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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.

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.

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.

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.

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.

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.

Background: Activation and dysregulation of innate, adaptive and resident immune cells in response to damage determine the pathophysiology of demyelinating disorders. Among the plethora of involved cells, microglia/macrophages and astrocytes play an important role in the pathogenesis of demyelinating disorders. The in-depth investigation of the spatio-temporal profile of these cell types in vivo may inform about the exact disease state and localization as well as may allow to monitor therapeutic modulation of the components of the neuroinflammatory response during the course of multiple sclerosis (MS). In this study, we aimed to non-invasively decipher the degree and temporal profile of neuroinflammation (TSPO - [F-18]DPA-714 PET) in relation to selected magnetic resonance imaging (MRI) parameters (T-2 maps) in the cuprizone (CPZ)-induced model of demyelination. Methods: C57Bl6 (n=30) mice were fed with a standard chow mixed with 0.2% (w/w) CPZ for 4 (n=10; demyelination) and 6 weeks (n=10; spontaneous remyelination). The degree of neuroinflammation at de- and remyelination was assessed by [F-18]DPA-714 PET, multi-echo T-2 MRI, autoradiography and immunohistochemistry. Results: CPZ-induced brain alterations were confirmed by increase of T-2 relaxation times in both white and grey matter after 3 and 5 weeks of CPZ. Peak [F-18]DPA-714 was found in the corpus callosum (CC, white matter), the hippocampus (HC, grey matter) and thalamus (grey matter) after 4 weeks of CPZ treatment and declined after 6 weeks of CPZ. Ex vivo autoradiography and dedicated immunofluorescence showed demyelination/remyelination with corresponding increased/decreased TSPO levels in the CC and hippocampus, confirming the spatial distribution of [F-18]DPA-714 in vivo. The expression of TSPO microglia and astrocytes is time-dependent in this model. Microglia predominantly express TSPO at demyelination, while the majority of astrocytes express TSPO during remyelination. The combination of PET- and MRI-based imaging biomarkers demonstrated the regional and temporal development of the CPZ model-associated neuroinflammatory response in grey and white matter regions. Conclusions: The combination of [F-18]DPA-714 PET and T-2 mapping may allow to further elucidate the regional and temporal profile of inflammatory signals depending on the myelination status, although the underlying inflammatory microenvironment changes. A combination of the described imaging biomarkers may facilitate the development of patient-tailored strategies for immunomodulatory and neuro-restorative therapies in MS.

Detrimental inflammatory responses in the central nervous system are a hallmark of various brain injuries and diseases. With this study we provide evidence that lentiviral vector-mediated expression of the immune-modulating cytokine interleukin 13 (IL-13) induces an alternative activation program in both microglia and macrophages conferring protection against severe oligodendrocyte loss and demyelination in the cuprizone mouse model for multiple sclerosis (MS). First, IL-13 mediated modulation of cuprizone induced lesions was monitored using T2 -weighted magnetic resonance imaging and magnetization transfer imaging, and further correlated with quantitative histological analyses for inflammatory cell influx, oligodendrocyte death, and demyelination. Second, following IL-13 immune gene therapy in cuprizone-treated eGFP+ bone marrow chimeric mice, we provide evidence that IL-13 directs the polarization of both brain-resident microglia and infiltrating macrophages towards an alternatively activated phenotype, thereby promoting the conversion of a pro-inflammatory environment toward an anti-inflammatory environment, as further evidenced by gene expression analyses. Finally, we show that IL-13 immune gene therapy is also able to limit lesion severity in a pre-existing inflammatory environment. In conclusion, these results highlight the potential of IL-13 to modulate microglia/macrophage responses and to improve disease outcome in a mouse model for MS. GLIA 2016;64:2181-2200.

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.

The cuprizone mouse model allows the investigation of the complex molecular mechanisms behind nonautoimmune-mediated demyelination and spontaneous remyelination. While it is generally accepted that oligodendrocytes are specifically vulnerable to cuprizone intoxication due to their high metabolic demands, a comprehensive overview of the etiology of cuprizone-induced pathology is still missing to date. In this review we extensively describe the physico-chemical mode of action of cuprizone and discuss the molecular and enzymatic mechanisms by which cuprizone induces metabolic stress, oligodendrocyte apoptosis, myelin degeneration and eventually axonal and neuronal pathology. In addition, we describe the dual effector function of the immune system which tightly controls demyelination by effective induction of oligodendrocyte apoptosis, but in contrast also paves the way for fast and efficient remyelination by the secretion of neurotrophic factors and the clearance of cellular and myelinic debris. Finally, we discuss the many clinical symptoms that can be observed following cuprizone treatment, and how these strengthened the cuprizone model as a useful tool to study human multiple sclerosis, schizophrenia and epilepsy.

