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.

In Vivo PET Detection of Lung Micrometastasis in Mice by Targeting Endothelial VCAM-1 Using a Dual-Contrast PET/MRI Probe.

Current clinical diagnostic imaging methods for lung metastases are sensitive only to large tumours (1-2 mm cross-sectional diameter), and early detection can dramatically improve treatment. We have previously demonstrated that an antibody-targeted MRI contrast agent based on microparticles of iron oxide (MPIO; 1 µm diameter) enables the imaging of endothelial vascular cell adhesion molecule-1 (VCAM-1). Using a mouse model of lung metastasis, upregulation of endothelial VCAM-1 expression was demonstrated in micrometastasis-associated vessels but not in normal lung tissue, and binding of VCAM-MPIO to these vessels was evident histologically. Owing to the lack of proton MRI signals in the lungs, we modified the VCAM-MPIO to include zirconium-89 (89Zr, t1/2 = 78.4 h) in order to allow the in vivo detection of lung metastases by positron emission tomography (PET). Using this new agent (89Zr-DFO-VCAM-MPIO), it was possible to detect the presence of micrometastases within the lung in vivo from ca. 140 µm in diameter. Histological analysis combined with autoradiography confirmed the specific binding of the agent to the VCAM-1 expressing vasculature at the sites of pulmonary micrometastases. By retaining the original VCAM-MPIO as the basis for this new molecular contrast agent, we have created a dual-modality (PET/MRI) agent for the concurrent detection of lung and brain micrometastases.

Imaging angiogenesis in an intracerebrally induced model of brain macrometastasis using αv ß3 -targeted iron oxide microparticles.

Brain metastasis is responsible for a large proportion of cancer mortality, and there are currently no effective treatments. Moreover, the impact of treatments, particularly antiangiogenic therapeutics, is difficult to ascertain using current magnetic resonance imaging (MRI) methods. Imaging of the angiogenic vasculature has been successfully carried out in solid tumours using microparticles of iron oxide (MPIO) conjugated to a Arg-Gly-Asp peptide (RGD) targeting integrin αv ß3 . The aim of this study was to determine whether RGD-MPIO could be used to identify angiogenic blood vessels in brain metastases in vivo. A mouse model of intracerebrally implanted brain macrometastasis was established through intracerebral injection of 4T1-GFP cells. T2 *-weighted imaging was used to visualise MPIO-induced hypointense voxels in vivo, and Prussian blue staining was used to visualise MPIO and endogenous iron histologically ex vivo. The RGD-MPIO showed target-specific binding in vivo, but the sensitivity of the agent for visualising angiogenic vessels per se was reduced by the presence of endogenous iron-laden macrophages in larger metastases, resulting in pre-existing hypointense areas within the tumour. Further, our data suggest that peptide-targeted MPIO, but not antibody-targeted MPIO, are taken up by perivascular macrophages within the macrometastatic microenvironment, resulting in additional nonspecific contrast. While pre-MPIO imaging will circumvent the issues surrounding pre-existing hypointensities and enable detection of specific contrast, our preliminary findings suggest that the use of antibodies rather than peptides as the targeting ligand may represent a preferable route forward for new angiogenesis-targeted molecular MRI agents.

Quantitative chemical exchange saturation transfer imaging of nuclear overhauser effects in acute ischemic stroke.

PURPOSE: In chemical exchange saturation transfer imaging, saturation effects between - 2 to - 5 ppm (nuclear Overhauser effects, NOEs) have been shown to exhibit contrast in preclinical stroke models. Our previous work on NOEs in human stroke used an analysis model that combined NOEs and semisolid MT; however their combination might feasibly have reduced sensitivity to changes in NOEs. The aim of this study was to explore the information a 4-pool Bloch-McConnell model provides about the NOE contribution in ischemic stroke, contrasting that with an intentionally approximate 3-pool model. METHODS: MRI data from 12 patients presenting with ischemic stroke were retrospectively analyzed, as well as from six animals induced with an ischemic lesion. Two Bloch-McConnell models (4 pools, and a 3-pool approximation) were compared for their ability to distinguish pathological tissue in acute stroke. The association of NOEs with pH was also explored, using pH phantoms that mimic the intracellular environment of naïve mouse brain. RESULTS: The 4-pool measure of NOEs exhibited a different association with tissue outcome compared to 3-pool approximation in the ischemic core and in tissue that underwent delayed infarction. In the ischemic core, the 4-pool measure was elevated in patient white matter ( 1.20±0.20 ) and in animals ( 1.27±0.20 ). In the naïve brain pH phantoms, significant positive correlation between the NOE and pH was observed. CONCLUSION: Associations of NOEs with tissue pathology were found using the 4-pool metric that were not observed using the 3-pool approximation. The 4-pool model more adequately captured in vivo changes in NOEs and revealed trends depending on tissue pathology in stroke.

