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Background In recent years, the ability of conventional magnetic resonance imaging (MRI), including T1 contrast-enhanced (CE) MRI, to monitor high-efficacy therapies and predict long-term disability in multiple sclerosis (MS) has been challenged. Therefore, non-invasive methods to improve MS lesions detection and monitor therapy response are needed. Methods We studied the combined cuprizone and experimental autoimmune encephalomyelitis (CPZ-EAE) mouse model of MS, which presents inflammatory-mediated demyelinated lesions in the central nervous system as commonly seen in MS patients. Using hyperpolarized 13C MR spectroscopy (MRS) metabolic imaging, we measured cerebral metabolic fluxes in control, CPZ-EAE and CPZ-EAE mice treated with two clinically-relevant therapies, namely fingolimod and dimethyl fumarate. We also acquired conventional T1 CE MRI to detect active lesions, and performed ex vivo measurements of enzyme activities and immunofluorescence analyses of brain tissue. Last, we evaluated associations between imaging and ex vivo parameters. Results We show that hyperpolarized [1-13C]pyruvate conversion to lactate is increased in the brain of untreated CPZ-EAE mice when compared to the control, reflecting immune cell activation. We further demonstrate that this metabolic conversion is significantly decreased in response to the two treatments. This reduction can be explained by increased pyruvate dehydrogenase activity and a decrease in immune cells. Importantly, we show that hyperpolarized 13C MRS detects dimethyl fumarate therapy, whereas conventional T1 CE MRI cannot. Conclusions In conclusion, hyperpolarized MRS metabolic imaging of [1-13C]pyruvate detects immunological responses to disease-modifying therapies in MS. This technique is complementary to conventional MRI and provides unique information on neuroinflammation and its modulation.

Traumatic brain injury (TBI) is a major public health problem. Despite considerable research deciphering injury pathophysiology, precision therapies remain elusive. Here, we present large-scale data sharing and machine intelligence approaches to leverage TBI complexity. The Open Data Commons for TBI (ODC-TBI) is a community-centered repository emphasizing Findable, Accessible, Interoperable, and Reusable data sharing and publication with persistent identifiers. Importantly, the ODC-TBI implements data sharing of individual subject data, enabling pooling for high-sample-size, feature-rich data sets for machine learning analytics. We demonstrate pooled ODC-TBI data analyses, starting with descriptive analytics of subject-level data from 11 previously published articles (N = 1250 subjects) representing six distinct pre-clinical TBI models. Second, we perform unsupervised machine learning on multi-cohort data to identify persistent inflammatory patterns across different studies, improving experimental sensitivity for pro- versus anti-inflammation effects. As funders and journals increasingly mandate open data practices, ODC-TBI will create new scientific opportunities for researchers and facilitate multi-data-set, multi-dimensional analytics toward effective translation.

Table S1. GS-2 orthotopic tumor model: in vivo single voxel 1H MRS; Table S2. GS-2 orthotopic tumor model: ex vivo 1H HR-MAS MR Spectroscopy; Table S3. U87-MG orthotopic tumor model: ex vivo 1H HR-MAS MR Spectroscopy.

PDF file - 3201K, Supplementary Figure S1. Real time RT PCR analysis of BRGs compared to paired pre-treatment GBMs; Supplementary Figure S2. FISH reveals no change in c-Met amplification after bevacizumab resistance as compared to before; Supplementary Figure S3. Changes in Pearson correlation coefficients to 3 previously described molecular GBM subtypes in BRGs and bevacizumab-na�ve GBMs after recurrence; Supplementary Figure S4. C-Met phosphorylation increases after bevacizumab resistance; Supplementary Figure S5. Increased expression of phosphorylated FAK, a downstream effector of c-Met, in bevacizumab-resistant glioblastomas (BRGs) compared to before treatment; Supplementary Figure S6. Increased expression of phosphorylated STAT3, a downstream effector of c-Met, in bevacizumab-resistant glioblastomas (BRGs) compared to before treatment; Supplementary Figure S7. Hypoxia after bevacizumab resistance versus bevacizumab-na�ve recurrence; Supplementary Figure S8. Endothelial cells do not contribute to increased c-Met expression after bevacizumab resistance; Supplementary Figure S9. Hypoxia increases HGF expression in U87-IgG and U87-BevR cells; Supplementary Figure S10. Xenografts established from BRGs analyzed by immunohistochemistry; Supplementary Figure S11. Immunofluorescent staining of xenografts established from bevacizumab-na�ve GBMs; Supplementary Figure S12. Hematoxylin and Eosin staining of xenografts established from bevacizumab-na�ve GBMs; Supplementary Figure S13. Comparison of xenografts from BRGs versus xenografts from bevacizumab-na�ve GBMs; Supplementary Figure S14. Murine MRI of intracranial BRG-derived xenografts reveals loss of the ability of VEGF blockade to reduce vascular permeability and fractional blood volume; Supplementary Figure S15. Reduced c-Met expression after transducing BRG-derived SF7796 xenograft cells with c-Met shRNA; Supplementary Figure S16. Effects of c-Met knockdown on brain slice invasion in U87-BevR cells; Supplementary Figure S17. Altered morphology in BRG cells after transduction with shRNA targeting c-Met; Supplementary Figure S18. C-Met knockdown alters brain slice invasion in BRG-derived SF7796 cells; Supplementary Figure S19. Hematoxylin and eosin stainings of intracranial SF7796/shControl and SF7796/shCmet1 xenografts; Supplementary Figure S20. Transduction of U87 with retrovirus derived from pBABE-Puro-Tpr-met leads to increased Tpr-met phosphorylation.

