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Inhibition of peripheral TNF can block the malaise associated with CNS inflammatory diseases.
Circulating cytokine levels are elevated in many neuropathologies and may be a cause of the associated malaise and depression. Using a rat model, we demonstrate that sickness behaviors generated by microinjection of IL-1beta into the anterior hypothalamus are adopted by naive recipient animals following plasma transfer. We further show that neutralizing peripheral TNF by etanercept (a p75 TNF receptor/Fc fusion protein) prior to the IL-1beta microinjection inhibits certain IL-1beta-mediated sickness behaviors, such as the depression of open-field activity and reduced glucose consumption. IL-1beta-induced central lesions induce peripheral TNF as part of the acute-phase response, and this appears to be the principal target of the etanercept. Thus behavioral changes induced by CNS lesions may result from peripheral expression of cytokines that can be targeted with drugs which do not need to cross the blood-brain barrier to be efficacious.
Liver Kupffer cells control the magnitude of the inflammatory response in the injured brain and spinal cord.
The CNS inflammatory response is regulated by hepatic chemokine synthesis, which promotes leukocytosis and facilitates leukocyte recruitment to the site of injury. To understand the role of the individual cell populations in the liver during the hepatic response to acute brain injury, we selectively depleted Kupffer cells (KC), using clodronate-filled liposomes, and assessed the inflammatory response following a microinjection of IL-1beta into the rat brain or after a compression injury in the spinal cord. We show by immunohistochemistry that KC depletion reduces neutrophil infiltration into the IL-1beta-injected brain by 70% and by 50% into the contusion-injured spinal cord. qRT-PCR analysis of hepatic chemokine mRNA expression showed that chemokine expression in the liver after brain injury is not restricted to a single cell population. In non-depleted rats, CXCL-10, IL-1beta, CCL-2, and MIP-1alpha mRNAs were increased up to sixfold more than in KC depleted rats. However, CXCL-1 and MIP-1beta were not significantly affected by KC depletion. The reduction in chemokine mRNA expression by the liver was not associated with decreased neutrophil mobilisation as might have been expected. These findings suggest that in response to CNS injury, KC mediated mechanisms are responsible for increasing neutrophil entry to the site of CNS injury, but that neutrophil mobilisation is dependent on other non-KC mediated events. However, the suppression of KC activity may prevent secondary damage after acute brain injury.
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.
Altered chemokine expression in the spinal cord and brain contributes to differential interleukin-1beta-induced neutrophil recruitment.
The pattern of neutrophil recruitment that accompanies inflammation in the CNS depends on the site of injury and the stage of development. The adult brain parenchyma is refractory to neutrophil recruitment and associated damage as compared to the spinal cord or juvenile brain. Using quantitative Taqman RT-PCR and enzyme-liked immunosorbent assay (ELISA), we compared mRNA and protein expression of the rat neutrophil chemoattractant chemokines (CINC) in spinal cord and brain of adult and juvenile rats to identify possible association with the observed differences in neutrophil recruitment. Interleukin-1beta (IL-1beta) injection resulted in up-regulated chemokine expression in both brain and spinal cord. CINC-3 mRNA was elevated above CINC-1 and CINC-2alpha, with expression levels for each higher in spinal cord than in brain. By ELISA, IL-1beta induced greater CINC-1 and CINC-2alpha expression compared to CINC-3, with higher protein levels in spinal cord than in brain. In the juvenile brain, significantly higher levels of CINC-2alpha protein were observed in response to IL-1beta injection than in the adult brain following an equivalent challenge. Correspondingly, neutrophil recruitment was observed in the juvenile brain and adult spinal cord, but not in the adult brain. No expression of CINC-2beta mRNA was detected. Thus differential chemokine induction may contribute to variations in neutrophil recruitment in during development and between the different CNS compartments.
Loss of the tight junction proteins occludin and zonula occludens-1 from cerebral vascular endothelium during neutrophil-induced blood-brain barrier breakdown in vivo.
The tight junctions found between cerebral vascular endothelial cells form the basis of the blood-brain barrier. Breakdown of the blood-brain barrier is a feature of a variety of CNS pathologies that are characterized by extensive leucocyte recruitment, such as multiple sclerosis and stroke. The molecular mechanisms associated with opening of the blood-brain barrier and leucocyte recruitment in vivo are currently poorly understood. We have used an in vivo rat model to investigate the molecular response of the CNS endothelium to neutrophil adhesion and migration. Injection of interleukin-1 beta into the striatum of juvenile brains results in a neutrophil-dependent increase in vessel permeability at 4 h. Only a subset of blood vessels were associated with neutrophil recruitment. These particular vessels displayed an increase in phosphotyrosine staining, loss of the tight junctional proteins, occludin and zonula occludens-1, and apparent redistribution of the adherens junction protein vinculin. Examination of these vessels under the electron microscope indicated that the cell-cell adhesions in such vessels are morphologically different from normal junctions. This study provides the first direct evidence in vivo that leucocyte recruitment can trigger signal transduction cascades leading to junctional disorganization and blood-brain barrier breakdown. Our results have established an endothelial cell molecular profile associated with leucocyte-induced blood-brain barrier breakdown in vivo, and the relevance of different in vitro cell culture models may now be viewed more objectively.
