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) ¹³ C 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 [1-¹³ C] 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 [1-¹³ C] pyruvate and HP ¹³ C urea. 2D chemical shift imaging following simultaneous intravenous injection of HP [1-¹³ C] pyruvate and HP ¹³ C 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 [1-¹³ C] 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 ¹³ C 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 ¹³ C MRS, in alignment with increased numbers of microglia/macrophages and astrocytes. This study demonstrates that HP ¹³ C spectroscopy has substantial potential for providing noninvasive information on neuroinflammation.

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.

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.

Proinflammatory mononuclear phagocytes (MPs) play a crucial role in the progression of multiple sclerosis (MS) and other neurodegenerative diseases. Despite advances in neuroimaging, there are currently limited available methods enabling noninvasive detection of MPs in vivo. Interestingly, upon activation and subsequent differentiation toward a proinflammatory phenotype MPs undergo metabolic reprogramming that results in increased glycolysis and production of lactate. Hyperpolarized (HP) ¹³C magnetic resonance spectroscopic imaging (MRSI) is a clinically translatable imaging method that allows noninvasive monitoring of metabolic pathways in real time. This method has proven highly useful to monitor the Warburg effect in cancer, through MR detection of increased HP [1-¹³C]pyruvate-to-lactate conversion. However, to date, this method has never been applied to the study of neuroinflammation. Here, we questioned the potential of ¹³C MRSI of HP [1-¹³C]pyruvate to monitor the presence of neuroinflammatory lesions in vivo in the cuprizone mouse model of MS. First, we demonstrated that ¹³C MRSI could detect a significant increase in HP [1-¹³C]pyruvate-to-lactate conversion, which was associated with a high density of proinflammatory MPs. We further demonstrated that the increase in HP [1-¹³C]lactate was likely mediated by pyruvate dehydrogenase kinase 1 up-regulation in activated MPs, resulting in regional pyruvate dehydrogenase inhibition. Altogether, our results demonstrate a potential for ¹³C MRSI of HP [1-¹³C]pyruvate as a neuroimaging method for assessment of inflammatory lesions. This approach could prove useful not only in MS but also in other neurological diseases presenting inflammatory components.

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.

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.

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'-¹⁸F-fluoro-9-β-d-arabinofuranosylguanine (¹⁸F-FAraG) PET imaging to noninvasively 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 to reproducibly induce 2 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 before disease induction, during demyelination with high levels of innate immune cells, and after 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 the brain and spinal cord at the time point of T cell infiltration. ¹⁸F-FAraG signal from white matter (corpus callosum) and gray 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 the brain and spinal cord of fingolimod-treated mice; T1-weighted contrast-enhanced MRI revealed intact BBB, whereas T2-weighted MRI findings remained unchanged. Conclusion: The combination of MRI and ¹⁸F-FAraG PET enables detection of inflammatory demyelination and T cell infiltration in an MS mouse model, providing a new way to evaluate lesion heterogeneity during disease progression and after DMT. On clinical translation, these methods hold great potential for stratifying patients, monitoring MS progression, and determining therapy responses.

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.

Many biomarkers in clinical neuroscience lack pathological certification. This issue is potentially a significant contributor to the limited success of neuroprotective and neurorestorative therapies for human neurological disease-and is evident even in areas with therapeutic promise such as myelin repair. Despite the identification of promising remyelinating candidates, biologically validated methods to demonstrate therapeutic efficacy or provide robust preclinical evidence of remyelination in the CNS are lacking. Therapies with potential to remyelinate the CNS constitute one of the most promising and highly anticipated therapeutic developments in the pipeline to treat multiple sclerosis and other demyelinating diseases. The optic nerve has been proposed as an informative pathway to monitor remyelination in animals and human subjects. Recent clinical trials using visual evoked potential have had promising results, but without unequivocal evidence about the cellular and molecular basis for signal changes on visual evoked potential, the interpretation of these trials is constrained. The visual evoked potential was originally developed and used in the clinic as a diagnostic tool but its use as a quantitative method for assessing therapeutic response requires certification of its biological specificity. Here, using the tools of experimental pathology we demonstrate that quantitative measurements of myelination using both histopathological measures of nodal structure and ultrastructural assessments correspond to visual evoked potential latency in both inflammatory and chemical models of demyelination. Visual evoked potential latency improves after treatment with a tool remyelinating compound (clemastine), mirroring both quantitative and qualitative myelin assessment. Furthermore, clemastine does not improve visual evoked potential latency following demyelinating injury when administered to a transgenic animal incapable of forming new myelin. Therefore, using the capacity for therapeutic enhancement and biological loss of function we demonstrate conclusively that visual evoked potential measures myelin status and is thereby a validated tool for preclinical verification of remyelination.

Hyperpolarization techniques significantly enhance the sensitivity of magnetic resonance (MR) and thus present fascinating new directions for research and applications with in vivo MR imaging and spectroscopy (MRI/S). Hyperpolarized ¹³C MRI/S, in particular, enables real-time non-invasive assessment of metabolic processes and holds great promise for a diverse range of clinical applications spanning fields like oncology, neurology, and cardiology, with a potential for improving early diagnosis of disease, patient stratification, and therapy response assessment. Despite its potential, technical challenges remain for achieving clinical translation. This paper provides an overview of the discussions that took place at the international workshop "New Horizons in Hyperpolarized ¹³C MRI," in March 2023 at the Bavarian Academy of Sciences and Humanities, Munich, Germany. The workshop covered new developments, as well as future directions, in topics including polarization techniques (particularly focusing on parahydrogen-based methods), novel probes, considerations related to data acquisition and analysis, and emerging clinical applications in oncology and other fields.