Purpose: To identify mediators of glioblastoma antiangiogenic therapy resistance and target these mediators in xenografts.Experimental Design: We conducted microarray analysis comparing bevacizumab-resistant glioblastomas (BRG) with pretreatment tumors from the same patients. We established novel xenograft models of antiangiogenic therapy resistance to target candidate resistance mediator(s).Results: BRG microarray analysis revealed upregulation versus pretreatment of receptor tyrosine kinase c-Met, which underwent further investigation because of its prior biologic plausibility as a bevacizumab resistance mediator. BRGs exhibited increased hypoxia versus pretreatment in a manner correlating with their c-Met upregulation, increased c-Met phosphorylation, and increased phosphorylation of c-Met–activated focal adhesion kinase and STAT3. We developed 2 novel xenograft models of antiangiogenic therapy resistance. In the first model, serial bevacizumab treatment of an initially responsive xenograft generated a xenograft with acquired bevacizumab resistance, which exhibited upregulated c-Met expression versus pretreatment. In the second model, a BRG-derived xenograft maintained refractoriness to the MRI tumor vasculature alterations and survival-promoting effects of bevacizumab. Growth of this BRG-derived xenograft was inhibited by a c-Met inhibitor. Transducing these xenograft cells with c-Met short hairpin RNA inhibited their invasion and survival in hypoxia, disrupted their mesenchymal morphology, and converted them from bevacizumab-resistant to bevacizumab-responsive. Engineering bevacizumab-responsive cells to express constitutively active c-Met caused these cells to form bevacizumab-resistant xenografts.Conclusion: These findings support the role of c-Met in survival in hypoxia and invasion, features associated with antiangiogenic therapy resistance, and growth and therapeutic resistance of xenografts resistant to antiangiogenic therapy. Therapeutically targeting c-Met could prevent or overcome antiangiogenic therapy resistance. Clin Cancer Res; 19(7); 1773–83. ©2012 AACR.

PDF file - 1353K, Supplementary Table S1. Patient and tumor characteristics from the 22 bevacizumab-resistant versus 22 bevacizumab-na�ve glioblastomas from whom paired pre-treatment tissue was available. Supplementary Table S2. Altered transcripts in 15 bevacizumab-resistant glioblastomas compared to before treatment. Supplementary Table S3. Altered transcripts in 15 bevacizumab-resistant glioblastomas compared to before treatment. Supplementary Table S4. Altered transcripts in bevacizumab-naive recurrent glioblastomas compared to their paired glioblastomas at diagnosis. Supplemetary Table S5. FISH Results. Supplementary Table S6. Patient and tumor characteristics from 22 bevacizumab-na�ve recurrent glioblastomas (control group) from whom paired pre-treatment tissue was available.

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Effect of 2-HG on cell proliferation of IDH1 wild-type cells for the U87 (A) and NHA (B) models.

SuppFigure3. Stable suppression of PKM2 expression in G55 cells significantly alters intra-cellular concentrations of pyruvate and lactate. (*, p

Comparison of PDH activity in the various models used in this study.

Neurovascular coupling (NVC), a vital physiological process that rapidly and precisely directs localized blood flow to the most active regions of the brain, is accomplished in part by the vast network of cerebral capillaries acting as a sensory web capable of detecting increases in neuronal activity and orchestrating the dilation of upstream parenchymal arterioles. Here, we report aCol4a1mutant mouse model of cerebral small vessel disease (cSVD) with age-dependent defects in capillary-to-arteriole dilation, functional hyperemia in the brain, and memory. The fundamental defect in aged mutant animals was the depletion of the minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) in brain capillary endothelial cells, leading to the loss of inwardly rectifier K+(Kir2.1) channel activity. Blocking phosphatidylinositol-3-kinase (PI3K), an enzyme that diminishes the bioavailability of PIP2by converting it to phosphatidylinositol (3,4,5)-trisphosphate (PIP3), restored Kir2.1 channel activity, capillary-to-arteriole dilation, and functional hyperemia. In longitudinal studies, chronic PI3K inhibition also improved the memory function of agedCol4a1mutant mice. Our data suggest that PI3K inhibition is a viable therapeutic strategy for treating defective NVC and cognitive impairment associated with cSVD.One-sentence summaryPI3K inhibition rescues neurovascular coupling defects in cerebral small vessel disease.

Humans and mice with mutations inCOL4A1andCOL4A2manifest hallmarks of cerebral small vessel disease (cSVD), but the pathogenic mechanisms are largely unknown. Mice with a missense mutation inCol4a1at amino acid 1344 (Col4a1+/G1344D) exhibited age-dependent intracerebral hemorrhage (ICH) and brain lesions. Here we report that this pathology was associated with the loss of myogenic vasoconstriction, an intrinsic vascular response essential for the autoregulation of cerebral blood flow. Electrophysiological analyses showed that the loss of myogenic constriction resulted from blunted pressure-induced smooth muscle cell (SMC) membrane depolarization. Further, we found that dysregulation of membrane potential was associated with impaired Ca2+-dependent activation of large-conductance Ca2+-activated K+(BK) and transient receptor potential melastatin 4 (TRPM4) cation channels linked to disruptions in sarcoplasmic reticulum (SR) Ca2+signaling. TreatingCol4a1+/G1344Dmice with 4-phenylbutyrate, a compound that promotes the trafficking of misfolded proteins and alleviates SR stress, restored SR Ca2+signaling, BK and TRPM4 channel activity, prevented loss of myogenic tone, and reduced ICH. We conclude that alterations in SR Ca2+handling that impair membrane potential regulating ion channel activity result in dysregulation of SMC membrane potential and loss of myogenic tone contributing to age-related cSVD inCol4a1+/G1344Dmice.Graphical Abstract