Study of common quantification methods of amide proton transfer magnetic resonance imaging for ischemic stroke detection.

PURPOSE: To assess the correlation and differences between common amide proton transfer (APT) quantification methods in the diagnosis of ischemic stroke. METHODS: Five APT quantification methods, including asymmetry analysis and its variants as well as two Lorentzian model-based methods, were applied to data acquired from six rats that underwent middle cerebral artery occlusion scanned at 9.4T. Diffusion and perfusion-weighted images, and water relaxation time maps were also acquired to study the relationship of these conventional imaging modalities with the different APT quantification methods. RESULTS: The APT ischemic area estimates had varying sizes (Jaccard index: 0.544 ≤ J ≤ 0.971) and had varying correlations in their distributions (Pearson correlation coefficient: 0.104 ≤ r ≤ 0.995), revealing discrepancies in the quantified ischemic areas. The Lorentzian methods produced the highest contrast-to-noise ratios (CNRs; 1.427 ≤ CNR ≤ 2.002), but generated APT ischemic areas that were comparable in size to the cerebral blood flow (CBF) deficit areas; asymmetry analysis and its variants produced APT ischemic areas that were smaller than the CBF deficit areas but larger than the apparent diffusion coefficient deficit areas, though having lower CNRs (0.561 ≤ CNR ≤ 1.083). CONCLUSION: There is a need to further investigate the accuracy and correlation of each quantification method with the pathophysiology using a larger scale multi-imaging modality and multi-time-point clinical study. Future studies should include the magnetization transfer ratio asymmetry results alongside the findings of the study to facilitate the comparison of results between different centers and also the published literature.

Imaging of translocator protein upregulation is selective for pro-inflammatory polarized astrocytes and microglia.

Translocator protein (TSPO) expression is increased in activated glia, and has been used as a marker of neuroinflammation in PET imaging. However, the extent to which TSPO upregulation reflects a pro- or anti-inflammatory phenotype remains unclear. Our aim was to determine whether TSPO upregulation in astrocytes and microglia/macrophages is limited to a specific inflammatory phenotype. TSPO upregulation was assessed by flow cytometry in cultured astrocytes, microglia, and macrophages stimulated with lipopolysaccharide (LPS), tumor necrosis factor (TNF), or interleukin-4 (Il-4). Subsequently, mice were injected intracerebrally with either a TNF-inducing adenovirus (AdTNF) or IL-4. Glial expression of TSPO and pro-/anti-inflammatory markers was assessed by immunohistochemistry/fluorescence and flow cytometry. Finally, AdTNF or IL-4 injected mice underwent PET imaging with injection of the TSPO radioligand 18 F-DPA-713, followed by ex vivo autoradiography. TSPO expression was significantly increased in pro-inflammatory microglia/macrophages and astrocytes both in vitro, and in vivo after AdTNF injection (p < .001 vs. control hemisphere), determined both histologically and by FACS. Both PET imaging and autoradiography revealed a significant (p < .001) increase in 18 F-DPA-713 binding in the ipsilateral hemisphere of AdTNF-injected mice. In contrast, no increase in either TSPO expression assessed histologically and by FACS, or ligand binding by PET/autoradiography was observed after IL-4 injection. Taken together, these results suggest that TSPO imaging specifically reveals the pro-inflammatory population of activated glial cells in the brain in response to inflammatory stimuli. Since the inflammatory phenotype of glial cells is critical to their role in neurological disease, these findings may enhance the utility and application of TSPO imaging.

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.

A Critical Role for Astrocytes in Hypercapnic Vasodilation in Brain.

