Inflammatory bowel disease induces pathological α-synuclein aggregation in the human gut and brain.

AIMS: According to Braak's hypothesis, it is plausible that Parkinson's disease (PD) originates in the enteric nervous system (ENS) and spreads to the brain through the vagus nerve. In this work, we studied whether inflammatory bowel diseases (IBDs) in humans can progress with the emergence of pathogenic α-synuclein (α-syn) in the gastrointestinal tract and midbrain dopaminergic neurons. METHODS: We have analysed the gut and the ventral midbrain from subjects previously diagnosed with IBD and form a DSS-based rat model of gut inflammation in terms of α-syn pathology. RESULTS: Our data support the existence of pathogenic α-syn in both the gut and the brain, thus reinforcing the potential role of the ENS as a contributing factor in PD aetiology. Additionally, we have analysed the effect of a DSS-based rat model of gut inflammation to demonstrate (i) the appearance of P-α-syn inclusions in both Auerbach's and Meissner's plexuses (gut), (ii) an increase in α-syn expression in the ventral mesencephalon (brain) and (iii) the degeneration of nigral dopaminergic neurons, which all are considered classical hallmarks in PD. CONCLUSION: These results strongly support the plausibility of Braak's hypothesis and emphasise the significance of peripheral inflammation and the gut-brain axis in initiating α-syn aggregation and transport to the substantia nigra, resulting in neurodegeneration.

SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development.

Skp1-Cul1-F-box protein (SCF) ubiquitin ligases direct cell survival decisions by controlling protein ubiquitylation and degradation. Sufu (Suppressor of fused) is a central regulator of Hh (Hedgehog) signaling and acts as a tumor suppressor by maintaining the Gli (Glioma-associated oncogene homolog) transcription factors inactive. Although Sufu has a pivotal role in Hh signaling, the players involved in controlling Sufu levels and their role in tumor growth are unknown. Here, we show that Fbxl17 (F-box and leucine-rich repeat protein 17) targets Sufu for proteolysis in the nucleus. The ubiquitylation of Sufu, mediated by Fbxl17, allows the release of Gli1 from Sufu for proper Hh signal transduction. Depletion of Fbxl17 leads to defective Hh signaling associated with an impaired cancer cell proliferation and medulloblastoma tumor growth. Furthermore, we identify a mutation in Sufu, occurring in medulloblastoma of patients with Gorlin syndrome, which increases Sufu turnover through Fbxl17-mediated polyubiquitylation and leads to a sustained Hh signaling activation. In summary, our findings reveal Fbxl17 as a novel regulator of Hh pathway and highlight the perturbation of the Fbxl17-Sufu axis in the pathogenesis of medulloblastoma.

An automated method for segmentation and quantification of blood vessels in histology images.

Alzheimer's disease (AD) is a chronic neuro-degenerative disease that adversely affect many people on a global scale. Despite different diagnostic and therapeutic treatment, there is no cure for AD. The brain is one of the most complex organ and researchers are still trying to understand so as to find a cure. OBJECTIVE: To complement the efforts of clinical researchers engaged in research in alzheimer's disease, accurate segmentation and quantification of blood vessels in brain images is required. METHOD: For robust segmentation of blood vessels even in the presence of colour variation, we introduce a fully automated morphological tool that can extract and quantify vessels from haematoxylin and diaminobenzidine stained histology brain image. The method, exploits saturation channel of stained image slides, ISODATA threshold method is applied to obtain a binary image. This helps in eliminating background and remaining with only blood vessels. A one-stage procedure that includes eliminating small artefacts is performed on the binary mask. The intensity of the image is transformed. Joining is performed to deal with fragmentation of intact blood vessels on the images, and artefactual appearance of the blood vessel structures. The artefactual fragments based on measured incoherence with neighbouring tissue are removed. The vessels are then labelled to facilitate quantification. Morphometric measurements are used during the vessel quantification assess both vessels with lumen and vessels without lumen. We have quantified the diameter of blood vessels. RESULTS: The image processing technique is developed in close collaboration with neuroscientist experts to help clinician. We have evaluated our proposed approach qualitatively. The method was validated against their manual quantification results. Qualitative results show that the method can indeed segment the blood vessels in the presence of colour variations and artefacts. The quantitative method produces fairly better results.

Molecular MRI enables early and sensitive detection of brain metastases.