Lymphocytes and innate immune cells are key drivers of multiple sclerosis (MS) and are the main target of MS disease modifying therapies (DMT). Ex vivo analyses of MS lesions have revealed cellular heterogeneity and variable T-cell levels, which may have important implication for patient stratification and choice of DMT. Although magnetic resonance imaging (MRI) has proven valuable to monitor DMT efficacy, its lack of specificity for cellular subtypes highlights the need for complementary methods to improve lesion characterization. Here, we evaluated the potential of 2’-deoxy-2’-[18F]fluoro-9-β-D-arabinofuranosylguanine (18F-FAraG) PET imaging to non-invasively assess infiltrating T-cells and to provide, in combination with MRI, a novel tool to determine lesion types. Methods: We used a novel MS mouse model that combines cuprizone and experimental autoimmune encephalomyelitis (EAE) to reproducibly induce two brain inflammatory lesion types, differentiated by their T-cell content. 18F-FAraG PET imaging, T2-weighted MRI, and T1-weighted contrast-enhanced MRI were performed prior to disease induction, during demyelination with high levels of innate immune cells, and following T-cell infiltration. Fingolimod immunotherapy was used to evaluate the ability of PET and MRI to detect therapy response. Ex vivo immunofluorescence analyses for T-cells, microglia/macrophages, myelin and blood brain barrier (BBB) integrity were performed to validate the in vivo findings. Results:18F-FAraG signal was significantly increased in brain and spinal cord at the time point of T-cell infiltration. 18F-FAraG signal from white matter (corpus callosum), and grey matter (cortex, hippocampus) further correlated with T-cell density. T2-weighted MRI detected white matter lesions independently of T-cells. T1-weighted contrast-enhanced MRI indicated BBB disruption at the time point of T-cell infiltration. Fingolimod treatment prevented motor deficits and decreased T-cell and microglia/macrophage levels. In agreement, 18F-FAraG signal was decreased in brain and spinal cord of Fingolimod-treated mice, T1-weighted contrast-enhanced MRI revealed intact BBB, while T2-weighted MRI remained unchanged. Conclusion: The combination of MRI and 18F-FAraG PET enables detection of inflammatory demyelination and T-cell infiltration in a MS mouse model, providing a new way to evaluate lesion heterogeneity during disease progression and following DMT. Upon clinical translation, these methods hold great potential for patient stratification, monitoring MS progression and therapy responses.

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.

Glutamine synthetase (GS) is a key enzyme that metabolizes glutamate into glutamine. While GS is highly enriched in astrocytes, expression in other glial lineages has been noted. Using a combination of reporter mice and cell type-specific markers, we show that GS is expressed in myelinating oligodendrocytes (OL) but not oligodendrocyte progenitor cells of the mouse and human ventral spinal cord. To investigate the role of GS in mature OL, we used a conditional knockout (cKO) approach to selectively delete GS-encoding gene (Glul) in OL, which caused a significant decrease in glutamine levels on mouse spinal cord extracts. GS cKO mice (CNP-cre+ :Glulfl/fl ) showed no differences in motor neuron numbers, size or axon density; OL differentiation and myelination in the ventral spinal cord was normal up to 6months of age. Interestingly, GS cKO mice showed a transient and specific decrease in peak force while locomotion and motor coordination remained unaffected. Last, GS expression in OL was increased in chronic pathological conditions in both mouse and humans. We found a disease-stage dependent increase of OL expressing GS in the ventral spinal cord of SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Moreover, we showed that GLUL transcripts levels were increased in OL in leukocortical tissue from multiple sclerosis but not control patients. These findings provide evidence towards OL-encoded GS function in spinal cord sensorimotor axis, which is dysregulated in chronic neurological diseases.

Macrophage activation, first generalized to the M1/M2 dichotomy, is a complex and central process of the innate immune response. Simply, M1 describes the classical proinflammatory activation, leading to tissue damage, and M2 the alternative activation promoting tissue repair. Given the central role of macrophages in multiple diseases, the ability to noninvasively differentiate between M1 and M2 activation states would be highly valuable for monitoring disease progression and therapeutic responses. Since M1/M2 activation patterns are associated with differential metabolic reprogramming, we hypothesized that hyperpolarized 13C magnetic resonance spectroscopy (HP 13C MRS), an innovative metabolic imaging approach, could distinguish between macrophage activation states noninvasively. The metabolic conversions of HP [1-13C]pyruvate to HP [1-13C]lactate, and HP [1-13C]dehydroascorbic acid to HP [1-13C]ascorbic acid were monitored in live M1 and M2 activated J774a.1 macrophages noninvasively by HP 13C MRS on a 1.47 Tesla NMR system. Our results show that both metabolic conversions were significantly increased in M1 macrophages compared to M2 and nonactivated cells. Biochemical assays and high resolution 1H MRS were also performed to investigate the underlying changes in enzymatic activities and metabolite levels linked to M1/M2 activation. Altogether, our results demonstrate the potential of HP 13C MRS for monitoring macrophage activation states noninvasively.

Traumatic brain injury (TBI) is a major unsolved public health problem worldwide with considerable preclinical research dedicated to recapitulating clinical TBI, deciphering the underlying pathophysiology, and developing therapeutics. However, the heterogeneity of clinical TBI and correspondingly in preclinical studies have made translation from bench to bedside difficult. Here, we present the potential of data sharing, data aggregation, and multivariate analytics to integrate heterogeneity and empower researchers. We introduce the Open Data Commons for Traumatic Brain Injury (ODC-TBI.org) as a user-centered web platform and cloudbased repository focused on preclinical TBI research that enables data citation with persistent identifiers, promotes data element harmonization, and follows FAIR data sharing principles. Importantly, the ODC-TBI implements data sharing at the level of individual subjects, thus enabling data reuse for granular big data analytics and data-hungry machine learning approaches. We provide use cases applying descriptive analytics and unsupervised machine learning on pooled ODC-TBI data. Descriptive statistics included subject-level data for 11 published papers (N = 1250 subjects) representing six distinct TBI models across mice and rats (implementing controlled cortical impact, closed head injury, fluid percussion injury, and CHIMERA TBI modalities). We performed principal component analysis (PCA) on cohorts of animals combined through the ODC-TBI to identify persistent inflammatory patterns across different experimental designs. Our workflow ultimately improved the sensitivity of our analyses in uncovering patterns of pro- vs anti-inflammation and oxidative stress without the multiple testing problems of univariate analyses. As the practice of open data becomes increasingly required by the scientific community, ODC-TBI provides a foundation that creates new scientific opportunities for researchers and their work, facilitates multi-dataset and multidimensional analytics, and drives collaboration across molecular and computational biologists to bridge preclinical research to the clinic.

