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Chronic glutamate-mediated excitotoxicity has been suggested to contribute to the pathogenesis of Multiple Sclerosis (MS). Recent data suggest that inhibition of glutamate neurotransmission via specific interaction with glutamate receptors (GluRs) might be interesting for inhibition of disease progression and early symptomatic treatment in MS. The aim of our investigation was to study the role of NMDA receptors and group I mGluRs in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We tested the effect of MPEP (2-methyl-6-(phenylethynyl)-pyridine), the mGluR5 antagonist, in dose of 5 mg/kg b.w./day, and amantadine (the uncompetitive NMDA receptor antagonist) in dose of 100 mg/kg b.w./day on development of neurological deficits in EAE rats. Both drugs were administered intraperitoneally ones daily into EAE rats during 7 days, starting from day 5 to 12 post immunization. The neurological symptoms of EAE started at 10-11 days post immunization and peaked after 12-13 days. We noted the changes in body weight during the course of EAE. Until day 8 p.i. the body weight of rats in control and treated groups was in the same range. Starting from day 8 p.i. rats in all groups showed a progressive weight loss by about 20-30% until day 14 p.i. Application of amantadine was found to be effective and significantly reduced neurological symptoms of EAE. We did not observed any neuroprotective effects of MPEP. The level of mGluR5 protein did not increase in early phase of EAE (4 day p.i.). However, starting from day 8 p.i. to day 25 p.i. we observed its significant elevation. The difference between control and examined group reached 20% at 25 day p.i. We did not observe significant differences in mGluR5 level between three experimental groups: EAE rats (control), MPEP-treated and amantadine-treated rats. Our results confirm the involvement of glutamate into pathogenesis of EAE. Although we noted changes in the expression of mGluR 5 during the course of EAE, MPEP was ineffective in reducing the symptoms of the disease. Results suggest the main role of NMDA glutamate receptors in the pathogenesis of EAE. Research was supported by funds from grant nr: NN401620038 from Polish Ministry of Scientific and Higher Education
BACKGROUND AND AIMS: Multiple sclerosis (MS) is an autoimmune, neuroinflammatory disease resulting in progressive demyelinization. Glial cells are important players during development of inflammationwithin central nervoussystem.Underinflammatory signals astroglia and microglia change their morphology and up-regulate expression of GFAP and Iba1 proteins, respectively. Both of these cell populations express purinergic receptor P2X7. The aim of the study was to investigate the effect of P2X7R antagonist (Brilliant Blue G, BBG) on the course of experimental autoimmune encephalomyelitis (EAE) with the focus on the influence of BBG-treatment on the reactive gliosis. METHODS: EAE, an animal model of MS, was evoked by the immunization of Lewis rats with guinea pigs’ spinal cord homogenate with complete Freund’s adjuvant. BBG (50 mg/kg) was administered daily from day 0 to day 6 post immunization through the jugular catheter. RESULTS: Immunofluorescent analysis of brain slices showed the activation of astroglial and microglial cells in EAE rats, but not in EAE rats treated with BBG. The expression of GFAP was 5,5 times higher in EAE than in EAE+BBG group at the asymptomatic phase and 3.1 times higher at symptomatic phase of the disease. These results were confirmed by western blot analysis. Expression of Iba1 was 5.4 times and 3.6 times higher in the EAE than in EAE+BBG group at the asymptomatic and symptomatic phases, respectively. The effects of BBG treatment on the expression of proinflammatory cytokines such as IL-1β, IL-6, TNF-α were determined by western blot method. The level of these cytokines was significantly lower in EAE+BBG group than in EAE animals and remained similar to that observed in control rats. CONCLUSIONS: Blockade of P2X7R during the course of EAE results in: (1) improved condition of Animals; (2) decreased activation of astro- and microglia; (3) decreased level of proinflamatory cytokines Supported by Polish National Science Centre, grant nr: DEC-2012- /05/N/NZ4/02191.