Metastasis to the brain is a leading cause of cancer mortality. The current diagnostic method of gadolinium-enhanced MRI is sensitive only to larger tumors, when therapeutic options are limited. Earlier detection of brain metastases is critical for improved treatment. We have developed a targeted MRI contrast agent based on microparticles of iron oxide that enables imaging of endothelial vascular cell adhesion molecule-1 (VCAM-1). Our objectives here were to determine whether VCAM-1 is up-regulated on vessels associated with brain metastases, and if so, whether VCAM-1-targeted MRI enables early detection of these tumors. Early up-regulation of cerebrovascular VCAM-1 expression was evident on tumor-associated vessels in two separate murine models of brain metastasis. Metastases were detectable in vivo using VCAM-1-targeted MRI 5 d after induction (<1,000 cells). At clinical imaging resolutions, this finding is likely to translate to detection at tumor volumes two to three orders of magnitude smaller (0.3-3 × 10(5) cells) than those volumes detectable clinically (10(7)-10(8) cells). VCAM-1 expression detected by MRI increased significantly (P < 0.0001) with tumor progression, and tumors showed no gadolinium enhancement. Importantly, expression of VCAM-1 was shown in human brain tissue containing both established metastases and micrometastases. Translation of this approach to the clinic could increase therapeutic options and change clinical management in a substantial number of cancer patients.

Structural and functional effects of metastases in rat brain determined by multimodal MRI.

Metastasis to the brain results in significant impairment of brain function and poor patient survival. Currently, magnetic resonance imaging (MRI) is under-utilised in monitoring brain metastases and their effects on brain function. Here, we sought to establish a model of focal brain metastasis in the rat that enables serial multimodal structural and functional MRI studies, and to assess the sensitivity of these approaches to metastatic growth. Female Berlin-Druckrey-IX rats were injected intracerebrally with metastatic ENU1564 cells in the ventroposterior medial nucleus (VPM) of the thalamus, a relay node of the whisker-to-barrel cortex pathway. Animals underwent multimodal structural and vascular MRI, as well as functional MRI of the cortical blood oxygenation level dependent (BOLD) responses to whisker pad stimulation. T2 , diffusion, magnetisation transfer and perfusion weighted MRI enabled differentiation between a central area of more advanced metastatic growth and penumbral regions of co-optive perivascular micrometastatic growth, with magnetisation transfer MRI being the most sensitive to micrometastatic growth. Areas of cortical BOLD activation in response to whisker pad stimulation were significantly reduced in the hemisphere containing metastases in the VPM. The reduction in BOLD response correlated with metastatic burden in the thalamus, and was sensitive to the presence of smaller metastases than currently detectable clinically. Our findings suggest that multimodal MRI provides greater sensitivity to tumour heterogeneity and micrometastatic growth than single modality contrast-enhanced MRI. Understanding the relationships between these MRI parameters and the underlying pathology may greatly enhance the utility of MRI in diagnosis, staging and monitoring of brain metastasis.

Anti-IL-17A treatment reduces clinical score and VCAM-1 expression detected by in vivo magnetic resonance imaging in chronic relapsing EAE ABH mice.

IL-17 is argued to play an important role in the multiple sclerosis-like disease experimental autoimmune encephalitis (EAE). We investigated the therapeutic effects of anti-IL-17A in a chronic relapsing EAE ABH mouse model using conventional scoring, quantitative behavioral outcomes, and a novel vascular cell adhesion molecule 1 (VCAM-1)-targeted magnetic resonance imaging (MRI) contrast agent [anti-VCAM-microparticles of iron oxide (MPIO)] to identify conventionally undetectable neuropathology. Mice were administered prophylactic or treatment regimens of anti-IL-17A or IgG and two injections of anti-VCAM-MPIO before undergoing T2*-weighted three-dimensional and gadolinium-diethylenetriamine pentaacetic acid T1-weighted MRI. Rotarod, inverted screen, and open field motor function tests were performed, conventional clinical scores calculated, and central IL-17A mRNA expression quantified during acute disease, remission, and relapse. Prophylactic anti-IL-17A prevents acute disease and relapse and is associated with reduced clinical and functional severity. Treatment regimens delay relapse, improve functional scores, and are associated with reduced VCAM-MPIO lesions during remission. No significant alteration was detectable in levels of gadolinium-diethylenetriamine pentaacetic acid- or VCAM-MPIO-positive lesions during relapse. Prophylactic and treatment anti-IL-17A were therapeutically effective in chronic relapsing EAE, improving clinical and quantifiable functional outcomes. IL-17A expression seems significant during acute disease but less important chronically. Disease-related immunoneuropathology is more sensitively detected using VCAM-MPIO MRI, which may, therefore, be used to monitor therapy meaningfully.