Macrophage activation, first generalized to the M1/M2 dichotomy, is a complex and central process of the innate immune response. Simply, M1 describes the classical pro-inflammatory activation, leading to tissue damage, and M2 the alternative activation promoting tissue repair. Given the central role of macrophages in multiple diseases, the ability to non-invasively differentiate between M1 and M2 activation states would be highly valuable for monitoring disease progression and therapeutic responses. Since M1/M2 activation patterns are associated with differential metabolic reprogramming, we hypothesized that hyperpolarized 13C magnetic resonance spectroscopy (HP 13C MRS), an innovative metabolic imaging approach, could distinguish between macrophage activation states noninvasively. The metabolic conversions of HP [1-13C]pyruvate to HP [1-13C]lactate and HP [1-13C]dehydroascorbic acid to HP [1-13C]ascorbic acid were monitored in live M1 and M2 activated J774a.1 macrophages non-invasively by HP 13C MRS on a 1.47 Tesla NMR system. Our results show that both metabolic conversions were significantly increased in M1 macrophages compared to M2 and non-activated cells. Biochemical assays and high resolution 1H MRS were also performed to investigate the underlying changes in enzymatic activities and metabolite levels linked to M1/M2 activation. Altogether, our results demonstrate the potential of HP 13C MRS for monitoring macrophage activation states non-invasively.

Lymphocytes and innate immune cells are key drivers of multiple sclerosis (MS) and are the main target of MS disease-modifying therapies (DMT). Ex vivo analyses of MS lesions have revealed cellular heterogeneity and variable T cell levels, which may have important implications for patient stratification and choice of DMT. Although MRI has proven valuable to monitor DMT efficacy, its lack of specificity for cellular subtypes highlights the need for complementary methods to improve lesion characterization. Here, we evaluated the potential of 2'-deoxy-2'

Complex alterations in cerebral energetic metabolism arise after traumatic brain injury (TBI). To date, methods allowing for metabolic evaluation are highly invasive, limiting our understanding of metabolic impairments associated with TBI pathogenesis. We investigated whether 13C MRSI of hyperpolarized (HP) [1-13C] pyruvate, a non-invasive metabolic imaging method, could detect metabolic changes in controlled cortical injury (CCI) mice (n = 57). Our results show that HP [1-13C] lactate-to-pyruvate ratios were increased in the injured cortex at acute (12/24 hours) and sub-acute (7 days) time points after injury, in line with decreased pyruvate dehydrogenase (PDH) activity, suggesting impairment of the oxidative phosphorylation pathway. We then used the colony-stimulating factor-1 receptor inhibitor PLX5622 to deplete brain resident microglia prior to and after CCI, in order to confirm that modulations of HP [1-13C] lactate-to-pyruvate ratios were linked to microglial activation. Despite CCI, the HP [1-13C] lactate-to-pyruvate ratio at the injury cortex of microglia-depleted animals at 7 days post-injury remained unchanged compared to contralateral hemisphere, and PDH activity was not affected. Altogether, our results demonstrate that HP [1-13C] pyruvate has great potential for in vivo non-invasive detection of cerebral metabolism post-TBI, providing a new tool to monitor the effect of therapies targeting microglia/macrophages activation after TBI.

Alterations in myelin integrity are involved in many neurological disorders and demyelinating diseases, such as multiple sclerosis (MS). Although magnetic resonance imaging (MRI) is the gold standard method to diagnose and monitor MS patients, clinically available MRI protocols show limited specificity for myelin detection, notably in cerebral grey matter areas. Ultrashort echo time (UTE) MRI has shown great promise for direct imaging of lipids and myelin sheaths, and thus holds potential to improve lesion detection. In this study, we used a sequence combining magnetization transfer (MT) with UTE ("UTE-MT", TE​=​76​μs) and with short TE ("STE-MT", TE​=​3000​μs) to evaluate spatial and temporal changes in brain myelin content in the cuprizone mouse model for MS on a clinical 7​T scanner. During demyelination, UTE-MT ratio (UTE-MTR) and STE-MT ratio (STE-MTR) values were significantly decreased in most white matter and grey matter regions. However, only UTE-MTR detected cortical changes. After remyelination in subcortical and cortical areas, UTE-MTR values remained lower than baseline values, indicating that UTE-MT, but not STE-MT, imaging detected long-lasting changes following a demyelinating event. Next, we evaluated the potential correlations between imaging values and underlying histopathological markers. The strongest correlation was observed between UTE-MTR and percent coverage of myelin basic protein (MBP) immunostaining (r2​=​0.71). A significant, although lower, correlation was observed between STE-MTR and MBP (r2​=​0.48), and no correlation was found between UTE-MTR or STE-MTR and gliosis immunostaining. Interestingly, correlations varied across brain substructures. Altogether, our results demonstrate that UTE-MTR values significantly correlate with myelin content as measured by histopathology, not only in white matter, but also in subcortical and cortical grey matter regions in the cuprizone mouse model for MS. Readily implemented on a clinical 7​T system, this approach thus holds great potential for detecting demyelinating/remyelinating events in both white and grey matter areas in humans. When applied to patients with neurological disorders, including MS patient populations, UTE-MT methods may improve the non-invasive longitudinal monitoring of brain lesions, not only during disease progression but also in response to next generation remyelinating therapies.

Lipopolysaccharide (LPS) is a commonly used agent for induction of neuroinflammation in preclinical studies. Upon injection, LPS causes activation of microglia and astrocytes, whose metabolism alters to favor glycolysis. Assessing in vivo neuroinflammation and its modulation following therapy remains challenging, and new noninvasive methods allowing for longitudinal monitoring would be highly valuable. Hyperpolarized (HP) C-13 magnetic resonance spectroscopy (MRS) is a promising technique for assessing in vivo metabolism. In addition to applications in oncology, the most commonly used probe of [1-C-13] pyruvate has shown potential in assessing neuroinflammation-linked metabolism in mouse models of multiple sclerosis and traumatic brain injury. Here, we aimed to investigate LPS-induced neuroinflammatory changes using HP [1-C-13] pyruvate and HP C-13 urea. 2D chemical shift imaging following simultaneous intravenous injection of HP [1-C-13] pyruvate and HP C-13 urea was performed at baseline (day 0) and at days 3 and 7 post-intracranial injection of LPS (n = 6) or saline (n = 5). Immunofluorescence (IF) analyses were performed for Iba1 (resting and activated microglia/macrophages), GFAP (resting and reactive astrocytes) and CD68 (activated microglia/macrophages). A significant increase in HP [1-C-13] lactate production was observed at days 3 and 7 following injection, in the injected (ipsilateral) side of the LPS-treated mouse brain, but not in either the contralateral side or saline-injected animals. HP C-13 lactate/pyruvate ratio, without and with normalization to urea, was also significantly increased in the ipsilateral LPS-injected brain at 7 days compared with baseline. IF analyses showed a significant increase in CD68 and GFAP staining at 3 days, followed by increased numbers of Iba1 and GFAP positive cells at 7 days post-LPS injection. In conclusion, we can detect LPS-induced changes in the mouse brain using HP C-13 MRS, in alignment with increased numbers of microglia/macrophages and astrocytes. This study demonstrates that HP C-13 spectroscopy has substantial potential for providing noninvasive information on neuroinflammation.