Cerebral blood flow (CBF) is controlled by arterial blood pressure, arterial CO2, arterial O2, and brain activity and is largely constant in the awake state. Although small changes in arterial CO2 are particularly potent to change CBF (1 mmHg variation in arterial CO2 changes CBF by 3%-4%), the coupling mechanism is incompletely understood. We tested the hypothesis that astrocytic prostaglandin E2 (PgE2) plays a key role for cerebrovascular CO2 reactivity, and that preserved synthesis of glutathione is essential for the full development of this response. We combined two-photon imaging microscopy in brain slices with in vivo work in rats and C57BL/6J mice to examine the hemodynamic responses to CO2 and somatosensory stimulation before and after inhibition of astrocytic glutathione and PgE2 synthesis. We demonstrate that hypercapnia (increased CO2) evokes an increase in astrocyte [Ca2+]i and stimulates COX-1 activity. The enzyme downstream of COX-1 that synthesizes PgE2 (microsomal prostaglandin E synthase-1) depends critically for its vasodilator activity on the level of glutathione in the brain. We show that, when glutathione levels are reduced, astrocyte calcium-evoked release of PgE2 is decreased and vasodilation triggered by increased astrocyte [Ca2+]iin vitro and by hypercapnia in vivo is inhibited. Astrocyte synthetic pathways, dependent on glutathione, are involved in cerebrovascular reactivity to CO2 Reductions in glutathione levels in aging, stroke, or schizophrenia could lead to dysfunctional regulation of CBF and subsequent neuronal damage.SIGNIFICANCE STATEMENT Neuronal activity leads to the generation of CO2, which has previously been shown to evoke cerebral blood flow (CBF) increases via the release of the vasodilator PgE2 We demonstrate that hypercapnia (increased CO2) evokes increases in astrocyte calcium signaling, which in turn stimulates COX-1 activity and generates downstream PgE2 production. We demonstrate that astrocyte calcium-evoked production of the vasodilator PgE2 is critically dependent on brain levels of the antioxidant glutathione. These data suggest a novel role for astrocytes in the regulation of CO2-evoked CBF responses. Furthermore, these results suggest that depleted glutathione levels, which occur in aging and stroke, will give rise to dysfunctional CBF regulation and may result in subsequent neuronal damage.

Robust and high resolution hyperpolarized metabolic imaging of the rat heart at 7 T with 3D spectral-spatial EPI.

PURPOSE: Hyperpolarized metabolic imaging has the potential to revolutionize the diagnosis and management of diseases where metabolism is dysregulated, such as heart disease. We investigated the feasibility of imaging rodent myocardial metabolism at high resolution at 7 T. METHODS: We present here a fly-back spectral-spatial radiofrequency pulse that sidestepped maximum gradient strength requirements and enabled high resolution metabolic imaging of the rodent myocardium. A 3D echo-planar imaging readout followed, with centric ordered z-phase encoding. The cardiac gated sequence was used to image metabolism in rodents whose metabolic state had been manipulated by being fasted, fed, or fed and given the pyruvate dehydrogenase kinase inhibitor dichloroacetate. RESULTS: We imaged hyperpolarized metabolites with a spatial resolution of 2×2×3.8 mm(3) and a temporal resolution of 1.8 s in the rat heart at 7 T. Significant differences in myocardial pyruvate dehydrogenase flux were observed between the three groups of animals, concomitant with the known biochemistry. CONCLUSION: The proposed sequence was able to image in vivo metabolism with excellent spatial resolution in the rat heart. The field of view enabled the simultaneous multi-organ acquisition of metabolic information from the rat, which is of great utility for preclinical research in cardiovascular disease. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance.

Determination of an optimally sensitive and specific chemical exchange saturation transfer MRI quantification metric in relevant biological phantoms.

The purpose of this study was to develop realistic phantom models of the intracellular environment of metastatic breast tumour and naïve brain, and using these models determine an analysis metric for quantification of CEST MRI data that is sensitive to only labile proton exchange rate and concentration. The ability of the optimal metric to quantify pH differences in the phantoms was also evaluated. Novel phantom models were produced, by adding perchloric acid extracts of either metastatic mouse breast carcinoma cells or healthy mouse brain to bovine serum albumin. The phantom model was validated using 1 H NMR spectroscopy, then utilized to determine the sensitivity of CEST MRI to changes in pH, labile proton concentration, T1 time and T2 time; six different CEST MRI analysis metrics (MTRasym , APT*, MTRRex , AREX and CESTR* with and without T1 /T2 compensation) were compared. The new phantom models were highly representative of the in vivo intracellular environment of both tumour and brain tissue. Of the analysis methods compared, CESTR* with T1 and T2 time compensation was optimally specific to changes in the CEST effect (i.e. minimal contamination from T1 or T2 variation). In phantoms with identical protein concentrations, pH differences between phantoms could be quantified with a mean accuracy of 0.6 pH units. We propose that CESTR* with T1 and T2 time compensation is the optimal analysis method for these phantoms. Analysis of CEST MRI data with T1 /T2 time compensated CESTR* is reproducible between phantoms, and its application in vivo may resolve the intracellular alkalosis associated with breast cancer brain metastases without the need for exogenous contrast agents.

