Beata Dąbrowska-Bouta

Effects of antagonists of glutamate receptors on pro-inflammatory cytokines in the brain cortex of rats subjected to experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Inflammatory cytokines and glutamate neurotoxicity have been proposed as major determinants accompanying the demyelination and axonal degeneration observed during the course of MS. The present study using the animal model of MS known as experimental autoimmune encephalomyelitis (EAE) demonstrates that pharmacological inhibition of ionotropic NMDA glutamate receptors by their antagonists (amantadine and memantine) suppresses neurological symptoms of disease in EAE rats and reduces expression of pro-inflammatory cytokines in the brain. Conversely, antagonists of group I metabotropic glutamate receptors, mGluRs (LY 367385 and MPEP), do not affect the inflammatory process and the neurological condition of EAE rats.

DOES LEAD PROVOKE THE PEROXIDATION PROCESS IN RAT BRAIN SYNAPTOSOMES

Up to now there has been no information concerning the effect of lead on the peroxidation process in brain nerve endings. We have examined whether lead acetate (in chronic and acute models of toxicity in vivo and in vitro) affected the level of free radicals in synaptosomes obtained from rat brain. Simultaneously, we have checked the effect of peroxidation of Pb2+ on brain homogenates and microsomal fraction. Our results indicated that the lead level in synaptosomal fraction obtained from lead-treated rats was much higher than in controls. We did not observe induction of spontaneous and Fe(3+)-dependent peroxidation either in synaptosomes or in homogenates and brain microsomes after chronic and acute lead administration to the rats. Lead itself also did not enhance both processes when added in vitro to the control brain synaptosomes in micromolar concentrations. The lack of the lead effect on the peroxidation process in subcellular fractions of brain was rather surprising, because lead is known to be the accelerator of Fe(3+)-dependent peroxidation processes in liver. Additionally, livers from rats under the same toxicity conditions were examined. We have found that lead did not provoke spontaneous peroxidation in liver, but contrary to brain fractions, it drastically increased iron-dependent peroxidation in liver homogenates and microsomes. The lack of the effect of lead on inducing peroxidation processes in brain is probably the consequence of the brain having stronger protective mechanisms against its toxicity than the liver.

Modulation of glutamate transport and receptor binding by glutamate receptor antagonists in EAE rat brain.

The etiology of multiple sclerosis (MS) is currently unknown. However, one potential mechanism involved in the disease may be excitotoxicity. The elevation of glutamate in cerebrospinal fluid, as well as changes in the expression of glutamate receptors (iGluRs and mGluRs) and excitatory amino acid transporters (EAATs), have been observed in the brains of MS patients and animals subjected to experimental autoimmune encephalomyelitis (EAE), which is the predominant animal model used to investigate the pathophysiology of MS. In the present paper, the effects of glutamatergic receptor antagonists, including amantadine, memantine, LY 367583, and MPEP, on glutamate transport, the expression of mRNA of glutamate transporters (EAATs), the kinetic parameters of ligand binding to N-methyl-D-aspartate (NMDA) receptors, and the morphology of nerve endings in EAE rat brains were investigated. The extracellular level of glutamate in the brain is primarily regulated by astrocytic glutamate transporter 1 (GLT-1) and glutamate-aspartate transporter (GLAST). Excess glutamate is taken up from the synaptic space and metabolized by astrocytes. Thus, the extracellular level of glutamate decreases, which protects neurons from excitotoxicity. Our investigations showed changes in the expression of EAAT mRNA, glutamate transport (uptake and release) by synaptosomal and glial plasmalemmal vesicle fractions, and ligand binding to NMDA receptors; these effects were partially reversed after the treatment of EAE rats with the NMDA antagonists amantadine and memantine. The antagonists of group I metabotropic glutamate receptors (mGluRs), including LY 367385 and MPEP, did not exert any effect on the examined parameters. These results suggest that disturbances in these mechanisms may play a role in the processes associated with glutamate excitotoxicity and the progressive brain damage in EAE.