Imaging angiogenesis, inflammation, and metastasis in the tumor microenvironment with magnetic resonance imaging.

With the development of new imaging techniques, the potential for probing the molecular, cellular, and structural components of the tumor microenvironment in situ has increased dramatically. A multitude of imaging modalities have been successfully employed to probe different aspects of the tumor microenvironment, including expression of molecules, cell motion, cellularity, vessel permeability, vascular perfusion, metabolic and physiological changes, apoptosis, and inflammation. This chapter focuses on the most recent advances in magnetic resonance imaging methods, which offer a number of advantages over other methodologies, including high spatial resolution and the use of nonionizing radiation, as well as the use of such methods in the context of primary and secondary brain tumors. It also highlights how they can be used to assess the molecular and cellular changes in the tumor microenvironment in response to therapy.

A convenient model of serum-induced reactivity of human astrocytes to investigate astrocyte-derived extracellular vesicles.

Extracellular vesicles (EVs) are secreted by all cells in the CNS, including neurons and astrocytes. EVs are lipid membrane enclosed particles loaded with various bioactive cargoes reflecting the dynamic activities of cells of origin. In contrast to neurons, the specific role of EVs released by astrocytes is less well understood, partly due to the difficulty in maintaining primary astrocyte cultures in a quiescent state. The aim of this study was to establish a human serum-free astrocyte culture system that maintains primary astrocytes in a quiescent state to study the morphology, function, and protein cargoes of astrocyte-derived EVs. Serum-free medium with G5 supplement and serum-supplemented medium with 2% FBS were compared for the culture of commercially available human primary fetal astrocytes. Serum-free astrocytes displayed morphologies similar to in vivo astrocytes, and surprisingly, higher levels of astrocyte markers compared to astrocytes chronically cultured in FBS. In contrast, astrocyte and inflammatory markers in serum-free astrocytes were upregulated 24 h after either acute 2% FBS or cytokine exposure, confirming their capacity to become reactive. Importantly, this suggests that distinct signaling pathways are involved in acute and chronic astrocyte reactivity. Despite having a similar morphology, chronically serum-cultured astrocyte-derived EVs (ADEVs) were smaller in size compared to serum-free ADEVs and could reactivate serum-free astrocytes. Proteomic analysis identified distinct protein datasets for both types of ADEVs with enrichment of complement and coagulation cascades for chronically serum-cultured astrocyte-derived EVs, offering insights into their roles in the CNS. Collectively, these results suggest that human primary astrocytes cultured in serum-free medium bear similarities with in vivo quiescent astrocytes and the addition of serum induces multiple morphological and transcriptional changes that are specific to human reactive astrocytes and their ADEVs. Thus, more emphasis should be made on using multiple structural, molecular, and functional parameters when evaluating ADEVs as biomarkers of astrocyte health.

VCAM-1-targeted magnetic resonance imaging reveals subclinical disease in a mouse model of multiple sclerosis.

Diagnosis of multiple sclerosis (MS) currently requires lesion identification by gadolinium (Gd)-enhanced or T(2)-weighted magnetic resonance imaging (MRI). However, these methods only identify late-stage pathology associated with blood-brain barrier breakdown. There is a growing belief that more widespread, but currently undetectable, pathology is present in the MS brain. We have previously demonstrated that an anti-VCAM-1 antibody conjugated to microparticles of iron oxide (VCAM-MPIO) enables in vivo detection of VCAM-1 by MRI. Here, in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS, we have shown that presymptomatic lesions can be quantified using VCAM-MPIO when they are undetectable by Gd-enhancing MRI. Moreover, in symptomatic animals VCAM-MPIO binding was present in all regions showing Gd-DTPA enhancement and also in areas of no Gd-DTPA enhancement, which were confirmed histologically to be regions of leukocyte infiltration. VCAM-MPIO binding correlated significantly with increasing disability. Negligible MPIO-induced contrast was found in either EAE animals injected with an equivalent nontargeted contrast agent (IgG-MPIO) or in control animals injected with the VCAM-MPIO. These findings describe a highly sensitive molecular imaging tool that may enable detection of currently invisible pathology in MS, thus accelerating diagnosis, guiding treatment, and enabling quantitative disease assessment.