Mouse Models of Brain Metastasis for Unravelling Tumour Progression.

Secondary tumours in the brain account for 40 % of triple negative breast cancer patients, and the percentage may be higher at the time of autopsy. The use of in vivo models allow us to recapitulate the molecular mechanisms potentially used by circulating breast tumour cells to proliferate within the brain.Metastasis is a multistep process that depends on the success of several stages including cell evasion from the primary tumour, distribution and survival within the blood stream and cerebral microvasculature, penetration of the blood-brain barrier and proliferation within the brain microenvironment. Cellular adhesion molecules are key proteins involved in all of the steps in the metastatic process. Our group has developed two different in vivo models to encompass both seeding and colonisation stages of the metastatic process: (1) haematogenous dissemination of tumour cells by direct injection into the left ventricle of the heart, and (2) direct implantation of the tumour cells into the mouse brain.This chapter describes, in detail, the practical implementation of the intracerebral model, which can be used to analyse tumour proliferation within a specific area of the central nervous system and tumour-host cell interactions. We also describe the use of immunohistochemistry techniques to identify, at the molecular scale, tumour-host cell interactions, which may open new windows for brain metastasis therapy.

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.

Motor skill learning induces changes in white matter microstructure and myelination.

Learning a novel motor skill is associated with well characterized structural and functional plasticity in the rodent motor cortex. Furthermore, neuroimaging studies of visuomotor learning in humans have suggested that structural plasticity can occur in white matter (WM), but the biological basis for such changes is unclear. We assessed the influence of motor skill learning on WM structure within sensorimotor cortex using both diffusion MRI fractional anisotropy (FA) and quantitative immunohistochemistry. Seventy-two adult (male) rats were randomly assigned to one of three conditions (skilled reaching, unskilled reaching, and caged control). After 11 d of training, postmortem diffusion MRI revealed significantly higher FA in the skilled reaching group compared with the control groups, specifically in the WM subjacent to the sensorimotor cortex contralateral to the trained limb. In addition, within the skilled reaching group, FA across widespread regions of WM in the contralateral hemisphere correlated significantly with learning rate. Immunohistological analysis conducted on a subset of 24 animals (eight per group) revealed significantly increased myelin staining in the WM underlying motor cortex in the hemisphere contralateral (but not ipsilateral) to the trained limb for the skilled learning group versus the control groups. Within the trained hemisphere (but not the untrained hemisphere), myelin staining density correlated significantly with learning rate. Our results suggest that learning a novel motor skill induces structural change in task-relevant WM pathways and that these changes may in part reflect learning-related increases in myelination.

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.

In vivo magnetic resonance imaging of acute brain inflammation using microparticles of iron oxide.

Multiple sclerosis is a disease of the central nervous system that is associated with leukocyte recruitment and subsequent inflammation, demyelination and axonal loss. Endothelial vascular cell adhesion molecule-1 (VCAM-1) and its ligand, alpha4beta1 integrin, are key mediators of leukocyte recruitment, and selective inhibitors that bind to the alpha4 subunit of alpha4beta1 substantially reduce clinical relapse in multiple sclerosis. Urgently needed is a molecular imaging technique to accelerate diagnosis, to quantify disease activity and to guide specific therapy. Here we report in vivo detection of VCAM-1 in acute brain inflammation, by magnetic resonance imaging in a mouse model, at a time when pathology is otherwise undetectable. Antibody-conjugated microparticles carrying a large amount of iron oxide provide potent, quantifiable contrast effects that delineate the architecture of activated cerebral blood vessels. Their rapid clearance from blood results in minimal background contrast. This technology is adaptable to monitor the expression of endovascular molecules in vivo in various pathologies.

Role of the systemic immune system in brain metastasis.