In the CNS an intensive communication between neurons and glial cells occurs. Activation of astrocytes is observed under different pathological conditions, including multiple sclerosis, which results in overexpression of number of proteins, like GFAP and S-100β, which is involved in development of infl ammatory reaction. There is a growing number of evidence that different brain pathologies are characterized by very early active contribution of astrocytes to neurodegenerative axonal damage. Our study presents time-dependent analysis of astroglia-specifi c protein expression in different phases of EAE (from 4 to 25 days after immunization). The biphasic response of astroglia was observed ñ upregulation of both proteins, GFAP and S-100β, in the early stages of the disease and in the peak of the neurological defi cits in animals (10 dpi). Astrocytes build a network within the CNS and are connected by gap junctions, formed by connexins, mostly Cx43 which was shown to induce ATP release via hemichannels. In the cross-talk between astrocytes and neurons may be involved purinergic receptor P2X7, ATP-gated ion channel activated in pathological conditions and participating in regulation of infl ammatory response. In the EAE rats, in the early stages of the disease, we observed the enhanced level of Cx43 protein and P2X7R protein which was accompanied by changes in mRNA profi le. We conclude that early activation of astroglia in the inductive phase of EAE occurs which is connected with the overexpression of purinergic receptor P2X7. The results suggest that in MS/EAE pathology activation of astroglia in the preclinical stage, may contribute to the axonal damage and subsequent infl ammation, and that the purinergic signaling may play a role in both these phenomena.
BACKGROUND AND AIMS: Silver nanoparticles (AgNPs) are one of the most important class of nanomaterials used in a wide range of medical and industrial applications. However, the information about their toxicity to mammalsislimited. The aim of thisstudy was to investigate the effect of oral exposure to AgNPs on brain and liver of rats. The deposition of silver nanoparticles in these organs has been shown to induce hepatotoxicity and neurotoxicity. These toxic effects may include oxidative stress with subsequent inflammatory response. METHODS: Wistar rats were exposed orally to AgNPs (10±4 nm in diameter) or silver ions at a dose of 0.2 mg AgNPs or Ag+/ kg bw for 14 days. Then all animals were sacrificed 24 h after last exposure and tissues were collected for further studies. RESULTS: The presence of AgNPs in brain tissue was confirmed by using TEM technique. The level of free radicals and end-products of lipid peroxidation increased in both organs after exposure to both, silver nanoparticles or silver ions. However, the changes in inflammatory markers (based on IL-1β, IL-6 and TNF-α relative protein levels) were not statistically significant. CONCLUSIONS: These results show that following AgNPs or silver ions administration, oxidative stress is induced in brain and liver tissues. However, it seems that the time of exposure to both of silver forms was too short to cause the inflammatory response.
Blood-brain barrier (BBB) is a structure that maintains central nervous system (CNS) homeostasis by isolating it from the normal blood flow. In physiological conditions BBB prevents CNS penetration by blood-derived molecules and is a barrier for the immune system. BBB is built by tight junctions between endothelial cells of microvessels, pericytes, and astroglial end-feets. Pericytes are very important part of BBB showing a great impact on properties of endothelial cells and BBB tightness. In pathological conditions (i.e. inflammation) the structure of BBB is loosened and cells of the immune system have a free access to the brain and the spinal cord. That is the main mechanism of pathogenesis in both multiple sclerosis (MS) and the rodent model of the disease – experimental autoimmune encephalomyelitis (EAE). Overactivation of purinergic receptor P2X7, is a possible mechanism leading to neurodegeneration observed during the course of MS/EAE. This receptor has two distinct functions: it participates in maturation and release of proinflammatory cytokines or can polymerase to create transmembrane pores which can drive cell to death by apoptosis or necrosis. Thus, we hypothesized that overactivation of this receptor on pericytes may lead to cell damage and/or loss of the protective function towards BBB. In this study we first analyzed status of BBB which was determined by expression of claudin 5 – a marker of BBB tightness – in correlation with the expression of P2X7R in microvessels’ fractions and brain sections of rats subjected to EAE. Using immunoblots and confocal microscopic method we found negative correlation between P2X7R and claudin 5 expression which decreased significantly in all examined time points of the disease, reaching the minimum level (45% and 70% of control) at days 2 p.i. and 4 p.i., respectively. Additionally, we present the results of pericytes features and P2X7R expression in microvessels in early time after EAE induction. Condition of pericytes was visualized by immunofluorescent staining against PDGFRβ (a marker protein). Semiquantitative level of this protein was measured using Western blot analysis of brain homogenates and isolated microvessels fraction. The pattern of observed changes suggests contribution of pericyte-located P2X7R on BBB state and the involvement of this receptor into pathological mechanisms connected with development of EAE.
