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1.
Neuropathol Appl Neurobiol ; 34(2): 216-30, 2008 Apr.
Article in English | MEDLINE | ID: mdl-17983428

ABSTRACT

UNLABELLED: During neuroinflammation in multiple sclerosis (MS) fibrinogen, not normally present in the brain or spinal cord, enters the central nervous system through a compromised blood-brain barrier. Fibrin deposited on axons is ineffectively removed by tissue plasminogen activator (tPA), a key contributory factor being the upregulation of plasminogen activator inhibitor-1 (PAI-1). AIMS: This study investigated the role of PAI-1 during experimental neuroinflammatory disease. METHODS: Chronic relapsing experimental allergic encephalomyelitis (CREAE), a model of MS, was induced with spinal cord homogenate in PAI-1 knockout (PAI-1(-/-)) and wild type (WT) mice, backcrossed onto the Biozzi background. RESULTS: Disease incidence and clinical severity were reduced in PAI-1(-/-) mice, with animals developing clinical signs significantly later than WTs. Clinical relapses were absent in PAI-1(-/-) mice and the subsequent reduction in neuroinflammation was coupled with a higher capacity for fibrinolysis in spinal cord samples from PAI-1(-/-) mice, in association with increased tPA activity. Axonal damage was less apparent in PAI-1(-/-) mice than in WTs, implicating fibrin in both inflammatory and degenerative events during CREAE. CONCLUSIONS: PAI-1 is a potential target for therapy in neuroinflammatory degenerative diseases, allowing effective fibrin removal and potentially reducing relapse rate and axonal damage.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/metabolism , Encephalomyelitis, Autoimmune, Experimental/pathology , Fibrinolysis/physiology , Plasminogen Activator Inhibitor 1/metabolism , Animals , Axons/metabolism , Axons/pathology , Blotting, Western , Chronic Disease , Encephalomyelitis, Autoimmune, Experimental/immunology , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Inflammation/pathology , Mice , Mice, Knockout , Spinal Cord/immunology , Spinal Cord/metabolism , Spinal Cord/pathology , Tissue Plasminogen Activator/metabolism
2.
Neuropathol Appl Neurobiol ; 32(1): 15-22, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16409549

ABSTRACT

In multiple sclerosis (MS), the matrix metalloprotease (MMP) gelatinase B/MMP-9 and platelet endothelial cell adhesion molecule (PECAM)-1 have both been implicated in trans-endothelial infiltration of leucocytes into the brain, but their functional connection has not yet been investigated. We investigated the expression of gelatinase B and PECAM-1 in post mortem brains of MS patients by immunohistochemistry. Because increased soluble PECAM-1 serum levels have been observed in MS patients, we also tested in vitro whether this could be due to cleavage of PECAM-1 by gelatinase B or matrilysin-1/MMP-7. Constitutive expression of PECAM-1 was found on brain endothelial cells, whilst in active MS lesions cell-bound PECAM-1 was highly up-regulated on foamy macrophages in perivascular infiltrates and co-localized with gelatinase B. However, human THP-1 monocyte-bound or soluble recombinant PECAM-1 were both resistant to proteolytic cleavage by gelatinase B or matrilysin-1 in vitro, as demonstrated by Western blot analysis and flow cytometry. These results suggest that PECAM-1 and gelatinase B may complement each other during the transmigration of the blood-brain barrier by mononuclear cells.


Subject(s)
Brain/blood supply , Endothelial Cells/metabolism , Matrix Metalloproteinase 9/metabolism , Multiple Sclerosis/metabolism , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Blotting, Western , Brain/metabolism , Brain/pathology , Cell Movement/physiology , Cells, Cultured , Flow Cytometry , Humans , Immunohistochemistry , Leukocytes, Mononuclear/metabolism , Multiple Sclerosis/pathology
3.
Neuroscience ; 134(1): 261-8, 2005.
Article in English | MEDLINE | ID: mdl-15953683

ABSTRACT

Administered cannabinoids have been shown to ameliorate signs of CNS inflammatory disease in a number of animal models, including allergic encephalomyelitis. More recently, neuroprotective actions have been attributed to activation of the cannabinoid 1 receptor in a number of in vitro and in vivo models. One of these, chronic relapsing experimental allergic encephalomyelitis, is considered a robust analog of multiple sclerosis. In this study, spinal cord tissue from cannabinoid receptor 1 knockout mice was analyzed for neurofilament H and myelin basic protein content, as markers of neurons/axons and myelin respectively, during the course of chronic relapsing experimental allergic encephalomyelitis. Dephosphorylation of a neurofilament H epitope, immunoreactive to the SMI32 antibody, was assessed as a marker of axonal damage and levels of the endpoint cell death mediator caspase 3 were evaluated. It was found that both neurofilament and myelin basic protein levels decrease over the course of disease, indicating concomitant neuronal/axonal loss and demyelination. Loss of each marker was more severe in cannabinoid receptor 1 knockout animals. Increased SMI32 reactivity was observed as disease progressed. SMI32 reactivity was significantly increased in knockout animals over wildtype counterparts, an indication of greater axonal dephosphorylation and injury. Active caspase 3 levels were increased in all animals during disease, with knockout animals displaying highest levels, even in knockout animals prior to disease induction. These results indicate that lack of the cannabinoid receptor 1 is associated with increased caspase activation and greater loss and/or compromise of myelin and axonal/neuronal proteins. The increase of caspase 3 in knockout mice prior to disease induction indicates a latent physiological effect of the missing receptor. The data presented further strengthen the hypothesis of neuroprotection elicited via cannabinoid receptor 1 signaling.