Glyconanoparticles allow pre-symptomatic in vivo imaging of brain disease.

Initial recruitment of leukocytes in inflammation associated with diseases such as multiple sclerosis (MS), ischemic stroke, and HIV-related dementia, takes place across intact, but activated brain endothelium. It is therefore undetectable to symptom-based diagnoses and cannot be observed by conventional imaging techniques, which rely on increased permeability of the blood-brain barrier (BBB) in later stages of disease. Specific visualization of the early-activated cerebral endothelium would provide a powerful tool for the presymptomatic diagnosis of brain disease and evaluation of new therapies. Here, we present the design, construction and in vivo application of carbohydrate-functionalized nanoparticles that allow direct detection of endothelial markers E-/P-selectin (CD62E/CD62P) in acute inflammation. These first examples of MRI-visible glyconanoparticles display multiple copies of the natural complex glycan ligand of selectins. Their resulting sensitivity and binding selectivity has allowed acute detection of disease in mammals with beneficial implications for treatment of an expanding patient population suffering from neurological disease.

VCAM-1-targeted MRI Improves Detection of the Tumor-brain Interface.

PURPOSE: Despite optimal local therapy, tumor cell invasion into normal brain parenchyma frequently results in recurrence in patients with solid tumors. The aim of this study was to determine whether microvascular inflammation can be targeted to better delineate the tumor-brain interface through vascular cell adhesion molecule-1 (VCAM-1)-targeted MRI. EXPERIMENTAL DESIGN: Intracerebral xenograft rat models of MDA231Br-GFP (breast cancer) brain metastasis and U87MG (glioblastoma) were used to histologically examine the tumor-brain interface and to test the efficacy of VCAM-1-targeted MRI in detecting this region. Human biopsy samples of the brain metastasis and glioblastoma margins were examined for endothelial VCAM-1 expression. RESULTS: The interface between tumor and surrounding normal brain tissue exhibited elevated endothelial VCAM-1 expression and increased microvessel density. Tumor proliferation and stemness markers were also significantly upregulated at the tumor rim in the brain metastasis model. T2*-weighted MRI, following intravenous administration of VCAM-MPIO, highlighted the tumor-brain interface of both tumor models more extensively than gadolinium-DTPA-enhanced T1-weighted MRI. Sites of VCAM-MPIO binding, evident as hypointense signals on MR images, correlated spatially with endothelial VCAM-1 upregulation and bound VCAM-MPIO beads detected histologically. These findings were further validated in an orthotopic medulloblastoma model. Finally, the tumor-brain interface in human brain metastasis and glioblastoma samples was similarly characterized by microvascular inflammation, extending beyond the region detectable using conventional MRI. CONCLUSIONS: This work illustrates the potential of VCAM-1-targeted MRI for improved delineation of the tumor-brain interface in both primary and secondary brain tumors.

Review article: The aetiology of fatigue in inflammatory bowel disease and potential therapeutic management strategies.

BACKGROUND: Fatigue is the inability to achieve or maintain an expected work output resulting from central or peripheral mechanisms. The prevalence of inflammatory bowel disease (IBD) fatigue can reach 86% in active disease, persisting in 50%-52% of patients with mild to inactive disease. Fatigue is the commonest reason for work absence in IBD, and patients often report fatigue burden to be greater than that of primary disease symptoms. Relatively few evidence-based treatment options exist, and the aetiology is poorly understood. AIM: To review the available data and suggest a possible aetiology of IBD fatigue and to consider the efficacy of existing management strategies and highlight potential future interventions. METHODS: We reviewed fatigue-related literature in IBD using PubMed database. RESULTS: Disease related factors such as inflammation and pharmacological treatments negatively impact skeletal muscle and brain physiology, likely contributing to fatigue symptoms. Secondary factors such as malnutrition, anaemia, sleep disturbance and psychological comorbidity are potential determinants. Immune profile, faecal microbiota composition and physical fitness differ significantly between fatigued and non-fatigued patients, suggesting these may be aetiological factors. Solution-focused therapy, high-dosage thiamine supplementation and biological therapy may reduce fatigue perception in IBD. The effect of physical activity interventions is inconclusive. CONCLUSIONS: A multimodal approach is likely required to treat IBD fatigue. Established reversible factors like anaemia, micronutrient deficiencies and active disease should initially be resolved. Psychosocial intervention shows potential efficacy in reducing fatigue perception in quiescent disease. Restoring physical deconditioning by exercise training intervention may further improve fatigue burden.

