 C]lactate, hyperpolarized [1-13 C]alanine, and hyperpolarized [ 13 C]bicarbonate occurs within the time frame of the MR acquisition. These conversions are via lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and pyruvate dehydrogenase (PDH) respectively. Step 5: after data processing, MR data reflecting the in vivo metabolism of the injected hyperpolarized compound to its downstream metabolites can be visualized, for instance by displaying spectra, heatmaps, or kinetic analyses of signal:noise ratio (SNR) versus time.

Immunometabolism
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]. Therefore, hyperpolarized 13 C 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]. In vitro studies conducted in mouse macrophage cells using hyperpolarized [1-13 C]pyruvate showed that, after activation using the toxin lipopolysaccharide (LPS), hyperpolarized 13 C lactate:pyruvate was significantly increased compared with nontreated macrophages [13]. 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 [1-13 C]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 [1-13 C]lactate and hyperpolarized 13 C lactate:pyruvate at the site of injection [14]. These changes were in alignment with increased numbers of microglia/macrophages and astrocytes.
Activation of lymphocyte T cells and the resultant altered metabolism [15] have also been detected using hyperpolarized [1-13 C]pyruvate; activated CD4+ T cells showed a threefold higher hyperpolarized 13 C lactate:pyruvate compared with nonactivated cells [16].

Neurological Disorders
Energetic imbalance and abnormal energy metabolism have been implicated in most neurodegenerative disorders [17,18]. 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 13 C MRS/I approaches to assess metabolic impairment in the diseased brain outside the oncological field, and these are limited to hyperpolarized [1-13 C]pyruvate.
In models of traumatic brain injury (TBI), hyperpolarized 13 C lactate:pyruvate increased at the injury site within hours and remained elevated for several weeks following impact [19,20]. Furthermore, hyperpolarized 13 C bicarbonate:lactate was decreased at early time points post injury [20]. 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 13 C lactate:pyruvate, via use of a diet that specifically depletes the microglial population [19].
Further evidence that proinflammatory macrophages can be responsible for the observed increased hyperpolarized 13 C lactate:pyruvate was provided from two mouse models of multiple Trends in Neurosciences sclerosis (MS) [21,22]. MS is an autoimmune disease that leads to destruction of myelin sheaths and results in motor and cognitive deficits [23]. Increased hyperpolarized 13 C 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].
Hyperpolarized [1-13 C]pyruvate was successfully used to identify metabolic alterations in a model of ischemic stroke induced by intracranial injections of the vasoconstrictor endothelin-1 [25]. Hyperpolarized [1-13 C]pyruvate and hyperpolarized [1-13 C]lactate, but not hyperpolarized 13 C 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 [1-13 C]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 13 C lactate:pyruvate were described in the brains of mice fed a highfat diet for a 24-week period, and were correlated with decreased PDH activity, but not LDH activity, in the cortex and striatum [26]. Importantly, this study was the first to report a significant correlation between the hyperpolarized 13 C 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 13 C lactate:total 13 C was increased in the hippocampus [27]. 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, 13 C MRS/I of hyperpolarized [1-13 C]pyruvate revealed increased brain hyperpolarized 13 C lactate:pyruvate and hyperpolarized 13 C alanine:pyruvate before the appearance of clinical symptoms, and further increases of these ratios with disease progression [28]. In this case, it is not yet determined whether [1-13 C]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.

Developing New Hyperpolarized Probes to Target the Full Breadth of Metabolic Changes Occurring in Neurological Disorders
The broad applications of hyperpolarized [1-13 C]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 13 C 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 Trends in Neurosciences 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 [2-13 C]pyruvate differs from the widely used [1-13 C]pyruvate only by the position of the 13 C label; protocols for its use are identical. However, from a metabolic perspective, hyperpolarized [2-13 C]pyruvate enables investigation of tricarboxylic acid (TCA) cycle metabolism, because the 13 C label in the C2 position results in the MR-visible products of not only [2-13 C]lactate, but also [5-13 C]glutamate and [1-13 C]citrate [29]. Detection of metabolites besides [2-13 C]lactate is not a given, and may be dependent on multiple biological and technical factors [30]. Data acquired in the rat brain following injection of hyperpolarized [1-13 C]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 [1-13 C]pyruvate injection [31].