Subject(s)
Caspases/metabolism , Neurofilament Proteins/metabolism , Receptor, Cannabinoid, CB1/physiology , Spinal Cord/metabolism , Animals , Blotting, Western/methods , CD4 Antigens/metabolism , Caspase 3 , Diagnostic Imaging/methods , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/metabolism , Enzyme Activation , Enzyme-Linked Immunosorbent Assay/methods , Fluorescent Antibody Technique/methods , Mice , Mice, Knockout , Myelin Basic Protein/metabolism , Receptor, Cannabinoid, CB1/deficiency
4.
Eur J Neurosci ; 21(8): 2127-35, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15869509

ABSTRACT

Excessive nitric oxide (NO) production from the inducible isoform of nitric oxide synthase (iNOS) has been invoked as a causative factor in many neurodegenerative disorders, including multiple sclerosis. This hypothesis has been supported by in vitro studies showing that glial iNOS expression results in toxic NO concentrations (near 1 microm). To investigate the relevance of such findings, experiments were carried out ex vivo on optic nerves from rats with exacerbated experimental allergic encephalomyelitis, a model of multiple sclerosis. The nerves displayed characteristic immunopathology and expression of iNOS in macrophages and/or microglia and there was overt axonal damage in localized regions of the optic chiasm. The resulting NO levels in the optic nerve were sufficient to cause activation of guanylyl cyclase-coupled NO receptors, resulting in marked cGMP accumulation in axons throughout the nerve. Nevertheless, calibration of cGMP levels against those evoked by exogenous NO indicated that the nerves were not compromised metabolically and that their ambient NO concentration was only approximately 1 nm. Consistent with this observation, electrophysiological tests indicated that there was no ongoing malfunctioning of the type that can be elicited by high exogenous NO concentrations. It is concluded that, with iNOS expressed in physiological locations and levels, the tissue levels of NO remain at concentrations far lower than those shown to have toxic effects, despite continuous NO synthesis. The fact that NO can rise to much higher levels in dispersed cultures in vitro may be attributable to a deficiency in NO inactivation in such preparations.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/enzymology , Encephalomyelitis, Autoimmune, Experimental/pathology , Macrophages/enzymology , Nitric Oxide Synthase/metabolism , Optic Nerve/pathology , Ornithine/analogs & derivatives , 1-Methyl-3-isobutylxanthine/pharmacology , Action Potentials/drug effects , Animals , Arginine/pharmacology , Biomarkers/metabolism , CD11b Antigen/metabolism , CD2 Antigens/metabolism , Cyclic GMP/metabolism , DEET/pharmacology , Disease Models, Animal , Dose-Response Relationship, Drug , Electric Stimulation/methods , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Female , Guanylate Cyclase/metabolism , Guinea Pigs , Hydrazines/pharmacology , Immunohistochemistry/methods , Macrophages/pathology , Microscopy, Electron, Transmission/methods , Myelin Basic Protein/metabolism , Nitric Oxide/pharmacology , Nitric Oxide Synthase Type II , Optic Chiasm/pathology , Optic Chiasm/ultrastructure , Optic Nerve/drug effects , Optic Nerve/enzymology , Optic Nerve/ultrastructure , Ornithine/pharmacology , Phosphodiesterase Inhibitors/pharmacology , Rats , Rats, Inbred Lew , Time Factors
5.
J Neurol ; 250(11): 1293-301, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14648144

ABSTRACT

Different MRI techniques are used to investigate multiple sclerosis (MS) in vivo. The pathological specificity of these techniques is poorly understood, in particular their relationship to demyelination and axonal loss. The aim of this study was to evaluate the pathological substrate of high field MRI in post-mortem (PM) spinal cord (SC) of patients with MS. MRI was performed in PMSCs of four MS patients and a healthy subject on a 7 Tesla machine. Quantitative MRI maps (PD; T2; T1; magnetization transfer ratio, MTR; diffusion weighted imaging) were obtained. After scanning, the myelin content and the axonal density of the specimens were evaluated neuropathologically using quantitative techniques. Myelin content and axonal density correlated strongly with MTR, T1, PD, and diffusion anisotropy, but only moderately with T2 and weakly with the apparent diffusion coefficient. Quantitative MR measures provide a promising tool to evaluate components of MS pathology that are clinically meaningful. Further studies are warranted to investigate the potential of new quantitative MR measures to enable a distinction between axonal loss and demyelination and between demyelinated and remyelinated lesions.