We developed methods for the preparation of hyperpolarized (HP) sterile [2-(13)C]pyruvate to test its feasibility in first-ever human NMR studies following FDA-IND & IRB approval. Spectral results using this MR stable-isotope imaging approach demonstrated the feasibility of investigating human cerebral energy metabolism by measuring the dynamic conversion of HP [2-(13)C]pyruvate to [2-(13)C]lactate and [5-(13)C]glutamate in the brain of four healthy volunteers. Metabolite kinetics, signal-to-noise (SNR) and area-undercurve (AUC) ratios, and calculated [2-(13)C]pyruvate to [2-(13)C]lactate conversion rates (k(PL)) were measured and showed similar but not identical inter-subject values. The k(PL) measurements were equivalent with prior human HP [1-(13)C]pyruvate measurements.

Lipopolysaccharide (LPS) is a commonly used agent for induction of neuroinflammation in preclinical studies. Upon injection, LPS causes activation of microglia and astrocytes, whose metabolism alters to favor glycolysis. Assessingin vivoneuroinflammation and its modulation following therapy remains challenging, and new non-invasive methods allowing for longitudinal monitoring would be greatly valuable. Hyperpolarized (HP)13C magnetic resonance spectroscopy (MRS) is a promising technique for assessingin vivometabolism. In addition to applications in oncology, the most commonly used probe of [1-13C] pyruvate has shown potential in assessing neuroinflammation-linked metabolism in mouse models of multiple sclerosis and traumatic brain injury. Here, we wished to investigate LPS-induced neuroinflammatory changes using HP [1-13C] pyruvate and HP13C urea.2D chemical shift imaging following simultaneous intravenous injection of HP [1-13C] pyruvate and HP13C urea was performed at baseline (day 0), day 3 and day 7 post intracranial injection of LPS (n=6) or saline (n=5). Immunofluorescence (IF) analyses were performed for Iba1 (resting and activated microglia/macrophages), GFAP (resting and reactive astrocytes) and CD68 (activated microglia/macrophages).A significant increase in HP [1-13C] lactate production was observed in the injected (ipsilateral) side at 3 and 7 days of the LPS-treated mouse brain, but not in either the contralateral side or saline-injected animals. HP13C lactate/pyruvate ratio, without and with normalization to urea, was also significantly increased in the ipsilateral LPS-injected brain at 7 days compared to baseline. IF analyses showed a significant increase in CD68 and GFAP at 3 days, followed by increased numbers of Iba1 and GFAP positive cells at 7 days post-LPS injection.In conclusion, we can detect LPS-induced changes in the mouse brain using HP13C MRS, in alignment with increased numbers of microglia/macrophages and astrocytes. This study demonstrates that HP13C spectroscopy holds much potential for providing non-invasive information on neuroinflammation.

Traumatic brain injury (TBI) is of particular concern for the aging community since there is both increased incidence of TBI and decreased functional recovery in this population. In addition, TBI is the strongest environmental risk factor for development of Alzheimer&rsquo, s disease and other dementia-related neurodegenerative disorders. Critical changes that affect cognition take place over time following the initial insult. Our previous work identified immune system activation as a key contributor to cognitive deficits observed in aged animals. Using a focal contusion model in the current study, we demonstrate a brain lesion and cavitation formation, as well as prolonged blood&ndash, brain barrier breakdown. These changes were associated with a prolonged inflammatory response, characterized by increased microglial cell number and phagocytic activity 30 days post injury, corresponding to significant memory deficits. We next aimed to identify the injury-induced cellular and molecular changes that lead to chronic cognitive deficits in aged animals, and measured increases in complement initiation components C1q, C3, and CR3, which are known to regulate microglial&ndash, synapse interactions. Specifically, we found significant accumulation of C1q on synapses within the hippocampus, which was paralleled by synapse loss 30 days post injury. We used genetic and pharmacological approaches to determine the mechanistic role of complement initiation on cognitive loss in aging animals after TBI. Notably, both genetic and pharmacological blockade of the complement pathway prevented memory deficits in aged injured animals. Thus, therapeutically targeting early components of the complement cascade represents a significant avenue for possible clinical intervention following TBI in the aging population.

Traumatic brain injury (TBI) has long been identified as a precipitating risk factor for higher-order cognitive deficits associated with the frontal and prefrontal cortices (PFC). In addition, mild repetitive TBI (rTBI), in particular, is being steadily recognized to increase the risk of neurodegenerative disease. Thus, further understanding of how mild rTBI changes the pathophysiology of the brain to lead to cognitive impairment is warranted. The current models of rTBI lack knowledge regarding chronic higher-order cognitive functions and the underlying neuronal physiology, especially functions involving the PFC. Here, we establish that five repeated mild hits, allowing rotational acceleration of the head, lead to chronic deficits in PFC-dependent functions such as social behavior, spatial working memory, and environmental response with concomitant microgliosis and a small decrease in the adaptation rate of layer V pyramidal neurons in the medial PFC (mPFC). However, structural damage is not seen on in vivo T2-weighted magnetic resonance imaging (MRI), and extensive intrinsic excitability changes in layer V pyramidal neurons of the mPFC are not observed. Thus, this rTBI animal model can recapitulate chronic higher-order cognitive impairments without structural damage on MR imaging as observed in humans.