Hyperpolarized [1-13 C]lactate has itself been used as a probe; increased hyperpolarized [1-13 C] pyruvate:lactate was observed in the brain shortly after reperfusion of a middle cerebral artery Trends Trends in in Neurosciences Neurosciences Figure 2. Overview of Metabolic Reactions Currently Investigated Using Hyperpolarized Probes. Hyperpolarized probes (bold) and corresponding hyperpolarized downstream product(s) currently investigated in (i) the healthy brain (all items in the schematic except arginine); (ii) the field of immunometabolism (yellow box); and (iii) preclinical models of neurological disorders (red boxes). References to the corresponding study/studies are noted in square brackets above the reaction arrows [29][30][31][32]35,36,[40][41][42][43][44][45][46][47]. The relaxation times (T 1 ) of the hyperpolarized probes are ranked as long (40-60 s, green), average (20-40 s, orange) and short (5-20 s, red), from data at the clinical magnetic field strength of 3 Tesla (extrapolated from data at other field strengths for glucose, glutamate, acetate and ketoisocaproate). occlusion mouse model of stroke. No lesion was detected using conventional MRI [33]. Interestingly, therapeutic strategies have identified lactate as a possible neuroprotective agent to limit cerebral damage after stroke and, thus, hyperpolarized [1-13 C]lactate could be an effective theranostic agent, although use of hyperpolarized [1-13 C]lactate may be BBB limited [34].
Widening the scope further, hyperpolarized 2-keto[1-13 C]isocaproate (KIC) conversion to hyperpolarized [1-13 C]leucine by branched-chain amino acid aminotransferase (BCAT) has been imaged in the healthy rat brain [35], which could be informative with regards to the metabolism of glutamate, a key metabolite in astroglia, microglia, and neurons.
Hyperpolarized [1-13 C]glutamate and hyperpolarized [1-13 C]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 [1-13 C]glutamate has been shown to provide information on glutamate to glutamine conversion in the healthy rat brain, although it required BBB opening [36]. This probe is potentially useful for studying chronic excitotoxicity, a symptom associated with many neurodegenerative disorders [37], but probe administration may have adverse effects [38,39]. 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 [1-13 C]acetate in vivo resulted in observation of the TCA cycle intermediate α-ketoglutarate in the rat brain [40].
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 [1-13 C]dehydroascorbic acid (DHA) has been shown to cross the BBB and be converted to hyperpolarized [1-13 C]vitamin C alongside oxidation of glutathione (GSH). As such, the conversion of hyperpolarized [1-13 C] DHA to hyperpolarized [1-13 C]vitamin C could be used as a marker of oxidative stress [41,42]. In the rat brain, production of hyperpolarized [1-13 C]vitamin C from hyperpolarized [1-13 C]DHA was shown to be significantly reduced after treatment with diethyl maleate, a GSH-depleting agent [43]. Unfortunately, administration of this hyperpolarized probe causes respiratory and cardiac distress in animal models [44], and, thus, developments are required before this probe can progress to the clinic.
In looking to image the transient but vital oxidative status of the brain, hyperpolarized [1-13 C] acetoacetate must also be considered. Hyperpolarized [1-13 C]acetoacetate to hyperpolarized [1-13 C]beta-hydroxybutyrate (BHB) conversion is dependent on and, therefore, can be a biomarker for, the NAD + :NADH ratio, a proxy for the redox status of the cells. Detection of this conversion was recently shown to be feasible in the mouse brain [45]. Finally, conversion of hyperpolarized γ-glutamyl-[1-13 C]glycine to [1-13 C]glycine via γ-glutamyl-transferase (GGT) has been observed in healthy brain tissue [46]. GGT is responsible for maintaining cysteine levels for GSH production and, thus, changes in hyperpolarized γ-glutamyl-[1-13 C]glycine metabolism may be informative in times of oxidative stress and GSH depletion.