Subject(s)
Axons/pathology , Magnetic Resonance Imaging , Multiple Sclerosis/pathology , Myelin Sheath/pathology , Spinal Cord/pathology , Aged , Diffusion Magnetic Resonance Imaging , Female , Humans , Male , Multiple Sclerosis/diagnostic imaging , Radiography , Spinal Cord/diagnostic imaging
6.
Neuropathol Appl Neurobiol ; 29(5): 434-44, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14507335

ABSTRACT

Dense astrocytic scarring in chronic multiple sclerosis (MS) plaques produces an inhibitory environment which can impede tissue repair. Animal studies have shown that the antigenic phenotype of the most abundant cell type in the brain, the astrocyte, varies depending on astrocyte type and location. To identify the phenotype of scar astrocytes (SAs) in chronic lesions, markers of reactive astrocytes characterized in animal studies were investigated. To date these are the only established markers. Cerebral subventricular deep white matter from normal control, MS normal appearing white matter and lesions (acute, subacute and chronic) were examined by immunohistochemistry and immunoblotting. The antigenic profile of SAs revealed significant modification of astrocyte protein expression in chronic MS lesions. SAs express nestin, embryonic neural cell adhesion molecule, fibroblast growth factor receptor 4, epidermal growth factor receptor, nerve growth factor and a subpopulation of SAs also express basic fibroblast growth factor. These are in addition to the expected markers glial fibrillary acidic protein, vimentin, and the tenascins C and R. Therefore, an SA antigenic phenotype has now been defined. This knowledge may allow the development of therapeutic strategies that prevent scar formation and promote tissue repair.


Subject(s)
Astrocytes/physiology , Multiple Sclerosis/genetics , Adult , Aged , Animals , Antibodies, Monoclonal , Blotting, Western , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Cicatrix , Humans , Immunohistochemistry , Middle Aged , Multiple Sclerosis/pathology , Phenotype
7.
J Neurol Neurosurg Psychiatry ; 74(2): 197-202, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12531948

ABSTRACT

BACKGROUND: Beta(2) adrenoreceptor expression on peripheral blood mononuclear cells is increased in progressive multiple sclerosis. This increase has been correlated with disease activity in relapsing-remitting multiple sclerosis. OBJECTIVE: To determine the beta(2) adrenoreceptor expression in primary and secondary progressive multiple sclerosis in relation to findings on magnetic resonance imaging (MRI) and clinical disease activity. METHODS: 10 patients with multiple sclerosis were studied (five with primary progressive and five with secondary progressive forms of the disease) over a period of six months. Monthly clinical and MRI assessments of the brain and spinal cord were carried out. Beta(2) adrenoreceptor expression was assessed monthly using a ligand binding assay with [(125)I]iodocyanopindolol. Expression of beta(2) adrenoceptors on peripheral blood mononuclear cells was also assessed in five normal controls over a similar period. RESULTS: The mean (SEM) value of beta(2) adrenoreceptor density for the five normal controls was 1346 (183) sites/cell, with affinity Kd of 120 (40) pM. MRI disease activity in primary progressive multiple sclerosis was reported on two occasions and on those occasions the expression of beta(2) adrenoreceptors was increased in excess of 1900 sites/cell; in the remaining 28 observations beta(2) adrenoreceptor expression was within the normal range (800 to 1900 sites/cell). In patients with secondary progressive disease, MRI disease activity was observed on 16 occasions. In these patients expression of beta(2) adrenoreceptors was increased in excess of 2000 sites/cell in all measurements except in one subject who did not show MRI activity throughout the six months period of study. The affinity of the receptors was within the normal range in all cases. CONCLUSIONS: Increased expression of beta(2) adrenoreceptors was correlated with MRI disease activity in two patients with primary progressive multiple sclerosis. In secondary progressive multiple sclerosis, increased expression of beta(2) adrenoreceptors tended not to correlate with MRI disease activity. This may reflect a persistent Th1 immune reaction in the secondary progressive form of the disease.


Subject(s)
Monocytes/immunology , Multiple Sclerosis, Chronic Progressive/diagnosis , Multiple Sclerosis, Relapsing-Remitting/diagnosis , Receptors, Adrenergic, beta-2/blood , Adult , Brain/immunology , Brain/pathology , Female , Humans , Iodine Radioisotopes , Iodocyanopindolol , Longitudinal Studies , Magnetic Resonance Imaging , Male , Middle Aged , Multiple Sclerosis, Chronic Progressive/immunology , Multiple Sclerosis, Relapsing-Remitting/immunology , Radioligand Assay , Spinal Cord/immunology , Spinal Cord/pathology , Th1 Cells/immunology
8.
J Neurochem ; 82(5): 1179-91, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12358765

ABSTRACT

Regulation of microglial reactivity and neurotoxicity is critical for neuroprotection in neurodegenerative diseases. Here we report that microglia possess functional group II metabotropic glutamate receptors, expressing mRNA and receptor protein for mGlu2 and mGlu3, negatively coupled to adenylate cyclase. Two different agonists of these receptors were able to induce a neurotoxic microglial phenotype which was attenuated by a specific antagonist. Chromogranin A, a secretory peptide expressed in amyloid plaques in Alzheimer's disease, activates microglia to a reactive neurotoxic phenotype. Chromogranin A-induced microglial activation and subsequent neurotoxicity may also involve an underlying stimulation of group II metabotropic glutamate receptors since their inhibition reduced chromogranin A-induced microglial reactivity and neurotoxicity. These results show that selective inhibition of microglial group II metabotropic glutamate receptors has a positive impact on neuronal survival, and may prove a therapeutic target in Alzheimer's disease.


