Nicola R. Sibson

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, α4β1 integrin, are key mediators of leukocyte recruitment, and selective inhibitors that bind to the α4 subunit of α4β1 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.

The Vascular Basement Membrane as “Soil” in Brain Metastasis

Brain-specific homing and direct interactions with the neural substance are prominent hypotheses for brain metastasis formation and a modern manifestation of Pagets “seed and soil” concept. However, there is little direct evidence for this “neurotropic” growth in vivo. In contrast, many experimental studies have anecdotally noted the propensity of metastatic cells to grow along the exterior of pre-existing vessels of the CNS, a process termed vascular cooption. These observations suggest the “soil” for malignant cells in the CNS may well be vascular, rather than neuronal. We used in vivo experimental models of brain metastasis and analysis of human clinical specimens to test this hypothesis. Indeed, over 95% of early micrometastases examined demonstrated vascular cooption with little evidence for isolated neurotropic growth. This vessel interaction was adhesive in nature implicating the vascular basement membrane (VBM) as the active substrate for tumor cell growth in the brain. Accordingly, VBM promoted adhesion and invasion of malignant cells and was sufficient for tumor growth prior to any evidence of angiogenesis. Blockade or loss of the β1 integrin subunit in tumor cells prevented adhesion to VBM and attenuated metastasis establishment and growth in vivo. Our data establishes a new understanding of CNS metastasis formation and identifies the neurovasculature as the critical partner for such growth. Further, we have elucidated the mechanism of vascular cooption for the first time. These findings may help inform the design of effective molecular therapies for patients with fatal CNS malignancies.

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.

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.

Confounding effects of anesthesia on functional activation in rodent brain: a study of halothane and alpha-chloralose anesthesia.

Functional magnetic resonance imaging (fMRI) in animal models provides a platform for more extensive investigation of drug effects and underlying physiological mechanisms than is possible in humans. However, it is usually necessary for the animal to be anesthetized. In this study, we have used a rat model of direct cortical stimulation to investigate the effects of anesthesia in rodent fMRI. Specifically, we have sought to answer two questions (i) what is the relationship between baseline neuronal activity and the BOLD response to stimulation under halothane anesthesia? And (ii) how does the BOLD response change after transferring from halothane to the commonly used anesthetic alpha-chloralose? In the first set of experiments, we found no significant differences in the amplitude of the BOLD response at the different halothane doses studied, despite electroencephalography (EEG) recordings indicating a dose-dependent reduction in baseline neuronal activity with increasing halothane levels. In the second set of experiments, a reduction in the spatial extent of the BOLD response was apparent immediately after transfer from halothane to alpha-chloralose anesthesia, although no change in the peak signal change was evident. However, several hours after transfer to alpha-chloralose, a significant increase in both the spatial extent and peak height of the BOLD response was observed, as well as an increased sensitivity to secondary cortical and subcortical activation. These findings suggest that, although alpha-chloralose anesthesia is associated with a greater BOLD response for a fixed stimulus relative to halothane, there is substantial variation in the extent and magnitude of the response over time that could introduce considerable variability in studies using this anesthetic.

Does glutamate image your thoughts

Functional imaging methods exploit the relationship between neuronal activity, energy demand and cerebral blood flow to functionally map the brain. Despite the increasing use of these imaging tools in basic and clinical neuroscience, the neurobiological processes underlying the imaging signals remain unclear. Recently, interest has been focused on uncovering the signals that trigger the metabolic and vascular changes accompanying variations in neuronal activity. Advances in this field have demonstrated that release of the major excitatory neurotransmitter glutamate initiates diverse signaling processes between neurons and astrocytes, and that this signaling could be crucial for the occurrence of brain imaging signals. In this article we review the hypothesis that glutamate represents a common trigger for both neurometabolic and neurovascular coupling.

MRI detection of early endothelial activation in brain inflammation.

MRI is an increasingly important clinical tool, but it is clear that conventional imaging fails to identify the full extent of lesion load in certain conditions, such as multiple sclerosis. The aim of this study was to determine whether a novel contrast agent (Gd‐DTPA‐B(sLeX)A, which contains an sLeX mimetic moiety that enables it to bind to the adhesion molecule E‐selectin) can be used to identify endothelial activation in the brain. Microinjection of the proinflammatory cytokines IL‐1β or TNF‐α into the striatum of Wistar rats rapidly induces focal adhesion molecule expression on the endothelium in the absence of MRI‐visible changes. This phenomenon was used to investigate the potential of Gd‐DTPA‐B(sLeX)A to reveal MRI‐invisible brain pathology. T1‐weighted serial images were acquired in anesthetized animals before and after administration of Gd‐DTPA‐B(sLeX)A, 3–4 hr after cytokine was injected intracerebrally. Both TNF‐α and IL‐1β up‐regulated E‐selectin on the brain endothelium, which correlated with increased signal intensity observed after administration of the novel contrast agent. No enhancement was visible with the nonselective contrast agent Gd‐DTPA‐BMA, indicating that there was no leakage of the agent across the blood–brain barrier (BBB) or nonselective binding to the endothelium. These data demonstrate the potential of such contrast agents for the early detection of brain injury and inflammation. Magn Reson Med 51:248–252, 2004.

A contrast agent recognizing activated platelets reveals murine cerebral malaria pathology undetectable by conventional MRI

Human and murine cerebral malaria are associated with elevated levels of cytokines in the brain and adherence of platelets to the microvasculature. Here we demonstrated that the accumulation of platelets in the brain microvasculature can be detected with MRI, using what we believe to be a novel contrast agent, at a time when the pathology is undetectable by conventional MRI. Ligand-induced binding sites (LIBS) on activated platelet glycoprotein IIb/IIIa receptors were detected in the brains of malaria-infected mice 6 days after inoculation with Plasmodium berghei using microparticles of iron oxide (MPIOs) conjugated to a single-chain antibody specific for the LIBS (LIBS-MPIO). No binding of the LIBS-MPIO contrast agent was detected in uninfected animals. A combination of LIBS-MPIO MRI, confocal microscopy, and transmission electron microscopy revealed that the proinflammatory cytokine TNF-alpha, but not IL-1beta or lymphotoxin-alpha (LT-alpha), induced adherence of platelets to cerebrovascular endothelium. Peak platelet adhesion was found 12 h after TNF-alpha injection and was readily detected with LIBS-MPIO contrast-enhanced MRI. Temporal studies revealed that the level of MPIO-induced contrast was proportional to the number of platelets bound. Thus, the LIBS-MPIO contrast agent enabled noninvasive detection of otherwise undetectable cerebral pathology by in vivo MRI before the appearance of clinical disease, highlighting the potential of targeted contrast agents for diagnostic, mechanistic, and therapeutic studies.

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 LewisX. 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.

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 × 105 cells) than those volumes detectable clinically (107–108 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.