Anti-inflammatory macrophages, associated with repair functions, are characterized by their ability to upregulate arginase, which converts arginine into urea and ornithine, the latter being a substrate for the synthesis of polyamine and collagen. Hyperpolarized [6-13 C]arginine detected arginase activity in an in vitro study using primary mouse myeloid-derived suppressor cells (MDSCs) [47]. Hyperpolarized 13 C urea:arginine was significantly increased in MDSCs compared with control bone marrow cells. Although in vivo translation of hyperpolarized [6-13 C]arginine is challenged by a relatively short T 1 , new arginine preparation formulations and labeling strategies Trends in Neurosciences (e.g., [6-13 C, 15 N 3 ]arginine) have demonstrated improved arginine imaging, as exemplified in the rodent liver [48,49].
Translation to the Clinic: Hyperpolarized 13 C Pyruvate Studies and Clinical Trials Investigating Human Brain Metabolism The first-in-human study using hyperpolarized 13 C MRS/I, published in 2013, was conducted in patients with prostate cancer and proved the feasibility and safety of using hyperpolarized [1-13 C] pyruvate in the clinical setting [50]. A few years later, in 2018, the first hyperpolarized 13 C MRS/I studies in the human brain were performed in patients with glioma [51][52][53][54][55]. In addition to essential information on tumor metabolism (not discussed here [56]), these early studies provided the first hyperpolarized 13 C data on nontumor brain tissue. In this normal-appearing tissue, hyperpolarized [1-13 C]pyruvate was transported across the BBB and converted to the metabolic products hyperpolarized [1-13 C]lactate and hyperpolarized 13 C bicarbonate. Most studies agreed that hyperpolarized [1-13 C]pyruvate signal is highest in the vascular compartment, with the strongest signal intensity occurring in the superior sagittal sinus and transverse sinuses. Hyperpolarized [1-13 C]lactate production was observed throughout the brain [53,55], and appeared significantly higher in cortical/juxtacortical regions than in white matter tracts [51,53]. Hyperpolarized 13 C bicarbonate detection was more challenging [51], but showed higher signal in gray matter than in white matter [53]. Subsequent studies have focused on improving acquisition sequences [53], hardware configurations [54], and modeling of the kinetics of enzymatic pyruvate metabolism, to extract pseudo-rate constants for conversion of hyperpolarized [1-13 C]pyruvate to lactate (k PL ) and bicarbonate (k PB ) [55].
Given that all these studies were performed in patients with glioma who had undergone multiple treatments, one may wonder whether the normal-appearing brain tissue might exhibit an altered metabolism. However, interestingly, similar findings were recently shown in the brains of healthy volunteers. The first studies in human volunteers showed that hyperpolarized [1-13 C]pyruvate, lactate, and bicarbonate, and hyperpolarized 13 C lactate:pyruvate were significantly higher in gray matter compared with white matter. Furthermore, no significant difference was detected in k PL , k PB , or hyperpolarized 13 C bicarbonate:lactate between the two tissue types [57,58]. In an elegant study, in fact the largest study of hyperpolarized 13 C MRS in the healthy human brain thus far, with a sample size of 14 subjects, it was reported that, following injection of hyperpolarized [1-13 C]pyruvate [58], lactate topography of the brain was maintained across individuals, suggesting consistent region-specific lactate biology.
Feasibility of clinical injections of hyperpolarized [2-13 C]pyruvate has also been confirmed, showing the non-invasive detection of hyperpolarized [2-13 C]lactate and hyperpolarized [5-13 C]glutamate in the brains of healthy volunteers [59]. This detection of TCA-cycle-mediated glutamate production in the human brain mimics preclinical observations to some extent, although detection of other metabolites (e.g., citrate) was unconfirmed.
In a small but innovative ongoing early Phase I clinical trial (interventional, nonrandomized, NCT03502967) i , brain metabolism of both hyperpolarized [1-13 C] and hyperpolarized [2-13 C]pyruvate is being measured after TBI (N=5), for comparison to healthy controls (N=3). This study is an exciting first step into imaging neurological, non-oncological disease using hyperpolarized 13 C MRS/I, using the most established probes. It will complement ongoing preclinical studies, and hopefully highlight areas of improvement to ensure that future preclinical studies are as relevant as possible. Recent TBI research has highlighted the complex nuances of the disease, which cannot be observed by conventional imaging methods. Imaging metabolism using hyperpolarized [1-13 C]pyruvate in these cases may well be able to shine new light on early damage after injury.