Subject(s)
Alzheimer Disease/metabolism , Chromogranins/pharmacology , Microglia/metabolism , Neurons/cytology , Receptors, Metabotropic Glutamate/metabolism , Amyloid beta-Peptides/pharmacology , Animals , Apoptosis , Cells, Cultured , Chromogranin A , Coculture Techniques , Culture Media, Conditioned/toxicity , Microglia/cytology , Microglia/drug effects , Mitochondria/drug effects , Neurons/drug effects , Peptide Fragments/pharmacology , Phenotype , RNA, Messenger/biosynthesis , Rats , Rats, Wistar , Receptors, Metabotropic Glutamate/genetics , Reverse Transcriptase Polymerase Chain Reaction , Toxins, Biological/biosynthesis , Toxins, Biological/toxicity
9.
Brain ; 125(Pt 7): 1462-73, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12076997

ABSTRACT

Disease progression in multiple sclerosis occurs within the interface of glial activation and gliosis. This study aimed to investigate the relationship between biomarkers of different glial cell responses: (i) to disease dynamics and the clinical subtypes of multiple sclerosis; (ii) to disability; and (iii) to cross-validate these findings in a post-mortem study. To address the first goal, 51 patients with multiple sclerosis [20 relapsing remitting (RR), 21 secondary progressive (SP) and 10 primary progressive (PP)] and 51 neurological control patients were included. Disability was assessed using the ambulation index (AI), the Expanded Disability Status Scale score (EDSS) and the 9-hole PEG test (9HPT). Patients underwent lumbar puncture within 7 days of clinical assessment. Post-mortem brain tissue (12 multiple sclerosis and eight control patients) was classified histologically and adjacent sites were homogenized for protein analysis. S100B, ferritin and glial-fibrillary acidic protein (GFAP) were quantified in CSF and brain-tissue homogenate by ELISA (enzyme-linked immunosorbent assay) techniques developed in-house. There was a significant trend for increasing S100B levels from PP to SP to RR multiple sclerosis (P < 0.05). S100B was significantly higher in RR multiple sclerosis than in control patients (P < 0.01), whilst ferritin levels were significantly higher in SP multiple sclerosis than in control patients (P < 0.01). The S100B : ferritin ratio discriminated patients with RR multiple sclerosis from SP, PP or control patients (P < 0.05, P < 0.01 and P < 0.01, respectively). Multiple sclerosis patients with poor ambulation (AI > or =7) or severe disability (EDSS >6.5) had significantly higher CSF GFAP levels than less disabled multiple sclerosis or control patients (P < 0.01 and P < 0.001, respectively). There was a correlation between GFAP levels and ambulation in SP multiple sclerosis (r = 0.57, P < 0.01), and between S100B level and the 9HPT in PP multiple sclerosis patients (r = -0.85, P < 0.01). The post-mortem study showed significantly higher S100B levels in the acute than in the subacute plaques (P < 0.01), whilst ferritin levels were elevated in all multiple sclerosis lesion stages. Both GFAP and S100B levels were significantly higher in the cortex of multiple sclerosis than in control brain homogenate (P < 0.001 and P < 0.05, respectively). We found that S100B is a good marker for the relapsing phase of the disease (confirmed by post-mortem observation) as opposed to ferritin, which is elevated throughout the entire course. GFAP correlated with disability scales and may therefore be a marker for irreversible damage. The results of this study have broad implications for finding new and sensitive outcome measures for treatment trials that aim to delay the development of disability. They may also be considered in future classifications of multiple sclerosis patients.


Subject(s)
Multiple Sclerosis/pathology , Multiple Sclerosis/physiopathology , Neuroglia/pathology , S100 Proteins , Adult , Aged , Biomarkers/analysis , Brain Chemistry , Calcium-Binding Proteins/analysis , Calcium-Binding Proteins/cerebrospinal fluid , Cerebrospinal Fluid/chemistry , Enzyme-Linked Immunosorbent Assay , Female , Ferritins/analysis , Ferritins/cerebrospinal fluid , Glial Fibrillary Acidic Protein/analysis , Glial Fibrillary Acidic Protein/cerebrospinal fluid , Humans , Male , Middle Aged , Multiple Sclerosis/classification , Multiple Sclerosis, Chronic Progressive/pathology , Multiple Sclerosis, Chronic Progressive/physiopathology , Multiple Sclerosis, Relapsing-Remitting/pathology , Multiple Sclerosis, Relapsing-Remitting/physiopathology , Nerve Growth Factors/analysis , Nerve Growth Factors/cerebrospinal fluid , Neuroglia/metabolism , Predictive Value of Tests , S100 Calcium Binding Protein beta Subunit , Severity of Illness Index
10.
J Neurosci Res ; 66(6): 1173-8, 2001 Dec 15.
Article in English | MEDLINE | ID: mdl-11746450

ABSTRACT

An increased level of myelin basic protein (MBP) degradation peptide 80-89, representative of myelin breakdown, is detected in myelinating foetal rat brain aggregate cultures supplemented with peritoneal macrophages at a time coinciding with the onset of myelination. During the period of myelination, the proportion of activated macrophages/microglia in the aggregates decreases, accompanied by a reduction in the content of MBP degradation products. During the recovery period following a demyelinating episode, the rate of MBP synthesis in antibody-treated standard aggregates was greater than in their medium controls. However, the rate of MBP accumulation was not as efficient in macrophage-enriched aggregates and was associated with persistently raised MBP peptide levels. Thus, as occurs in multiple sclerosis lesions, attempts at remyelination appear to be counterbalanced by macrophage-mediated demyelination, with the continued presence of degraded myelin rendering a local environment that is not fully conducive to remyelination.