Multimodal evaluation of hypoxia in brain metastases of lung cancer and interest of hypoxia image-guided radiotherapy.

Lung cancer patients frequently develop brain metastases (BM). Despite aggressive treatment including neurosurgery and external-radiotherapy, overall survival remains poor. There is a pressing need to further characterize factors in the microenvironment of BM that may confer resistance to radiotherapy (RT), such as hypoxia. Here, hypoxia was first evaluated in 28 biopsies from patients with non­small cell lung cancer (NSCLC) BM, using CA-IX immunostaining. Hypoxia characterization (pimonidazole, CA-IX and HIF-1α) was also performed in different preclinical NSCLC BM models induced either by intracerebral injection of tumor cells (H2030-Br3M, H1915) into the cortex and striatum, or intracardial injection of tumor cells (H2030-Br3M). Additionally, [18F]-FMISO-PET and oxygen-saturation-mapping-MRI (SatO2-MRI) were carried out in the intracerebral BM models to further characterize tumor hypoxia and evaluate the potential of Hypoxia-image-guided-RT (HIGRT). The effect of RT on proliferation of BM ([18F]-FLT-PET), tumor volume and overall survival was determined. We showed that hypoxia is a major yet heterogeneous feature of BM from lung cancer both preclinically and clinically. HIGRT, based on hypoxia heterogeneity observed between cortical and striatal metastases in the intracerebrally induced models, showed significant potential for tumor control and animal survival. These results collectively highlight hypoxia as a hallmark of BM from lung cancer and the value of HIGRT in better controlling tumor growth.

Systemic inflammatory response reactivates immune-mediated lesions in rat brain.

The potential association between microbial infection and reactivation of a multiple sclerosis (MS) lesion is an important issue that remains unresolved, primarily because of the absence of suitable animal models and imaging techniques. Here, we have evaluated this question in an empirical manner using immunohistochemistry and magnetic resonance imaging (MRI), before and after the induction of a systemic inflammatory response in two distinct models of MS. In a pattern-II-type focal myelin oligodendrocyte glycoprotein-experimental autoimmune encephalomyelitis model, systemic endotoxin injection caused an increase in regional cerebral blood volume (rCBV) around the lesion site after 6 h, together with a reduction in the magnetization transfer ratio of the lesioned corpus callosum. These changes were followed by an increase in the diffusion of tissue water within the lesion 24 h after endotoxin challenge and new leukocyte recruitment as revealed both immunohistochemically and by MRI tracking of ultrasmall superparamagnetic iron oxide-labeled macrophages. Importantly, we detected in vivo expression of E- and P-selectin in quiescent lesions by MRI-detectable glyconanoparticles conjugated to sialyl Lewis(X). This finding may explain, at least in part, the ability of quiescent MS lesions to rapidly reinitiate the cell recruitment processes. In a pattern-I-type delayed-type hypersensitivity response model, a similar effect of endotoxin challenge on rCBV was observed, together with delayed breakdown of the blood-brain barrier, showing that systemic infection can alter the pathogenesis of MS-like lesions regardless of lesion etiology. These findings will have important implications for the management and monitoring of individuals with MS.

STAT3-Mediated Astrocyte Reactivity Associated with Brain Metastasis Contributes to Neurovascular Dysfunction.

