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1

Expression of purinergic P2X(7) receptor in rat brain during the symptomatic phase of experimental autoimmune encephalomyelitis and after recovery of neurological deficits

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Grygorowicz T., Sulejczak D., Struzynska L.
Acta Neurobiologiae Experimentalis
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2011
|
tom 71
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nr 1
Purinergic ionotropic P2X7 receptor is widely distributed in brain. Strong evidence suggests that this receptor is related to inflammatory and neurodegenerative changes in many pathological states of central nervous system (CNS), including multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) is the commonly used animal model of MS. In this study we investigate the expression of P2X7R protein in rat brain in the symptomatic phase of EAE (day 10 post immunization) and after reversion of neurological symptoms (day 20 p.i.). We found the increased level of P2X7R protein in brain homogenates of EAE rats in both examined time windows. Immunohistochemical study revealed enhanced receptor's immunoreactivity. Immunoblots done with isolated cellular brain fractions indicated that the P2X7R overexpression is related to synaptosomal fraction in the symptomatic phase and to the glial (GPV) fraction in the recovery phase of EAE. Concomitantly, we noticed overexpression of astroglial marker GFAP in brain homogenates and astroglial fraction (GPV), so as its enhanced immunoreactivity in brain sections (10 days p.i.) which did not decline to control values in the recovery phase, similarly to P2X7R expression. Results suggest the involvement of P2X7R-mediated signaling in the pathomechanisms of EAE with the possible relevance of astrocytic pool of cells.
2

The role of P2X7R in activation of glial cells during the early phase of experimental autoimmune encephalomyelitis

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Grygorowicz T., Welniak-Kaminska M., Lenkiewicz A., Struzynska L.
Acta Neurobiologiae Experimentalis
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2015
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tom 75
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nr Supl.
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.
3

Influence of DALBK-an antagonist of kinin receptor B1R-on the course of experimental autoimmune encephalomyelitis in rats

100%
Podsiadlo K., Grygorowicz T., Struzynska L.
Acta Neurobiologiae Experimentalis
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2015
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tom 75
|
nr Supl.
BACKGROUND AND AIMS: Experimental autoimmune encephalomyelitis (EAE) has been used for several decades as an animal model of multiple sclerosis (MS), an inflammatory demyelinating disease. According to the previous studies mammalian central nervous system presents all components of the kallikrein-kinin system. Biological activity of kinin is mediated by two types of G proteinbound receptors – B1 and B2. Therefore, there are reasons to investigate the participation of B1 receptor in enhancement of the BBB permeability during development of EAE. METHODS: One group of female Lewis rats was immunized by intradermal injection. The second group was injected ip with DALBK (B1R antagonist) after immunization. Control group was not immunized. Rats were monitored daily for clinical signs and loss of weight. Animals were sacrificed in different stages of the disease. Parts of brains were used for Western blotting analysis. Immunohistochemical study was also performed. RESULTS: We noticed the increased level of B1R protein in rat brain in the symptomatic phase of EAE. Animals treated with DALKB exhibited improvement of neurological symptoms and decreased level of B1R protein in most cases. Using a confocal microscope we assessed immunoreactivity of B1R and markers of individual components of glio-vascular unit (astrocytes, endothelial cells and pericytes). We also noticed changes in the level of astroglial markers GFAP and AQP4 during EAE, so as decreased expression of thight jounctions proteins (ZO-1, Occludin, Claudin 5), which were partially abolished by DALBK. CONCLUSIONS: Administration of kinin B1 receptor antagonist (DALBK) significantly improved the condition of animals by reducing the proinflammatory effects of kinins. The results show that the kinin receptor B1 may play a role in pathomechanisms operating during the course of EAE and appears to be a potential therapeutic target. This work was supported by funds from KNOW 2013-2017 project.
4

Cerebral microvessels in rat subjected to EAE: Focus on pericytes and purinergic receptor P2X7

100%
Grygorowicz T., Lenkiewicz A., Struzynska L.
Acta Neurobiologiae Experimentalis
|
2013
|
tom 73
|
nr 1
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.
5

Glial expression of purinergic P2X7 receptor in rat brain during the course of experimental autoimmune encephalomyelitis