Subject(s)
Antigens, CD , Antigens, Neoplasm , Antigens, Surface , Avian Proteins , Blood Proteins , Brain/embryology , Macrophages/metabolism , Multiple Sclerosis/metabolism , Myelin Basic Protein/metabolism , Myelin Sheath/metabolism , Nerve Regeneration/immunology , Phagocytosis/immunology , Adult , Animals , Antibodies/pharmacology , Basigin , Brain/immunology , Brain/metabolism , Cell Count , Cell Size/immunology , Cells, Cultured/drug effects , Cells, Cultured/immunology , Cells, Cultured/metabolism , Ectodysplasins , Fetus , Humans , Immunohistochemistry , Macrophages/drug effects , Macrophages/immunology , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Middle Aged , Multiple Sclerosis/immunology , Multiple Sclerosis/physiopathology , Myelin Basic Protein/immunology , Myelin Proteins , Myelin Sheath/immunology , Myelin Sheath/pathology , Myelin-Associated Glycoprotein/antagonists & inhibitors , Myelin-Associated Glycoprotein/immunology , Myelin-Associated Glycoprotein/metabolism , Myelin-Oligodendrocyte Glycoprotein , Peptide Fragments/immunology , Peptide Fragments/metabolism , Rats , Rats, Sprague-Dawley
11.
Brain ; 124(Pt 10): 1978-88, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11571216

ABSTRACT

Components of the plasminogen activator (PA) and matrix metalloprotease (MMP) cascade have been characterized in multiple sclerosis lesions by immunohistochemistry, enzyme-linked immunosorbent assay and enzyme activity assays in order to establish a functional role for the enzyme sequence in lesion development. Highly significant quantitative increases in urokinase PA (uPA), urokinase receptor (uPAR) and plasminogen activator inhibitor-1 were detected in acute multiple sclerosis lesions (P < 0.0001) and in uPAR in normal-appearing white matter (P < 0.0001) compared with control tissue. All three proteins were immunolocalized to mononuclear cells in perivascular cuffs and to macrophages in the lesion parenchyma. MMP-9 and the tissue inhibitor of metalloprotease-1 also increased during lesion development but the enzyme was present largely in the inactive pro-form. In contrast to uPA, the concentration and activity of tissue PA (tPA), the most abundant plasminogen activator in normal control brain, were reduced in multiple sclerosis specimens. In acute lesions tPA co-localized with fibrin(ogen) on large diameter axons also stained with SMI-32, an immunohistochemical marker of axonal damage. The uPA-uPAR complex, concentrated on inflammatory cells in the perivascular zone of the evolving lesion, may facilitate cellular infiltration into the CNS which is amplified by MMP- mediated degradation of blood vessel matrix. tPA localization on injured axons may be a marker of axonal damage or represent a protective mechanism aimed at removal of fibrin deposits and restoration of axonal function.


Subject(s)
Axons/enzymology , Axons/pathology , Multiple Sclerosis/enzymology , Multiple Sclerosis/pathology , Plasminogen Activators/metabolism , Adult , Aged , Female , Humans , Inflammation/enzymology , Inflammation/pathology , Male , Matrix Metalloproteinases/metabolism , Middle Aged , Plasminogen Activator Inhibitor 1/metabolism , Receptors, Cell Surface/metabolism , Receptors, Urokinase Plasminogen Activator , Tissue Plasminogen Activator/metabolism , Urokinase-Type Plasminogen Activator/metabolism
12.
Am J Pathol ; 158(6): 2127-38, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11395390

ABSTRACT

Lewis rats, on recovery from monophasic clinical experimental allergic encephalomyelitis (EAE), can be induced to develop repeated paralytic relapses with a graded reduction in clinical severity following intraperitoneal administration of IL-12. By the time of the third relapse, the number and size of inflammatory cuffs in the spinal cord were reduced with the makeup of the cellular infiltrate shifting to a significantly increased number of B cells. Serum levels of myelin basic protein (MBP)-specific IgG1 and IgG2b were found to rise over time while MBP and MBP peptide-positive macrophages and microglia became evident in perivascular cuffs and in spinal cord parenchyma, indicative of myelin phagocytosis. Axonal death was observed in semithin and EM sections of spinal cord in third relapse animals in association with iNOS and tPA immunostaining throughout gray and white matter. These neurotoxic or excitotoxic agents may contribute to axonal damage directly or indirectly by activated microglia and macrophages, leading to limited damage of the axonal-myelin unit.


Subject(s)
Axons/pathology , Encephalomyelitis, Autoimmune, Experimental/pathology , Myelin Sheath/pathology , Acute Disease , Animals , Autoantibodies/biosynthesis , Axons/chemistry , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Encephalomyelitis, Autoimmune, Experimental/metabolism , Female , Immunoglobulin G/biosynthesis , Immunoglobulin G/chemistry , Interleukin-12 , Interleukins/blood , Leukocyte Count , Macrophage Activation , Myelin Basic Protein/immunology , Paralysis/chemically induced , Rats , Rats, Inbred Lew , Recurrence , Spinal Cord/pathology , Tissue Plasminogen Activator/analysis , Transforming Growth Factor beta/blood , Transforming Growth Factor beta1
13.
Proc Natl Acad Sci U S A ; 97(21): 11598-602, 2000 Oct 10.
Article in English | MEDLINE | ID: mdl-11027357