In the wake of the TBI clinical trial, further applications will likely grow out of those other areas investigated preclinically, namely MS and cognitive impairment. In MS, in addition to wellestablished MRI protocols, hyperpolarized [1-13 C]pyruvate imaging could provide valuable data on metabolic alterations both in lesioned tissue and in normal-appearing brain matter. Given the potential of [1-13 C]pyruvate to detect inflammatory processes, this could represent a valuable tool to monitor the efficacy of immunomodulatory treatments in MS and help refine therapeutic regimens. In the field of dementia and AD, there have been some clinical trial disappointments following targeting of ß-amyloid, and the presence of inflammation in the brain in AD is increasingly being investigated [60,61]. Given the early preclinical data demonstrating an ability to visualize inflammation using hyperpolarized [1-13 C]pyruvate (discussed earlier), metabolic imaging could be an exciting new source of information for diagnosis and treatment monitoring once translated to the clinic.

Current Technical and Biological Challenges for Preclinical and Clinical Metabolic Imaging, and Ongoing Development of Solutions
Improvements in hyperpolarization coupled with development of metabolic probes with a longer relaxation time are essential if the potential of preclinical research (and clinical translation) is to be fully realized.
Technical advancements for increased polarization levels are underway [62] ii . These, while improving the data achieved using hyperpolarized [1-13 C]pyruvate, will also provide greater capacity for new probes. The rapid loss of hyperpolarization through T 1 relaxation following probe dissolution remains one of the largest challenges for the field. From a technical standpoint, dedicated rapid acquisition schemes are required to enable rapid data acquisition and to use the limited hyperpolarized signal as efficiently as possible. While most preclinical studies are performed at higher field strength (3-14.1 Tesla), hyperpolarized 13 C MRS/I does in fact benefit from lower field strength due to the increased relaxation times of most probes at lower field, showing optimal results on clinical MR systems (1.5-3 Tesla or even less) [5]. Following data acquisition, developments in data processing could also improve data quality, exemplified by the recent development of a denoising algorithm, providing a possible increase in signal:noise ratios of about an order of magnitude [63].
In terms of biological interpretation, one major area of discussion, briefly mentioned earlier, is to what extent hyperpolarized [1-13 C]pyruvate crosses the BBB within the time-frame of the hyperpolarized experiment [64]. Alternatives, such as a more lipophilic version of pyruvate, have been explored to increased BBB crossing [31]. Use of probes other than pyruvate may also be limited in their crossing of the BBB, such as lactate [34]. In combination with all the metabolic probes in development, co-injection with metabolically inert hyperpolarized probes, such as [ 13 C]urea [14], [ 13 C]hydroxymethyl cyclopropane, or [ 13 C]t-butanol [65], can ensure that data are not skewed by changes in perfusion. To enable investigation of brain cellular metabolism without, for example, the concerns over BBB transport, live excised rat brain slices can be studied using hyperpolarized 13 C MRS in a bioreactor system to maintain cellular viability [66]. Once issues with tissue slice motion during hyperpolarized injection are resolved, this technique may prove useful in future studies of the diseased brain.
Variations in the amount of metabolic product seen may be anesthesia [67,68], species [69], or strain [34] dependent. Most preclinical hyperpolarized 13 C MRS/I studies have been performed with the animals under anesthesia. Metabolic data were successfully acquired from the liver following injection of hyperpolarized [6-13 C, 15 N 3 ]arginine in an awake, restrained mouse, in response to reported toxicity of this probe in isoflurane-anesthetized mice [48]. No such toxicity was seen in the awake animal. In preclinical studies, imaging awake animals requires extensive training of the animals, and it would be necessary to ensure that stress does not interfere with the study goals. However, if testing in awake animals could become the standard protocol, it would be valuable in eliminating the confounder of type and depth of anesthetic, and enhance the relevance to human studies.