Differential matrix metalloproteinase expression in cases of multiple sclerosis and stroke.
Multiple sclerosis (MS) and stroke pathology are characterized blood-brain barrier breakdown, leucocyte emigration, and tissue destruction. Each process is thought to involve the matrix metalloproteinases (MMP), but little is known of their expression. We undertook to investigate whether MMP expression is dependent on the nature of the CNS lesion and whether expression would coincide with the histopathology. MS or cerebral-infarct tissue was examined for the presence of gelatinase-A, gelatinase-B, matrilysin and stromelysin-1. Gelatinases A and B and matrilysin expression was found to be up-regulated in microglia/macrophages within acute MS lesions. In active-chronic MS lesions, matrilysin and gelatinase-A expression was pronounced in the active borders. In chronic MS lesions, the expression of matrilysin was confined to macrophages within perivascular cuffs. The pattern of MMP expression in infarct lesions differed considerably. Gelatinase-B was strongly expressed by neutrophils in tissue from patients up to 1 week after an infarct, whereas gelatinase-A and matrilysin staining was much less marked. From 1 week to 5 years, neutrophils were absent and the large number of macrophages present were expressing matrilysin and gelatinase A. Only a low level of gelatinase-A and matrilysin expression was observed in normal brain controls. Thus, MMPs are expressed in inflammatory lesions in the CNS, but their individual expression is dependent on the nature and chronicity of the lesion. However, the general pattern of expression, in perivascular cuffs and in active lesions, supports a role for these enzymes as mediators of blood-brain barrier breakdown and tissue destruction, both in MS and in cerebral ischaemia.
NMR-Based Metabolomics Separates the Distinct Stages of Disease in a Chronic Relapsing Model of Multiple Sclerosis.
Relapsing experimental allergic encephalomyelitis (Cr-EAE) is commonly used to explore the pathogenesis and efficacy of new therapies for MS, but it is unclear whether the metabolome of Cr-EAE is comparable to human multiple sclerosis (MS). For MS, the diagnosis and staging can be achieved by metabolomics on blood using a combination of magnetic resonance spectroscopy and partial least squares discriminant analysis (PLS-DA). Here, we sought to discover whether this approach could be used to differentiate between sequential disease states in Cr-EAE and whether the same metabolites would be discriminatory. Urine and plasma samples were obtained at different time-points from a clinically relevant model of MS. Using PLS-DA modelling for the urine samples furnished some predictive models, but could not discriminate between all disease states. However, PLS-DA modelling of the plasma samples was able to distinguish between animals with clinically silent disease (day 10, 28) and animals with active disease (day 14, 38). We were also able to distinguish Cr-EAE mice from naive mice at all-time points and control mice, treated with complete Freund's adjuvant alone, at day 14 and 38. Key metabolites that underpin these models included fatty acids, glucose and taurine. Two of these metabolites, fatty acids and glucose, were also key metabolites in separating relapsing-remitting MS from secondary-progressive MS in the human study. These results demonstrate the sensitivity of this metabolomics approach for distinguishing between different disease states. Furthermore, some, but not all, of the changes in metabolites were conserved in humans and the mouse model, which could be useful for future drug development.
Anti-CD20 inhibits T cell-mediated pathology and microgliosis in the rat brain.
OBJECTIVE: The mechanism of action of anti-B cell therapy in multiple sclerosis (MS) is not fully understood. Here, we compared the effect of anti-CD20 therapy on microglial activation in two distinct focal rat models of MS. METHODS: The effect of anti-CD20 therapy on lesion formation and extralesional microglial activation was evaluated in the fDTH-EAE (experimental allergic encephalomyelitis) model, which is a focal demyelinating type-IV delayed-type hypersensitivity lesion. For comparison, effects were also assessed in the focal humoral MOG model induced by intracerebral injection of cytokine in myelin oligodendrocyte glycoprotein immunized rats. Microglial activation was assessed in situ and in vivo using the TSPO SPECT ligand [(125)I]DPA-713, and by immunostaining for MHCII. The effect of treatment on demyelination and lymphocyte recruitment to the brain were evaluated. RESULTS: Anti-CD20 therapy reduced microglial activation, and lesion formation in the humoral model, but it was most effective in the antibody-independent fDTH-EAE. Immunohistochemistry for MHCII also demonstrated a reduced volume of microglial activation in the brains of anti-CD20-treated fDTH-EAE animals, which was accompanied by a reduction in T-cell recruitment and demyelination. The effect anti-CD20 therapy in the latter model was similarly strong as compared to the T-cell targeting MS compound FTY720. INTERPRETATION: The suppression of lesion development by anti-CD20 treatment in an antibody-independent model suggests that B-cells play an important role in lesion development, independent of auto-antibody production. Thus, CD20-positive B-cell depletion has the potential to be effective in a wider population of individuals with MS than might have been predicted from our knowledge of the underlying histopathology.