Under different pathological conditions activation of astrocytes of neuroprotective or neurotoxic nature is observed. There is a growing number of evidence that many pathological states of brain are characterized by very early active contribution of astrocytes to neurodegenerative axonal damage. Astroglia posses defense mechanisms against glutamate excitotoxicity (transporter systems) but may also contribute to the enhanced release of this potentially toxic amino acid trough exocytosis, P2X7 purinergic receptors, hemichannels or reversing of glutamate transporters. These cells are also a main source of ATP, active signaling molecule, which activates many purinergic receptors in brain, including P2X7R, which participates in development of infl ammation and neurodegeneration phenomena. The aim of this study was to investigate the expression of astroglia-specifi c proteins during the course of EAE using immunochemical and immunohistochemical analysis. We observed early activation of astroglia in the inductive phase of EAE (4 day p.i.) which was connected with overexpression of GFAP and S-100β. Expression of Cx43, protein that forms hemichannels was also enhanced so as the expression of P2X7R. Additionally, the level of GLT-1 glutamate transporter’s protein increase signifi cantly. The results suggest that in EAE pathology very early activation of astroglia takes place in the preclinical stage of the disease. The exact nature of this activation will be investigated.
Experimental autoimmune encephalomyelitis (EAE) is an animal model that mimics many aspects of multiple sclerosis (MS). Chronic or relapsing inflammation of the central nervous system results in the destruction of myelin sheath and cytokines play an important role in the pathogenesis of both MS and EAE. Myelin, oligodendrocytes and neurons are lost due to an inflammatory attack by leukocytes infiltrating the central nervous system (CNS) and releasing cytotoxic cytokines, anti CNS antibodies and large amounts of the excitatory neurotransmitter glutamate. Pharmacological studies have suggested that glutamate receptors mediate white matter injury in a variety of CNS diseases, including multiple sclerosis (MS). Memantine and amantadine are ionotropic glutamate receptors (iGluRs) antagonists. Memantine, a clinically applied drug with N-methyl-D-aspartate (NMDA) receptor antagonistic effects, dose-dependently ameliorates neurological deficits in Lewis rats subjected to experimental autoimmune encephalomyelitis (EAE). The aim of the present study was to investigate the effects of memantine and amantadine on the expression of proinflammatory cytokines such interleukin 1beta (IL-1β), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α) and various chemokines in the brain of EAE rats. Real-time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western Blot were used to analyze the cytokine profile. We noticed increased expression of array of cytokines in experimental group when compared to the control. Dramatic increase of IL-1β, IL-6, TNF-α, and chemokines concentration corresponding to the intensity of neurological symptoms and loss of weight was observed in EAE rats. Administration of iGluR antagonists at an advanced stage of unremitting EAE resulted in amelioration of the disease. Cytokine analysis revealed that memantine significantly decreased the expression of interleukins: IL-6 (65%), IL-1β (60%) and TNF-α (45%) whereas treatment with amantadine reduced only the expression of IL-6 (60%) and TNF-α (15%) when compared to EAE animals. These results show that antagonists of iGlu receptors modulate the course of the disease by reducing the expression of proinflammatory cytokines thereby confirming the involvement of glutamate receptors into pathological mechanisms operating during EAE. This study was supported by grant nr NN401620038 from Polish Ministry of Science and Higher Education