Vorinostat is a histone deacetylase (HDAC) inhibitor that inhibits cell proliferation and induces apoptosis in solid tumors and is in clinical trials for the treatment of glioblastoma (GBM). The goal of this study was to assess whether hyperpolarized (13)C Magnetic Resonance Spectroscopy (MRS) and Magnetic Resonance Spectroscopic Imaging (MRSI) can detect HDAC inhibition in GBM models. First, we confirmed HDAC inhibition in U87 GBM cells and evaluated real-time dynamic metabolic changes using a bioreactor system with live Vorinostat-treated or control cells. We found a significant 40% decrease in the (13)C MRS-detectable ratio of hyperpolarized [1-(13)C]lactate to hyperpolarized [1-(13)C]pyruvate, [1-(13)C]Lac/Pyr, and a 37% decrease in the pseudo-rate constant, k(PL), for hyperpolarized [1-(13)C]lactate production, in Vorinostat-treated cells compared to controls. To understand the underlying mechanism for this finding, we assessed the expression and activity of lactate dehydrogenase (LDH), which catalyzes the pyruvate to lactate conversion, its associated cofactor nicotinamide adenine dinucleotide (NADH), the expression of monocarboxylate transporters (MCTs) MCT1 and MCT4, that shuttle pyruvate and lactate in and out of the cell, and intracellular lactate levels. We found that the most likely explanation for our finding that hyperpolarized lactate is reduced in treated cells, is a 30% reduction in intracellular lactate levels that occurs as a result of increased expression of both MCT1 and MCT4 in Vorinostat-treated cells. In vivo (13)C MRSI studies of orthotopic tumors in mice also showed a significant 52% decrease in hyperpolarized [1-(13)C]Lac/Pyr when comparing Vorinostat-treated U87 GBM tumors to controls, and, as in the cell studies, this metabolic finding was associated with increased MCT1 and MCT4 expression in HDAC-inhibited tumors. Thus, the (13)C MRSI-detectable decrease in hyperpolarized [1-(13)C]lactate production could serve as a biomarker of response to HDAC inhibitors.

Metabolic imaging of brain tumors using 13C Magnetic Resonance Spectroscopy (MRS) of hyperpolarized [1-13C] pyruvate is a promising neuroimaging strategy which, after a decade of preclinical success in glioblastoma (GBM) models, is now entering clinical trials in multiple centers. Typically, the presence of GBM has been associated with elevated hyperpolarized [1-13C] lactate produced from [1-13C] pyruvate, and response to therapy has been associated with a drop in hyperpolarized [1-13C] lactate. However, to date, lower grade gliomas had not been investigated using this approach. The most prevalent mutation in lower grade gliomas is the isocitrate dehydrogenase 1 (IDH1) mutation, which, in addition to initiating tumor development, also induces metabolic reprogramming. In particular, mutant IDH1 gliomas are associated with low levels of lactate dehydrogenase A (LDHA) and monocarboxylate transporters 1 and 4 (MCT1, MCT4), three proteins involved in pyruvate metabolism to lactate. We therefore investigated the potential of 13C MRS of hyperpolarized [1-13C] pyruvate for detection of mutant IDH1 gliomas and for monitoring of their therapeutic response. We studied patient-derived mutant IDH1 glioma cells that underexpress LDHA, MCT1 and MCT4, and wild-type IDH1 GBM cells that express high levels of these proteins. Mutant IDH1 cells and tumors produced significantly less hyperpolarized [1-13C] lactate compared to GBM, consistent with their metabolic reprogramming. Furthermore, hyperpolarized [1-13C] lactate production was not affected by chemotherapeutic treatment with temozolomide (TMZ) in mutant IDH1 tumors, in contrast to previous reports in GBM. Our results demonstrate the unusual metabolic imaging profile of mutant IDH1 gliomas, which, when combined with other clinically available imaging methods, could be used to detect the presence of the IDH1 mutation in vivo., Graphical abstract Image 1, Highlights • Metabolic imaging of mutant IDH1 gliomas using hyperpolarized 13C MRS is described. • In contrast to GBM, mutant IDH1 gliomas produce no hyperpolarized [1-13C] lactate. • Hyperpolarized [1-13C] lactate is not reduced by treatment in mutant IDH1 tumors. • Mutant IDH1 gliomas present an unusual metabolic imaging profile.

This review is focused on describing the use of magnetic resonance (MR) spectroscopy for metabolic imaging of brain tumors. We will first review the MR metabolic imaging findings generated from preclinical models, focusing primarily on in vivo studies, and will then describe the use of metabolic imaging in the clinical setting. We will address relatively well-established (1) H MRS approaches, as well as (31) P MRS, (13) C MRS and emerging hyperpolarized (13) C MRS methodologies, and will describe the use of metabolic imaging for understanding the basic biology of glioma as well as for improving the characterization and monitoring of brain tumors in the clinic.

Mutant isocitrate dehydrogenase 1 (IDH1) catalyzes the production of 2-hydroxyglutarate but also elicits additional metabolic changes. Levels of both glutamate and pyruvate dehydrogenase (PDH) activity have been shown to be affected in U87 glioblastoma cells or normal human astrocyte (NHA) cells expressing mutant IDH1, as compared with cells expressing wild-type IDH1. In this study, we show how these phenomena are linked through the effects of IDH1 mutation, which also reprograms pyruvate metabolism. Reduced PDH activity in U87 glioblastoma and NHA IDH1 mutant cells was associated with relative increases in PDH inhibitory phosphorylation, expression of pyruvate dehydrogenase kinase-3, and levels of hypoxia inducible factor-1α. PDH activity was monitored in these cells by hyperpolarized 13C-magnetic resonance spectroscopy (13C-MRS), which revealed a reduction in metabolism of hyperpolarized 2-13C-pyruvate to 5-13C-glutamate, relative to cells expressing wild-type IDH1. 13C-MRS also revealed a reduction in glucose flux to glutamate in IDH1 mutant cells. Notably, pharmacological activation of PDH by cell exposure to dichloroacetate (DCA) increased production of hyperpolarized 5-13C-glutamate in IDH1 mutant cells. Furthermore, DCA treatment also abrogated the clonogenic advantage conferred by IDH1 mutation. Using patient-derived mutant IDH1 neurosphere models, we showed that PDH activity was essential for cell proliferation. Taken together, our results established that the IDH1 mutation induces an MRS-detectable reprogramming of pyruvate metabolism, which is essential for cell proliferation and clonogenicity, with immediate therapeutic implications. Cancer Res; 75(15); 2999–3009. ©2015 AACR.