Astrocytes are thought to play a pivotal role in coupling neural activity and cerebral blood flow. However, it has been shown that astrocytes undergo morphologic changes in response to brain metastasis, switching to a reactive phenotype, which has the potential to significantly compromise cerebrovascular function and contribute to the neurological sequelae associated with brain metastasis. Given that STAT3 is a key regulator of astrocyte reactivity, we aimed here to determine the impact of STAT3-mediated astrocyte reactivity on neurovascular function in brain metastasis. Rat models of brain metastasis and ciliary neurotrophic factor were used to induce astrocyte reactivity. Multimodal imaging, electrophysiology, and IHC were performed to determine the relationship between reactive astrocytes and changes in the cerebrovascular response to electrical and physiological stimuli. Subsequently, the STAT3 pathway in astrocytes was inhibited with WP1066 to determine the role of STAT3-mediated astrocyte reactivity, specifically, in brain metastasis. Astrocyte reactivity associated with brain metastases impaired cerebrovascular responses to stimuli at both the cellular and functional level and disrupted astrocyte-endothelial interactions in both animal models and human brain metastasis samples. Inhibition of STAT3-mediated astrocyte reactivity in rats with brain metastases restored cerebrovascular function, as shown by in vivo imaging, and limited cerebrovascular changes associated with tumor growth. Together these findings suggest that inhibiting STAT3-mediated astrocyte reactivity may confer significant improvements in neurological outcome for patients with brain metastases and could potentially be tested in other brain tumors. SIGNIFICANCE: These findings demonstrate that selectively targeting STAT3-mediated astrocyte reactivity ameliorates the cerebrovascular dysfunction associated with brain metastasis, providing a potential therapeutic avenue for improved patient outcome.

Comparison of MRI signatures in pattern I and II multiple sclerosis models.

The majority of individuals with multiple sclerosis (MS) exhibit T-cell- and macrophage-dominated lesions (patterns I and II; as opposed to III and IV). These lesions, in turn, may be distinguished on the basis of whether or not there are immunoglobulin and complement depositions at the sites of active myelin destruction; such depositions are found exclusively in pattern II lesions. The main aim of this study was to determine whether pattern I and pattern II MS lesions exhibit distinct MRI signatures. We have used a recently described focal MOG-induced EAE model of the rat brain, which recapitulates many of the hallmarks of pattern II MS; we compared this with our previous work in a delayed type hypersensitivity model of a pattern I type lesion in the rat brain. Demyelinating lesions with extensive inflammation were generated, in which the T2-weighted signal was increased. Magnetisation transfer ratio (MTR) maps revealed loss and subsequent incomplete recovery of the structure of the corpus callosum, together with changes in tissue water diffusion and an associated increase in ventricle size. Notably, the MTR changes preceeded histological demyelination and may report on the processes leading to demyelination, rather than demyelination per se. Immunohistochemically, these MRI-detectable signal changes correlated with both inflammatory cell infiltration and later loss of myelin. Breakdown of the blood-brain barrier and an increase in the regional cerebral blood volume were also evident in and around the lesion site at the early stage of the disease. Interestingly, however, the MRI signal changes in this pattern II type MS lesion were remarkably consistent with those previously observed in a pattern I lesion. These findings suggest that the observed signal changes reflect the convergent histopathology of the two models rather than the underlying mechanisms of the disease.

Development of Therapeutic Anti-JAGGED1 Antibodies for Cancer Therapy.

The role of Notch signaling and its ligand JAGGED1 (JAG1) in tumor biology has been firmly established, making them appealing therapeutic targets for cancer treatment. Here, we report the development and characterization of human/rat-specific JAG1-neutralizing mAbs. Epitope mapping identified their binding to the Notch receptor interaction site within the JAG1 Delta/Serrate/Lag2 domain, where E228D substitution prevented effective binding to the murine Jag1 ortholog. These antibodies were able to specifically inhibit JAG1-Notch binding in vitro, downregulate Notch signaling in cancer cells, and block the heterotypic JAG1-mediated Notch signaling between endothelial and vascular smooth muscle cells. Functionally, in vitro treatment impaired three-dimensional growth of breast cancer cell spheroids, in association with a reduction in cancer stem cell number. In vivo testing showed variable effects on human xenograft growth when only tumor-expressed JAG1 was targeted (mouse models) but a more robust effect when stromal-expressed Jag1 was also targeted (rat MDA-MB-231 xenograft model). Importantly, treatment of established triple receptor-negative breast cancer brain metastasis in rats showed a significant reduction in neoplastic growth. MRI imaging demonstrated that this was associated with a substantial improvement in blood-brain barrier function and tumor perfusion. Lastly, JAG1-targeting antibody treatment did not cause any detectable toxicity, further supporting its clinical potential for cancer therapy.

Close coupling between astrocytic and neuronal metabolisms to fulfill anaplerotic and energy needs in the rat brain.