Multiple sclerosis (MS) is an autoimmune, neurodegenerative disease which is one of the most frequent reasons of disabilities of young adults and a serious problem for modern medicine due to the unknown etiology. Experimental autoimmune encephalomyelitis (EAE) is a commonly used rodent model of MS. EAE is evoked by immunization of female Lewis rats with homogenate of guinea pigs’ spinal cord combined with complete Freund’s adjuvant and inactivated Mycobacterium tuberculosis. It is well known that during the development of MS and EAE, the immune system sensitizes against self myelin and the permeability of blood-brain barrier (BBB) increases what enables an inflow of immune cells into the central nervous system. The immune system attacks and destroys myelin in the brain and the spinal cord what further leads to degeneration of neurons. The aim of the study was to investigate the time-window of microglial activation, the level of proinflammatory cytokines (IL-1, IL-6, TNFα) and the status of BBB in the early stages of EAE. We correlated the results with the microglial and endothelial expression of purinergic P2X7 receptor which is known to play a role in inflammation due to a release of proinflammatory mediators. The results of microscopic analysis revealed the increased permeability of BBB. At day 2 and 4 p.i. we also observed decreased expression of claudin5 protein which is an important marker of BBB tightness. However, starting from day 6 p.i. we noticed significant upregulation of this protein expression. The early activation of microglial cells at day 4 post immunization (dpi), in asymptomatic phase of the disease, together with an increased level of proinflammatory cytokines were observed. These changes correlated temporary with overexpression of P2X7R which was noticed on microglial cells and pericytes of blood vessels in brains of EAE rats. The results suggest the critical role of this receptor in early events during EAE development.
Silver nanoparticles (AgNPs) demonstrate strong antimicrobial activity resulting in their wide-spread use in different applications, including medical ones. Despite of the fact that human exposure to nanosilver is constantly increasing, there is no many research dedicated for investigating their potential neurotoxic effects. Most of the previous studies on mechanisms of nanosilver neurotoxicity have used in vitro models. The aim of the present study was to determine whether this small-sized commercially available AgNPs induce ultrastructural and biochemical changes in brain of male adult rats. Rats were exposed orally to 10 ± 4 nm nanosilver in size for 14 days. Using transmission electron microscopy (TEM), nanosized granules were detected in brains of exposed rats. Besides, ultrastructural changes in cortical and hippocampal neurons were found. TBARs level was measured to assessed lipid peroxidation in homogenates from exposed animals and immunochemical analysis was used to measure the level of selected tissue markers of oxidative stress in brain. Supported by grant nr NN401619938.
Multiple sclerosis is a serious problem of medicine and one of the most frequent reasons of disabilities of young adults. EAE is a commonly used rodent model of MS. In physiological conditions the blood-brain barrier (BBB) maintains CNS homeostasis and prevents uncontrolled inflow of immune cells from the blood circuit. During the development of EAE, damaged BBB fails to protect CNS from autoreactive immune cells. In this study we try to investigate a possible role of P2X7R in pathological changes of BBB during EAE. Analyzing BBB tightness we observed decreased expression of Claudin5 (protein component of tight-junctions) in the isolated microvessels’ fraction using western blot technique. These data were confirmed by immunofluorescence staining of brain sections against Claudin5. To assess functional state of BBB we carried out immunohistochemical staining against albumin and we observed its extravasation in early phase of EAE. All these data suggest dysfunction of BBB at the early stage of the disease. In parallel we observed overexpression of P2X7R in microvessels’ fraction and noticed correlation between its expression and increased BBB permeability using antagonist of P2X7R. Supported by grant nr: 2012/05/N/NZ4/02191
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