Acetate has recently been identified as a major alternative source of nutrients for glioblastoma and brain metastases. After cellular uptake, acetate is converted to acetyl-CoA, a key metabolic intermediate that fuels the TCA cycle and is an essential building block for the biosynthesis of fatty acids. Interestingly, the potential role of acetate in lower-grade glioma harboring the isocitrate dehydrogenase 1 mutation has not yet been elucidated. The goal of this study was therefore to investigate the role of acetate in fatty acid biosynthesis using a well-characterized genetically-engineered cell model that overexpresses either wild-type IDH1 (IDHwt) or mutant IDH1 (IDHmut): an immortalized normal human astrocyte (NHA)-based model. We used 1H and 13C magnetic resonance spectroscopy to quantify the flux of [1,2-13C]-acetate to 13C fatty acids. Our results indicated that the total levels of fatty acids were not significantly different between IDHmut and IDHwt NHA cells. However, the flux of 13C-labeled acetate towards fatty acids was significantly reduced by ~60% in IDHmut NHA cells relative to IDHwt NHA cells. To investigate this disconnect and understand the underlying biological mechanisms, we performed cell biological assays. Surprisingly, this decrease in acetate metabolism was associated with a drop in fatty acid synthase and ATP citrate lyase expressions, two enzymes involved in fatty acid synthesis from acetyl-CoA, in IDHmut NHA cells, whereas expression of acetyl-CoA synthase (AceS1), the cytosolic enzyme converting acetate to acetyl-CoA, was not altered. Asignificant drop in lipid droplet accumulation was also observed in IDHmut NHA cells as indicated using a spectrophotometric assay. Taken together, this points to alternate sources for fatty acids (e.g. glucose, glutamine, uptake from serum) in IDH1mut cells and suggests that fatty acids are preferentially directed towards cell membrane assembly. It also highlights the unique metabolic reprogramming of mutant IDH1 cells.

Recent findings show that exposure to temozolomide (TMZ), a DNA-damaging drug used to treat glioblastoma (GBM), can suppress the conversion of pyruvate to lactate. To understand the mechanistic basis for this effect and its potential utility as a TMZ response biomarker, we compared the response of isogenic GBM cell populations differing only in expression of the DNA repair protein methyltransferase (MGMT), a TMZ-sensitivity determinant, after exposure to TMZ in vitro and in vivo. Hyperpolarized [1-(13)C]-pyruvate–based MRI was used to monitor temporal effects on pyruvate metabolism in parallel with DNA-damage responses and tumor cell growth. TMZ exposure decreased conversion of pyruvate to lactate only in MGMT-deficient cells. This effect coincided temporally with TMZ-induced increases in levels of the DNA-damage response protein pChk1. Changes in pyruvate to lactate conversion triggered by TMZ preceded tumor growth suppression and were not associated with changes in levels of NADH or lactate dehydrogenase activity in tumors. Instead, they were associated with a TMZ-induced decrease in the expression and activity of pyruvate kinase PKM2, a glycolytic enzyme that indirectly controls pyruvate metabolism. PKM2 silencing decreased PK activity, intracellular lactate levels, and conversion of pyruvate to lactate in the same manner as TMZ, and Chk1 silencing blocked the TMZ-induced decrease in PKM2 expression. Overall, our findings showed how TMZ-induced DNA damage is linked through PKM2 to changes in pyruvate metabolism, and how these changes can be exploited by MRI methods as an early sensor of TMZ therapeutic response. Cancer Res; 74(23); 7115–24. ©2014 AACR.

Purpose Hyperpolarized 13C magnetic resonance allows for the study of real-time metabolism in vivo, including significant hyperpolarized 13C lactate production in many tumors. Other studies have shown that aggressive and highly metastatic tumors rapidly transport lactate out of cells. Thus, the ability to not only measure the production of hyperpolarized 13C lactate but also understand its compartmentalization using diffusion-weighted MR will provide unique information for improved tumor characterization. Methods We used a bipolar, pulsed-gradient, double spin echo imaging sequence to rapidly generate diffusion-weighted images of hyperpolarized 13C metabolites. Our methodology included a simultaneously acquired B1 map to improve apparent diffusion coefficient (ADC) accuracy and a diffusion-compensated variable flip angle scheme to improve ADC precision. Results We validated this sequence and methodology in hyperpolarized 13C phantoms. Next, we generated ADC maps of several hyperpolarized 13C metabolites in a normal rat, rat brain tumor, and prostate cancer mouse model using both preclinical and clinical trial-ready hardware. Conclusion ADC maps of hyperpolarized 13C metabolites provide information about the localization of these molecules in the tissue microenvironment. The methodology presented here allows for further studies to investigate ADC changes due to disease state that may provide unique information about cancer aggressiveness and metastatic potential. Magn Reson Med 74:622–633, 2015. © 2014 Wiley Periodicals, Inc.

We developed methods for the preparation of hyperpolarized (HP) sterile [2-13C]pyruvate to test its feasibility in first-ever human NMR studies following FDA-IND & IRB approval. Spectral results using this MR stable-isotope imaging approach demonstrated the feasibility of investigating human cerebral energy metabolism by measuring the dynamic conversion of HP [2-13C]pyruvate to [2-13C]lactate and [5-13C]glutamate in the brain of four healthy volunteers. Metabolite kinetics, signal-to-noise (SNR) and area-under-curve (AUC) ratios, and calculated [2-13C]pyruvate to [2-13C]lactate conversion rates (kPL) were measured and showed similar but not identical inter-subject values. The kPL measurements were equivalent with prior human HP [1-13C]pyruvate measurements.