Carbon metabolism in the rat brain was studied in animals anesthetized with a light dose of pentobarbital and in awake animals under morphine, which were infused with either [1-13C]glucose+acetate or glucose+[2-13C]acetate for various periods of time. Brain amino-acid enrichments in tissue extracts were determined by nuclear magnetic resonance (NMR) spectroscopy and their time evolution was analyzed by automatic fitting. Acetyl-coenzyme A C2 enrichment and ratio between pyruvate carboxylase and pyruvate dehydrogenase activity (PC/PDH) were determined from glutamate and glutamine labeling. The following results were obtained: (i) amino-acid enrichment patterns implied metabolic compartmentation and occurrence of the glutamate-glutamine cycle; (ii) kinetics of aspartate, GABA, and glutamate labeling from [1-13C]glucose and of glutamine labeling from [2-13C]acetate indicated a twofold higher metabolic activity in awake than in anesthetized rat brain; (iii) evaluation of the contributions of the astrocytic and neuronal metabolisms to glutamine synthesis in both groups of rats indicated a coupling between neuronal tricarboxylic acid (TCA) cycle, glutamate-glutamine cycle and glial TCA cycle; and (iv) analyzing the extrapolations back to time zero and the steady-state values of PC/PDH indicated a close coupling between PC activity and both astrocytic and neuronal TCA cycles. All these results suggest a cooperative-like behavior of astrocytic and neuronal metabolisms to fulfill the anabolic and energy needs linked to brain activation.

13C Pyruvate Transport Across the Blood-Brain Barrier in Preclinical Hyperpolarised MRI.

Hyperpolarised MRI with Dynamic Nuclear Polarisation overcomes the fundamental thermodynamic limitations of conventional magnetic resonance, and is translating to human studies with several early-phase clinical trials in progress including early reports that demonstrate the utility of the technique to observe lactate production in human brain cancer patients. Owing to the fundamental coupling of metabolism and tissue function, metabolic neuroimaging with hyperpolarised [1-13C]pyruvate has the potential to be revolutionary in numerous neurological disorders (e.g. brain tumour, ischemic stroke, and multiple sclerosis). Through the use of [1-13C]pyruvate and ethyl-[1-13C]pyruvate in naïve brain, a rodent model of metastasis to the brain, or porcine brain subjected to mannitol osmotic shock, we show that pyruvate transport across the blood-brain barrier of anaesthetised animals is rate-limiting. We show through use of a well-characterised rat model of brain metastasis that the appearance of hyperpolarized [1-13C]lactate production corresponds to the point of blood-brain barrier breakdown in the disease. With the more lipophilic ethyl-[1-13C]pyruvate, we observe pyruvate production endogenously throughout the entire brain and lactate production only in the region of disease. In the in vivo porcine brain we show that mannitol shock permeabilises the blood-brain barrier sufficiently for a dramatic 90-fold increase in pyruvate transport and conversion to lactate in the brain, which is otherwise not resolvable. This suggests that earlier reports of whole-brain metabolism in anaesthetised animals may be confounded by partial volume effects and not informative enough for translational studies. Issues relating to pyruvate transport and partial volume effects must therefore be considered in pre-clinical studies investigating neuro-metabolism in anaesthetised animals, and we additionally note that these same techniques may provide a distinct biomarker of blood-brain barrier permeability in future studies.

Covalent assembly of nanoparticles as a peptidase-degradable platform for molecular MRI.

Ligand-conjugated microparticles of iron oxide (MPIO) have the potential to provide high sensitivity contrast for molecular magnetic resonance imaging (MRI). However, the accumulation and persistence of non-biodegradable micron-sized particles in liver and spleen precludes their clinical use and limits the translational potential of MPIO-based contrast agents. Here we show that ligand-targeted MPIO derived from multiple iron oxide nanoparticles may be coupled covalently through peptide linkers that are designed to be cleaved by intracellular macrophage proteases. The synthesized particles possess potential characteristics for targeted MRI contrast agents, including high relaxivity, unappreciable sedimentation, clearance from circulation and no overt toxicity. Importantly, we demonstrate that these particles are rapidly degraded both in vitro and in vivo, and that the targeted probes can be used for detection of inflammation in vivo using MRI. This approach provides a platform for molecular MRI contrast agents that is potentially more suitable for translation to humans.