One particularly interesting question is to what extent different cell types contribute to the observed in vivo signal, for example dominance of signal emanating from neurons, microglia, or astrocytes. Future work can begin to address this question, exploiting the flexibility of the hyperpolarized technique to use cell populations both in slurries [70] and bioreactor systems [71]. Developments in these systems, such as the use of micromagnetic resonance spectrometers [72] or co-culturing of multiple cell populations, will help decipher weights of contribution. Additionally, ex vivo biochemical analyses, the use of appropriate knockout/knock-in mouse models, and specific metabolic pathway inhibitors could also help address these questions [21].
Clinically, there are also both technical and biological considerations to overcome before the hyperpolarized 13 C MRS/I technology becomes standard clinical practice in neurological disorders, as discussed earlier. The clinical SpinLab polarizers are available at some cost, and dedicated specific pulse sequences are required. As with any new injectable, approval of an investigational new drug application (IND) from the US FDA (or equivalent) is required, and concentrations of new probes have to be tested for toxicity and good-quality data acquisition. It would be most valuable if data are comparable across clinical sites, although this is no small task given that standardization of data acquisition and processing will be needed. A full analysis of healthy brain metabolism using several hyperpolarized probes across multiple countries and, therefore, multiple volunteer populations would be optimal, to provide a diverse baseline dataset against which to assess disease states.
A detailed understanding of the biological meaning of clinical hyperpolarized data would be valuable, but is challenging given the limited ability to biopsy the imaged human tissue. Despite this, use of hyperpolarized pyruvate remains clinically valuable if it can provide distinctions between healthy, diseased, and treated brain tissue. In the short term, hyperpolarized [1-13 C]pyruvate can feasibly be used in clinical studies of neurological disorders because it is patient safe, and the hardware and data analysis are in place for brain studies in an increasing number of locations.

Concluding Remarks and Future Perspectives
Hyperpolarized [1-13 C]pyruvate has been recently applied, and shown to provide informative data, in multiple rodent models of neurological diseases and neuroinflammation. Furthermore, there are many hyperpolarized probes for in vivo enzymatic assessment of several metabolic pathways that have been developed and applied in the healthy brain. Thus, moving to models of neurological disease is a logical and feasible next step for such probes. Although challenges exist for hyperpolarized technology in the field of brain diseases (see Outstanding Questions), clinical translation is underway in patients with TBI and healthy volunteers and will likely follow in other patient populations in the coming years. Thus, it seems appropriate at this point to focus resources on improving hardware, acquisition capacity, processing pipelines, and hyperpolarized probes; efforts that will hopefully allow investigation of multiple metabolic pathways within the human brain in health and disease. An optimized methodology could improve the longitudinal monitoring of response to therapy. In combination with other imaging modalities, the hyperpolarized 13 C technology provides an exciting opportunity to enhance our understanding of in vivo brain metabolism, and in the future, to impact clinical care in a range of neurological disorders.

Outstanding Questions
Hyperpolarized 13 C MRS/I of [1-13 C] pyruvate can detect metabolic impairment in several preclinical models of neurological disorders. Can this metabolic imaging technique improve the diagnosis and evaluation of prognosis in patients with brain diseases?
Modulations in immune cell metabolism have been detected by hyperpolarized 13 C MRS/I of [1-13 C]pyruvate in ex vivo and in vivo preclinical models. Can this method be used to detect neuroinflammation in patients?
Can more sensitive acquisition and processing pipelines be developed to enable higher MR signal and better spatial resolution?
Can we establish a metabolic baseline dataset for healthy brain that is consistent across sites for disease state comparison?
Initial preclinical studies showed that hyperpolarized [1-13 C]pyruvate to [1-13 C]lactate conversion is sensitive to metabolic response to therapy in several models of neurological diseases. Can hyperpolarized 13 C MRS/I be used to monitor drug efficacy and response to therapy in neurological disorders clinically, and are changes in these parameters observed before changes in typical patient symptoms?
Several cerebral metabolic pathways have been shown to be accessible with hyperpolarized probes. Can hyperpolarized 13 C MRS/I be used to obtain a fuller picture of metabolic changes in neurological disorders using multiple, translatable hyperpolarized probes?