ABSTRACT

Clinical abnormalities in multiple sclerosis (MS) have classically been considered to be caused by demyelination and/or axonal degeneration; the possibility of molecular changes in neurons, such as the deployment of abnormal repertoires of ion channels that would alter neuronal electrogenic properties, has not been considered. Sensory Neuron-Specific sodium channel SNS displays a depolarized voltage dependence, slower activation and inactivation kinetics, and more rapid recovery from inactivation than classical "fast" sodium channels. SNS is selectively expressed in spinal sensory and trigeminal ganglion neurons within the peripheral nervous system and is not expressed within the normal brain. Here we show that sodium channel SNS mRNA and protein, which are not present within the cerebellum of control mice, are expressed within cerebellar Purkinje cells in a mouse model of MS, chronic relapsing experimental allergic encephalomyelitis. We also demonstrate SNS mRNA and protein expression within Purkinje cells from tissue obtained postmortem from patients with MS, but not in control subjects with no neurological disease. These results demonstrate a change in sodium channel expression in neurons within the brain in an animal model of MS and in humans with MS and suggest that abnormal patterns of neuronal ion channel expression may contribute to clinical abnormalities such as ataxia in these disorders.


Subject(s)
Brain/metabolism , Encephalomyelitis, Autoimmune, Experimental/metabolism , Multiple Sclerosis/metabolism , Neurons, Afferent/metabolism , Sodium Channels/metabolism , Animals , Brain/pathology , Encephalomyelitis, Autoimmune, Experimental/pathology , Humans , Immunohistochemistry , Mice , Multiple Sclerosis/pathology
14.
Brain ; 123 ( Pt 11): 2321-37, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11050032

ABSTRACT

This study identifies by microautoradiography activated microglia/macrophages as the main cell type expressing the peripheral benzodiazepine binding site (PBBS) at sites of active CNS pathology. Quantitative measurements of PBBS expression in vivo obtained by PET and [(11)C](R)-PK11195 are shown to correspond to animal experimental and human post-mortem data on the distribution pattern of activated microglia in inflammatory brain disease. Film autoradiography with [(3)H](R)-PK11195, a specific ligand for the PBBS, showed minimal binding in normal control CNS, whereas maximal binding to mononuclear cells was found in multiple sclerosis plaques. However, there was also significantly increased [(3)H](R)-PK11195 binding on activated microglia outside the histopathologically defined borders of multiple sclerosis plaques and in areas, such as the cerebral central grey matter, that are not normally reported as sites of pathology in multiple sclerosis. A similar pattern of [(3)H](R)-PK11195 binding in areas containing activated microglia was seen in the CNS of animals with experimental allergic encephalomyelitis (EAE). In areas without identifiable focal pathology, immunocytochemical staining combined with high-resolution emulsion autoradiography demonstrated that the cellular source of [(3)H](R)-PK11195 binding is activated microglia, which frequently retains a ramified morphology. Furthermore, in vitro radioligand binding studies confirmed that microglial activation leads to a rise in the number of PBBS and not a change in binding affinity. Quantitative [(11)C](R)-PK11195 PET in multiple sclerosis patients demonstrated increased PBBS expression in areas of focal pathology identified by T(1)- and T(2)-weighted MRI and, importantly, also in normal-appearing anatomical structures, including cerebral central grey matter. The additional binding frequently delineated neuronal projection areas, such as the lateral geniculate bodies in patients with a history of optic neuritis. In summary, [(11)C](R)-PK11195 PET provides a cellular marker of disease activity in vivo in the human brain.


Subject(s)
Antineoplastic Agents , Benzodiazepines/agonists , Brain/metabolism , Isoquinolines , Microglia/metabolism , Multiple Sclerosis/metabolism , Adult , Animals , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacokinetics , Binding Sites , Brain/diagnostic imaging , Brain/pathology , Carbon Radioisotopes , Encephalomyelitis, Autoimmune, Experimental/metabolism , Encephalomyelitis, Autoimmune, Experimental/pathology , Encephalomyelitis, Autoimmune, Experimental/physiopathology , Female , Humans , Isoquinolines/metabolism , Isoquinolines/pharmacokinetics , Magnetic Resonance Imaging , Male , Microglia/drug effects , Microglia/pathology , Middle Aged , Multiple Sclerosis/pathology , Multiple Sclerosis/physiopathology , Radioligand Assay , Rats , Rats, Inbred Lew , Tomography, Emission-Computed
15.
J Neuroimmunol ; 108(1-2): 192-200, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10900353

ABSTRACT

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) characterised by perivascular inflammatory cell infiltrates and plaques of demyelination. Chemokines have been shown to play an important role in the activation and directional migration of cells to sites of CNS inflammation. The action of chemokines requires the expression of their complementary chemokine receptors by their target cells. We have examined the expression of the beta-chemokine receptors CCR2, CCR3 and CCR5 in post-mortem MS CNS tissue using single- and double-labelling immunocytochemistry techniques. Low levels of CCR2, CCR3 and CCR5 were expressed by microglial cells throughout control CNS tissue. In chronic active MS lesions CCR2, CCR3 and CCR5 were associated with foamy macrophages and activated microglia. CCR2 and CCR5 were also present on large numbers of infiltrating lymphocytes. A smaller number of CCR3-positive lymphocytes were present, but we also noted CCR3 and CCR5 on astrocytes in five of the 14 cases of MS investigated, particularly associated with processes around vessels and at the glia limitans. Ligands for CCR2 and CCR3 include MCP-1 and MCP-3 which were co-localised around vessels with the infiltrating leukocytes, but were also present in unaffected areas of cortex. The elevated expression of CCR2, CCR3 and CCR5 in the CNS in MS suggests these beta-chemokine receptors and their ligands play a role in the pathogenesis of MS.