Metastatic disease in the central nervous system (CNS) is a cause of increasing mortality amongst cancer patients. As with other types of cancer, cells of the systemic immune system play a range of important roles in the development of metastatic lesions in the CNS, both repressing and promoting tumour growth. Recent advances in immunotherapy have changed the emphasis in cancer treatment away from conventional chemotherapy and radiotherapy for certain tumour types. Despite this, our understanding of systemic immune system involvement in CNS metastases remains poor. The blood-brain barrier prevents the majority of diagnostic and therapeutic agents from crossing into the brain parenchyma until the late stages of metastatic disease. Thus, the development of immunotherapy for CNS pathologies is particularly desirable. This review draws together our current understanding in the relationships between CNS metastases and circulating systemic immune cells. We discuss the roles that circulating systemic immune cells may play in the homing of metastatic cells to the perivascular space, and the pro-metastatic and antagonistic roles that infiltrating systemic immune cells may play at sites of metastasis. This article is part of a Special Issue entitled 'Neuroinflammation in neurodegeneration and neurodysfunction'.

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.

Targeted opening of the blood-brain barrier using VCAM-1 functionalised microbubbles and "whole brain" ultrasound.

Metastatic tumours in the brain now represent one of the leading causes of death from cancer. Current treatments are largely ineffective owing to the combination of late diagnosis and poor delivery of therapies across the blood-brain barrier (BBB). Conjugating magnetic resonance imaging (MRI) contrast agents with a monoclonal antibody for VCAM-1 (anti-VCAM1) has been shown to enable detection of micrometastases, two to three orders of magnitude smaller in volume than those currently detectable clinically. The aim of this study was to exploit this targeting approach to enable localised and temporary BBB opening at the site of early-stage metastases using functionalised microbubbles and ultrasound. Methods: Microbubbles functionalised with anti-VCAM1 were synthesised and shown to bind to VCAM-1-expressing cells in vitro. Experiments were then conducted in vivo in a unilateral breast cancer brain metastasis mouse model using Gadolinium-DTPA (Gd-DTPA) enhanced MRI to detect BBB opening. Following injection of Gd-DTPA and targeted microbubbles, the whole brain volume was simultaneously exposed to ultrasound (0.5 MHz, 10% duty cycle, 0.7 MPa peak negative pressure, 2 min treatment time). T1-weighted MRI was then performed to identify BBB opening, followed by histological confirmation via immunoglobulin G (IgG) immunohistochemistry. Results: In mice treated with targeted microbubbles and ultrasound, statistically significantly greater extravasation of Gd-DTPA and IgG was observed in the left tumour-bearing hemisphere compared to the right hemisphere 5 min after treatment. No acute adverse effects were observed. There was no investigation of longer term bioeffects owing to the nature of the study. Conclusion: The results demonstrate the feasibility of using targeted microbubbles in combination with low intensity ultrasound to localise opening of the BBB to metastatic sites in the brain. This approach has potential application in the treatment of metastatic tumours whose location cannot be established a priori with conventional imaging methods.

Complex spatiotemporal haemodynamic response following sensory stimulation in the awake rat.

Detailed understanding of the haemodynamic changes that underlie non-invasive neuroimaging techniques such as blood oxygen level dependent functional magnetic resonance imaging is essential if we are to continue to extend the use of these methods for understanding brain function and dysfunction. The use of animal and in particular rodent research models has been central to these endeavours as they allow in-vivo experimental techniques that provide measurements of the haemodynamic response function at high temporal and spatial resolution. A limitation of most of this research is the use of anaesthetic agents which may disrupt or mask important features of neurovascular coupling or the haemodynamic response function. In this study we therefore measured spatiotemporal cortical haemodynamic responses to somatosensory stimulation in awake rats using optical imaging spectroscopy. Trained, restrained animals received non-noxious stimulation of the whisker pad via chronically implanted stimulating microwires whilst optical recordings were made from the contralateral somatosensory cortex through a thin cranial window. The responses we measure from un-anaesthetised animals are substantially different from those reported in previous studies which have used anaesthetised animals. These differences include biphasic response regions (initial increases in blood volume and oxygenation followed by subsequent decreases) as well as oscillations in the response time series of awake animals. These haemodynamic response features do not reflect concomitant changes in the underlying neuronal activity and therefore reflect neurovascular or cerebrovascular processes. These hitherto unreported hyperemic response dynamics may have important implications for the use of anaesthetised animal models for research into the haemodynamic response function.