Myeloid-derived suppressor cells (MDSCs) are highly prevalent inflammatory cells that play a key role in tumor development and are considered therapeutic targets. MDSCs promote tumor growth by blocking T-cell-mediated anti-tumoral immune response through depletion of arginine that is essential for T-cell proliferation. To deplete arginine, MDSCs express high levels of arginase, which catalyzes the breakdown of arginine into urea and ornithine. Here, we developed a new hyperpolarized 13C probe, [6-13C]-arginine, to image arginase activity. We show that [6-13C]-arginine can be hyperpolarized and hyperpolarized [13C]-urea production from [6-13C]-arginine is linearly correlated with arginase concentration in vitro. Furthermore we show that we can detect a statistically significant increase in hyperpolarized [13C]-urea production in MDSCs when compared to control bone marrow cells. This increase was associated with an increase in intracellular arginase concentration detected using a spectrophotometric assay. Hyperpolarized [6-13C]-arginine could therefore serve to image tumoral MDSC function and more broadly M2-like macrophages.

The current standard of care for glioblastoma (GBM) is surgical resection, radiotherapy, and treatment with temozolomide (TMZ). However, resistance to current therapies and recurrence are common. To improve survival, agents that target the PI3K signaling pathway, which is activated in approximately 88% of GBM, are currently in clinical trials. A challenge with such therapies is that tumor shrinkage is not always observed. New imaging methods are therefore needed to monitor response to therapy and predict survival. The goal of this study was to determine whether hyperpolarized 13C magnetic resonance spectroscopic imaging (MRSI) and 1H magnetic resonance spectroscopy (MRS) can be used to monitor response to the second-generation dual PI3K/mTOR inhibitor voxtalisib (XL765, SAR245409), alone or in combination with TMZ. We investigated GS-2 and U87-MG GBM orthotopic tumors in mice, and used MRI, hyperpolarized 13C MRSI, and 1H MRS to monitor the effects of treatment. In our study, 1H MRS could not predict tumor response to therapy. However, in both our models, we observed a significantly lower hyperpolarized lactate-to-pyruvate ratio in animals treated with voxtalisib, TMZ, or combination therapy, when compared with controls. This metabolic alteration was observed prior to MRI-detectable changes in tumor size, was consistent with drug action, and was associated with enhanced animal survival. Our findings confirm the potential translational value of the hyperpolarized lactate-to-pyruvate ratio as a biomarker for noninvasively assessing the effects of emerging therapies for patients with GBM. Mol Cancer Ther; 15(5); 1113–22. ©2016 AACR.

The phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway is activated in more than88% of glioblastomas (GBM). New drugs targeting this pathway are currently in clinical trials. However, noninvasive assessment of treatment response remains challenging. By using magnetic resonance spectroscopy (MRS), PI3K/Akt/mTOR pathway inhibition was monitored in 3 GBM cell lines (GS-2, GBM8, and GBM6; each with a distinct pathway activating mutation) through the measurement of 2 mechanistically linked MR biomarkers: phosphocholine (PC) and hyperpolarized lactate.(31)P MRS studies showed that treatment with the PI3K inhibitor LY294002 induced significant decreases in PC to 34 %± 9% of control in GS-2 cells, 48% ± 5% in GBM8, and 45% ± 4% in GBM6. The mTOR inhibitor everolimus also induced a significant decrease in PC to 62% ± 14%, 57% ± 1%, and 58% ± 1% in GS-2, GBM8, and GBM6 cells, respectively. Using hyperpolarized (13)C MRS, we demonstrated that hyperpolarized lactate levels were significantly decreased following PI3K/Akt/mTOR pathway inhibition in all 3 cell lines to 51% ± 10%, 62% ± 3%, and 58% ± 2% of control with LY294002 and 72% ± 3%, 61% ± 2%, and 66% ± 3% of control with everolimus in GS-2, GBM8, and GBM6 cells, respectively. These effects were mediated by decreases in the activity and expression of choline kinase α and lactate dehydrogenase, which respectively control PC and lactate production downstream of HIF-1. Treatment with the DNA damaging agent temozolomide did not have an effect on either biomarker in any cell line. This study highlights the potential of PC and hyperpolarized lactate as noninvasive MR biomarkers of response to targeted inhibitors in GBM.

Vorinostat is a histone deacetylase (HDAC) inhibitor that inhibits cell proliferation and induces apoptosis in solid tumors, and is in clinical trials for the treatment of glioblastoma (GBM). The goal of this study was to assess whether hyperpolarized 13 C MRS and magnetic resonance spectroscopic imaging (MRSI) can detect HDAC inhibition in GBM models. First, we confirmed HDAC inhibition in U87 GBM cells and evaluated real-time dynamic metabolic changes using a bioreactor system with live vorinostat-treated or control cells. We found a significant 40% decrease in the 13 C MRS-detectable ratio of hyperpolarized [1-13 C]lactate to hyperpolarized [1-13 C]pyruvate, [1-13 C]Lac/Pyr, and a 37% decrease in the pseudo-rate constant, kPL , for hyperpolarized [1-13 C]lactate production, in vorinostat-treated cells compared with controls. To understand the underlying mechanism for this finding, we assessed the expression and activity of lactate dehydrogenase (LDH) (which catalyzes the pyruvate to lactate conversion), its associated cofactor nicotinamide adenine dinucleotide, the expression of monocarboxylate transporters (MCTs) MCT1 and MCT4 (which shuttle pyruvate and lactate in and out of the cell) and intracellular lactate levels. We found that the most likely explanation for our finding that hyperpolarized lactate is reduced in treated cells is a 30% reduction in intracellular lactate levels that occurs as a result of increased expression of both MCT1 and MCT4 in vorinostat-treated cells. In vivo 13 C MRSI studies of orthotopic tumors in mice also showed a significant 52% decrease in hyperpolarized [1-13 C]Lac/Pyr when comparing vorinostat-treated U87 GBM tumors with controls, and, as in the cell studies, this metabolic finding was associated with increased MCT1 and MCT4 expression in HDAC-inhibited tumors. Thus, the 13 C MRSI-detectable decrease in hyperpolarized [1-13 C]lactate production could serve as a biomarker of response to HDAC inhibitors.