Subject(s)
Central Nervous System/metabolism , Cytokines , Multiple Sclerosis/metabolism , Receptors, CCR5/analysis , Receptors, Chemokine/analysis , Adult , Aged , Aged, 80 and over , Antibody Specificity , Astrocytes/metabolism , Astrocytes/pathology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Central Nervous System/immunology , Central Nervous System/pathology , Chemokine CCL2/analysis , Chemokine CCL7 , Chemotaxis, Leukocyte , Chronic Disease , Disease Progression , Female , Humans , Immunohistochemistry , Inflammation/immunology , Inflammation/metabolism , Inflammation/pathology , Ligands , Macrophages/immunology , Macrophages/metabolism , Male , Matched-Pair Analysis , Microglia/metabolism , Microglia/pathology , Middle Aged , Monocyte Chemoattractant Proteins/analysis , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Receptors, CCR2 , Receptors, CCR3 , Recurrence
16.
Neurosci Lett ; 287(2): 146-50, 2000 Jun 23.
Article in English | MEDLINE | ID: mdl-10854733

ABSTRACT

One difficulty in generating in vitro models of neuropathogenesis lies in maintaining stable proportions of primary neurons within a mixed brain cell population. Rotation-mediated fetal brain aggregate culture has been modified to permit growth of human primary fetal brain cells containing 50 to 60% neurons. After 12 weeks cholinesterase, neuron specific enolase and microtubule-associated protein-2 were demonstrable by biochemical assay and immunocytochemical labelling of cryostat sections of human fetal brain aggregates. Upon exposure to the glutamate agonist; N-methyl-D-aspartate for 7 days at 35 days in vitro neuron specific enolase and cholinesterase decreased to 60 to 70% of untreated levels. Glial fibrillary acidic protein did not change significantly but swollen astrocytes were seen in labelled sections of treated aggregates. This method is useful to study human neurotoxicity and degeneration in mixed glial culture without astrocyte overgrowth.


Subject(s)
Astrocytes/cytology , Brain/cytology , Cell Culture Techniques/methods , Excitatory Amino Acid Agonists/toxicity , N-Methylaspartate/toxicity , Neurons/cytology , Astrocytes/drug effects , Cell Separation/methods , Cells, Cultured , Fetus/cytology , Humans , L-Lactate Dehydrogenase/analysis , Microscopy, Confocal , Nerve Degeneration/chemically induced , Neurons/drug effects , Neurons/enzymology , Phosphopyruvate Hydratase/analysis , Rotation
17.
Neuropathol Appl Neurobiol ; 26(2): 133-42, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10840276

ABSTRACT

The recruitment of leucocytes to sites of inflammation is an important feature of multiple sclerosis (MS) pathology. Chemokines are involved in the activation and specific directional migration of monocytes and T-lymphocytes to sites of inflammation. Using immunocytochemistry, the expression of the alpha-chemokines, interferon (IFN)-gamma-inducible protein-10 (IP-10) and monokine induced by IFN-gamma (Mig), and their receptor CXCR3 have been examined in post-mortem central nervous system (CNS) tissue from MS cases at different stages of lesion development. In actively demyelinating lesions both IP-10 and Mig protein were predominantly expressed by macrophages within the plaque and by reactive astrocytes in the surrounding parenchyma. CXCR3 was expressed by T cells and by astrocytes within the plaque. Interferon-gamma may stimulate glial cells to express IP-10 and Mig, which continue the local inflammatory response by selectively recruiting activated T-lymphocytes into the CNS.


Subject(s)
Chemokines, CXC/immunology , Intercellular Signaling Peptides and Proteins , Interferon-gamma/immunology , Multiple Sclerosis/immunology , Receptors, Chemokine/immunology , Adult , Aged , Antibody Specificity , Central Nervous System/chemistry , Central Nervous System/immunology , Chemokine CXCL10 , Chemokine CXCL9 , Chemokines, CXC/analysis , Chemokines, CXC/genetics , Chemokines, CXC/metabolism , Demyelinating Diseases/immunology , Female , Gene Expression/immunology , Humans , In Situ Hybridization , Interferon-gamma/analysis , Interferon-gamma/genetics , Male , Middle Aged , RNA, Messenger/analysis , Receptors, CXCR3 , Receptors, Chemokine/analysis , Receptors, Chemokine/genetics , T-Lymphocytes/immunology
18.
Glia ; 30(4): 342-51, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10797614

ABSTRACT

Myelinogenesis in rat brain aggregate cultures is associated with a pattern of growth factor mRNA expression comparable to that of the developing brain. The rate of increase in platelet-derived growth factor-AA (PDGF-AA) expression was greatest just before the detection of myelin basic protein (MBP) mRNA in the cultures and remained high thereafter, consistent with in vivo observations. Levels of fibroblast growth factor-2 (FGF-2) and of ciliary neurotrophic factor (CNTF) mRNA increased continuously over the period of MBP accumulation. High rates of transforming growth factor beta1 (TGF-beta1), insulin-like growth factor-I (IGF-I), and neurotrophin-3 (NT-3) expression at early time points during the culture gradually decreased over time, indicative of a key regulatory role during oligodendrocyte development. The addition of demyelinative anti-myelin oligodendrocyte glycoprotein (anti-MOG) antibody resulted in a significant increase in MBP peptide fragments with a C-terminus at phenylalanine 89 indicating proteolytic breakdown of MBP after myelin phagocytosis. Immediately after antibody treatment the expression of CNTF mRNA was significantly increased, compared with controls, while that of FGF-2 and IGF-I, and of PDGF-AA peaked during the early and later stages of recovery respectively. Thus, specific growth factors combine to regulate myelination and remyelination in the aggregates; these data have implications for demyelinating disease in which protective growth factor secretion may be central to regeneration.