Although MRI is the gold standard for the diagnosis and monitoring of multiple sclerosis (MS), current conventional MRI techniques often fail to detect cortical alterations and provide little information about gliosis, axonal damage and myelin status of lesioned areas. Diffusion tensor imaging (DTI) and diffusion kurtosis imaging (DKI) provide sensitive and complementary measures of the neural tissue microstructure. Additionally, specific white matter tract integrity (WMTI) metrics modelling the diffusion in white matter were recently derived. In the current study we used the well-characterized cuprizone mouse model of central nervous system demyelination to assess the temporal evolution of diffusion tensor (DT), diffusion kurtosis tensor (DK) and WMTI-derived metrics following acute inflammatory demyelination and spontaneous remyelination. While DT-derived metrics were unable to detect cuprizone induced cortical alterations, the mean kurtosis (MK) and radial kurtosis (RK) were found decreased under cuprizone administration, as compared to age-matched controls, in both the motor and somatosensory cortices. The MK remained decreased in the motor cortices at the end of the recovery period, reflecting long lasting impairment of myelination. In white matter, DT, DK and WMTI-derived metrics enabled the detection of cuprizone induced changes differentially according to the stage and the severity of the lesion. More specifically, the MK, the RK and the axonal water fraction (AWF) were the most sensitive for the detection of cuprizone induced changes in the genu of the corpus callosum, a region less affected by cuprizone administration. Additionally, microgliosis was associated with an increase of MK and RK during the acute inflammatory demyelination phase. In regions undergoing severe demyelination, namely the body and splenium of the corpus callosum, DT-derived metrics, notably the mean diffusion (MD) and radial diffusion (RD), were among the best discriminators between cuprizone and control groups, hence highlighting their ability to detect both acute and long lasting changes. Interestingly, WMTI-derived metrics showed the aptitude to distinguish between the different stages of the disease. Both the intra-axonal diffusivity (Da) and the AWF were found to be decreased in the cuprizone treated group, Da specifically decreased during the acute inflammatory demyelinating phase whereas the AWF decrease was associated to the spontaneous remyelination and the recovery period. Altogether our results demonstrate that DKI is sensitive to alterations of cortical areas and provides, along with WMTI metrics, information that is complementary to DT-derived metrics for the characterization of demyelination in both white and grey matter and subsequent inflammatory processes associated with a demyelinating event.

Conventional MRI is frequently used during the diagnosis of multiple sclerosis but provides only little additional pathological information. Proton MRS (H-1-MRS), however, provides biochemical information on the lesion pathology by visualization of a spectrum of metabolites. In this study we aimed to better understand the changes in metabolite concentrations following demyelination of the white matter. Therefore, we used the cuprizone model, a wellestablished mouse model to mimic type III human multiple sclerosis demyelinating lesions. First, we identified CX(3)CL1/CX(3)CR1 signaling as a major regulator of microglial activity in the cuprizone mouse model. Compared with control groups (heterozygous CX(3)CR1(+/-) C57BL/6 mice and wild type CX3CR1(+/+) C57BL/6 mice), microgliosis, astrogliosis, oligodendrocyte cell death and demyelination were shown to be highly reduced or absent in CX3CR1(-/-) C57BL/6 mice. Second, we show that 1H-MRS metabolite spectra are different when comparing cuprizone-treated CX3CR1(-/-) mice showing mild demyelination with cuprizone-treated CX3CR1(+/+) mice showing severe demyelination and demyelination-associated inflammation. Following cuprizone treatment, CX3CR1(+/+) mice show a decrease in the Glu, tCho and tNAA concentrations as well as an increased Tau concentration. In contrast, following cuprizone treatment CX3CR1(-/-) mice only showed a decrease in tCho and tNAA concentrations. Therefore, H-1-MRS might possibly allow us to discriminate demyelination from demyelination-associated inflammation via changes in Tau and Glu concentration. In addition, the observed decrease in tCho concentration in cuprizoneinduced demyelinating lesions should be further explored as a possible diagnostic tool for the early identification of human MS type III lesions. Copyright (C) 2015 John Wiley & Sons, Ltd. This work was supported by research grants G.0136.11 and G.0130.11 (granted to ZB, AVdL and PP) of the Fund for Scientific Research - Flanders (FWO - Vlaanderen, Belgium) and in addition by an "FWO - Krediet aan Navorsers" grant of the Fund for Scientific Research - Flanders (FWO -Vlaanderen, Belgium, granted to MV); next, by a Methusalem research grant from the Flemish government (granted to ZB) and in part by funding received from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement 278850 (INMiND) (granted to AVdL). Caroline Guglielmetti is holder of an IWT PhD grant (Institute for the Promotion of Innovation through Science and Technology in Flanders, IWT - Vlaanderen). Firat Kara is holder of an "FWO - Postdoc" grant of the Fund for Scientific Research - Flanders (FWO - Vlaanderen, Belgium). MRI equipment was funded by the Flemish Impulse funding for heavy scientific equipment (granted to AVdL).

Non-invasive measures of well-known pathological hallmarks of multiple sclerosis (MS) such as demyelination, inflammation and axonal injury would serve as useful markers to monitor disease progression and evaluate potential therapies. To this end, in vivo localized proton magnetic resonance spectroscopy ((1)H-MRS) provides a powerful means to monitor metabolic changes in the brain and may be sensitive to these pathological hallmarks. In our study, we used the cuprizone mouse model to study pathological features of MS, such as inflammation, de- and remyelination, in a highly reproducible manner. C57BL/6J mice were challenged with a 0.2% cuprizone diet for 6-weeks to induce demyelination, thereafter the mice were put on a cuprizone free diet for another 6weeks to induce spontaneous remyelination. We employed in vivo (1)H-MRS to longitudinally monitor metabolic changes in the corpus callosum of cuprizone-fed mice during the demyelination (weeks 4 and 6) and spontaneous remyelination (week 12) phases. The MRS spectra were quantified with LCModel and since the total creatine (tCr) levels did not change over time or between groups, metabolite concentrations were expressed as ratios relative to tCr. After 4 and 6weeks of cuprizone treatment a significant increase in taurine/tCr and a significant reduction in total N-acetylaspartate/tCr, total choline-containing compounds/tCr and glutamate/tCr could be observed compared to mice under normal diet. At week 12, when almost full remyelination was established, no statistically significant metabolic differences were present between the control and cuprizone group. Our results suggest that these metabolic changes may represent sensitive markers for cuprizone induced demyelination, axonal injury and inflammation. To the best of our knowledge, this is the first longitudinal in vivo (1)H-MRS study that monitored biochemical changes in the corpus callosum of cuprizone fed mice.