100%
Grygorowicz T., Sulejczak D., Sulkowski G., Struzynska L.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr 1
Multiple sclerosis is a common neurodegenerative disease with prevalence in Poland about 15 per 10000 people. It is characterized by inflamed lesions in myelin sheaths surrounding axons in the white matter of the brain and spinal cord. These changes lead to the damage of axons and, in consequence, to a broad spectrum of neurological symptoms. Experimental autoimmune encephalomyelitis (EAE) is the well known and commonly used animal model of MS. In the present study the temporal pattern of glial activation (microglia and astroglia) together with P2X7R expression were investigated in brain of Lewis rats during the course of EAE. This receptor, activated under pathological conditions, may participate in the regulation of inflammatory response and cell death. It was shown to induce the release of inflammatory mediators like Il-1b and TNF-a in different types of glial cells. It is also pathologically involved in the release of potentially cytotoxic substances like glutamate and ATP. Western blot analysis was used to assess the relative concentration of P2X7R protein in glial fraction whereas its cellular localization was studied by immunohistochemical method. Tissue was labeled with the specific markers (Iba1 - microglial marker, GFAP - astroglial marker) and examined in the different stages post immunization (2, 4, 6, 8, 10 days). We observed the early overexpression of P2X7R protein (2-4 d.p.i.) in glial fraction obtained from brains of EAE rats with parallel enhancement of glial markers. Double immunofluorescent labeling showed colocalization of the receptor with glial markers. The results revealed that activation of both astroglia and microglia takes place very early post immunization, well before the neurological symptoms of the disease occur and is temporary connected with the overexpression of P2X7R. This suggests the involvement of P2X7R-mediated signals into the early pathological mechanisms operating during the disease.
6

Astroglial reaction in the early asymptomatic phase of experimental autoimmune encephalomyelitis

88%
Grygorowicz T., Sulkowski G., Sulejczak D., Lenkiewicz A., Struzynska L.
Acta Neurobiologiae Experimentalis
|
2009
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tom 69
|
nr 3
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.
7

Overexpression of P2X7R and early activation of cerebral microglia during experimental autoimmune encephalomyelitis

88%
Grygorowicz T., Sulejczak D., Rafalowska J., Lenkiewicz A., Struzynska L.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr S
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.
8

Ultrastructural and biochemical changes in brain of rat chronically exposed to silver nanoparticles

76%
Widynska J., Dabrowska-Bouta B., Frontczak-Baniewicz M., Lenkiewicz A., Grygorowicz T., Struzynska L.
Acta Neurobiologiae Experimentalis
|
2013
|
tom 73
|
nr Suppl.1
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.
9

Possible role of microvessel-located P2X7R in BBB function during the course of EAE

76%
Grygorowicz T., Rafalowska J., Lenkiewicz A., Chrzanowska H., Wojda R., Szopinski R., Struzynska L.
Acta Neurobiologiae Experimentalis
|
2013
|
tom 73
|
nr Suppl.1
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
10

Peripheral inflammation affects function of trigeminal ganglion neurons

76%
Kuzawinska O., Lis K., Grygorowicz T., Cudna A., Dabrowska M., Mirowska-Guzel D., Balkowiec-Iskra E.
Acta Neurobiologiae Experimentalis
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2015
|
tom 75
|
nr Supl.
BACKGROUND AND AIMS: Our previous studies showed that inflammatory reaction in the area of trigeminal ganglion (TG) nociceptive endings affects neurochemical properties of TG, causing increase in concentration of both brain-derived neurotrophic factor (BDNF) and calcitonin gene-related peptide (CGRP). In the present study we have employed CFA-induced TG inflammation model to begun investigating the underlying molecular mechanisms of the inflammation-induced changes in trigeminal neurons properties. METHODS: A model of orofacial inflammation was obtained by local injection of CFA to the mice whisker pad. Pain reaction was assessed every day by von-Frey filaments. 7 and 14 days after CFA injection mice were euthanized. Both TG were removed for ELISA and quantitative PCR analysis of differences in concentration and expression of BDNF, CGRP and selected proinflammatory cytokines, both in male and female mice. After perfusion with 4% paraformaldehyde, TG were removed from the sculls, crioprotected and cut on the 20 μm sections. Tissue was immunostained using primary antibodies against BDNF and TRPV1 and then with secondary antibodies conjugated with Alexa Fluor. RESULTS: Expression of proinflammatory cytokines, BDNF and CGRP was different in male and female mice. Our results indicate that response of TG to peripheral inflammatory reaction is genderdependent, what may explain differences in frequency and severity of trigeminal nerve-associated disorders observed between women and men. CONCLUSIONS: TG neurons in female mice showed increased expression of CGRPa, this neuropeptide may act as the main mediator of trigeminal signaling during migraine. This project has been supported by the Polish National Science Center, based on the Decision No. DEC-2012/05/B/NZ4/02385. Research subject implemented with CePT infrastructure financed by the European Union – the European Regional Development Fund within the Operational Programme “Innovative economy” for 2007-2013”
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