Subject(s)
Brain/metabolism , Demyelinating Diseases/metabolism , Growth Substances/genetics , Growth Substances/metabolism , Myelin Sheath/metabolism , RNA, Messenger/biosynthesis , Animals , Antibodies , Brain/cytology , Brain/drug effects , Cells, Cultured , Ciliary Neurotrophic Factor/metabolism , Demyelinating Diseases/chemically induced , Fibroblast Growth Factor 2/metabolism , Insulin-Like Growth Factor I/metabolism , Myelin Basic Protein/metabolism , Platelet-Derived Growth Factor/metabolism , Rats
19.
J Immunol ; 164(8): 4359-66, 2000 Apr 15.
Article in English | MEDLINE | ID: mdl-10754336

ABSTRACT

In the development of multiple sclerosis (MS), (re)activation of infiltrating T cells by myelin-derived Ags is considered to be a crucial step. Previously, alpha B-crystallin has been shown to be an important myelin Ag to human T cells. Since alpha B-crystallin is an intracellular heat shock protein, the question arises at what stage, if any, during lesional development in MS this Ag becomes available for CD4+ T cells. In 3 of 10 active MS lesions, alpha B-crystallin could be detected inside phagocytic vesicles of perivascular macrophages, colocalizing with myelin basic protein and myelin oligodendrocyte glycoprotein (MOG). Although the detectability of MOG in phagosomes is considered as a marker for very recent demyelination, MOG was detected in more macrophages and in more lesions than alpha B-crystallin. The disappearance of alpha B-crystallin from macrophages even before MOG was confirmed by in vitro studies; within 6 h after myelin-uptake alpha B-crystallin disappears from the phagosomes. Alpha B-crystallin-containing macrophages colocalized with infiltrating T cells and they were characterized by expression of MHC class II, CD40, and CD80. To examine functional presentation of myelin Ags to T cells, purified macrophages were pulsed in vitro with whole myelin membranes. These macrophages activated both myelin-primed and alpha B-crystallin-primed T cells in terms of proliferation and IFN-gamma secretion. In addition, alpha B-crystallin-pulsed macrophages activated myelin-primed T cells to the same extent as myelin-pulsed macrophages, whereas myelin basic protein-pulsed macrophages triggered no response at all. These data indicate that, in active MS lesions, alpha B-crystallin is available for functional presentation to T cells early during inflammatory demyelination.


Subject(s)
Antigen Presentation/immunology , Crystallins/immunology , Crystallins/metabolism , Demyelinating Diseases/immunology , Demyelinating Diseases/pathology , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , T-Lymphocytes/immunology , Autoantigens/immunology , Autoantigens/metabolism , B7-1 Antigen/metabolism , CD40 Antigens/metabolism , Cathepsins/metabolism , Cell Movement/immunology , Demyelinating Diseases/metabolism , Histocompatibility Antigens Class II/metabolism , Humans , Hydrolysis , Inflammation/immunology , Inflammation/metabolism , Inflammation/pathology , Macrophages/enzymology , Macrophages/immunology , Macrophages/metabolism , Macrophages/pathology , Multiple Sclerosis/metabolism , Myelin Proteins/metabolism , Myelin Sheath/immunology , Myelin Sheath/metabolism , Phagocytosis , T-Lymphocytes/metabolism , T-Lymphocytes/pathology
20.
Neuropathol Appl Neurobiol ; 25(3): 207-14, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10417662

ABSTRACT

In the present study the distribution of the inhibitory extracellular molecules tenascin-R (TN-R) and tenascin- C (TN-C) was examined by immunocytochemistry during evolution of the multiple sclerosis (MS) lesion, in which astrogliosis is a prominent feature. Sections were cut from five control cases and from 22 blocks containing lesions representing different pathological stages in 18 cases of secondary progressive MS. Widespread expression of TN-R was found in the normal human central nervous system (CNS), while that of TN-C was in general restricted to white matter. In acute MS plaques however, there was a similar striking loss of both TN-R and TN-C up to the edge of the lesion, where the macrophage density is greatest, extending into the apparently normal white matter. In subacute lesions a TN-C and/or TN-R-immunopositive reactive astrocyte subpopulation was prominent, reflecting synthesis of extracellular matrix molecules. Both tenascins were expressed throughout chronic MS plaques at levels similar to those seen in adjacent white matter. The loss of TN-R and TN-C in acute plaques is indicative of enzyme-mediated breakdown of the matrix which may be a marker of blood-brain barrier breakdown and leucocyte extravasation. Subsequent production of tenascins by reactive astrocytes may result in glial scar formation impeding remyelination and axonal repair in MS lesions.


Subject(s)
Cell Adhesion Molecules/metabolism , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Multiple Sclerosis/metabolism , Multiple Sclerosis/pathology , Tenascin/metabolism , Adult , Astrocytes/metabolism , Astrocytes/pathology , Astrocytes/ultrastructure , Central Nervous System/pathology , Central Nervous System/ultrastructure , Female , Humans , Immunoenzyme Techniques , Immunohistochemistry , Male , Middle Aged
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