Modulation of Neurological Deficits and Expression of Glutamate Receptors during Experimental Autoimmune Encephalomyelitis after Treatment with Selected Antagonists of Glutamate Receptors

The aim of our investigation was to characterize the role of group I mGluRs and NMDA receptors in pathomechanisms of experimental autoimmune encephalomyelitis (EAE), the rodent model of MS. We tested the effects of LY 367385 (S-2-methyl-4-carboxyphenylglycine, a competitive antagonist of mGluR1), MPEP (2-methyl-6-(phenylethynyl)-pyridine, an antagonist of mGluR5), and the uncompetitive NMDA receptor antagonists amantadine and memantine on modulation of neurological deficits observed in rats with EAE. The neurological symptoms of EAE started at 10-11 days post-injection (d.p.i.) and peaked after 12-13 d.p.i. The protein levels of mGluRs and NMDA did not increase in early phases of EAE (4 d.p.i.), but starting from 8 d.p.i. to 25 d.p.i., we observed a significant elevation of mGluR1 and mGluR5 protein expression by about 20% and NMDA protein expression by about 10% over the control at 25 d.p.i. The changes in protein levels were accompanied by changes in mRNA expression of group I mGluRs and NMDARs. During the late disease phase (20–25 d.p.i.), the mRNA expression levels reached 300% of control values. In contrast, treatment with individual receptor antagonists resulted in a reduction of mRNA levels relative to untreated animals.

Influence of a low dose of silver nanoparticles on cerebral myelin and behavior of adult rats.

Nanoscale particles have large surface to volume ratio that significantly enhances their chemical and biological reactivity. Although general toxicity of nano silver (nanoAg) has been intensively studied in both in vitro and in vivo models, its neurotoxic effects are poorly known, especially those of low-dose exposure. In the present study we assess whether oral administration of nanoAg influences behavior of exposed rats and induces changes in cerebral myelin. We examine the effect of prolonged exposure of adult rats to small (10nm) citrate-stabilized nanoAg particles at a low dose of 0.2mg/kg b.w. (as opposed to the ionic silver) in a comprehensive behavioral analysis. Myelin ultrastructure and the expression of myelin-specific proteins are also investigated. The present study reveals slight differences with respect to behavioral effects of Ag(+)- but not nanoAg-treated rats. A weak depressive effect and hyperalgesia were observed after Ag(+) exposure whereas administration of nanoAg was found to specifically increase body weight and body temperature of animals. Both nanoAg and Ag(+) induce morphological disturbances in myelin sheaths and alter the expression of myelin-specific proteins CNP, MAG and MOG. These results suggest that the CNS may be a target of low-level toxicity of nanoAg.

Markers of oxidative stress in hepatopancreas of crayfish (Orconectes limosus, raf) experimentally exposed to nanosilver.

Silver nanoparticles, chemically neutral particles in the size range of 1–100 nm, express strong antimicrobial activity and therefore have a broad range of applications. The increased use of consumer products with nanosilver (nanoAg) may result in its release into the environment, and may particularly affect aquatic systems. The mechanisms of the harmful effects of nanoAg against aquatic organisms are unclear. Therefore, in the present study we investigate the pro‐oxidative potential of these nanoparticles in experimentally exposed crayfish Orconectes limosus. Markers of oxidative stress and parameters of the antioxidant cell defense system such as total glutathione, glutathione reductase and the level of sulfhydryl groups were examined in the hepatopancreas of both sexes of O. limosus collected seasonally from Białe Lake (Poland) and subsequently exposed to nanoAg particles for 2 weeks. Exposure to nanoAg led to a high concentration‐dependent increase in the rate of lipid peroxidation and a decrease of protein‐bound SH groups which indicates protein oxidation. These markers of oxidative stress were accompanied by decreased levels of thiols and reduced activity of glutathione reductase. These results indicate a deficiency of reduced glutathione and suggest that the exposed organisms have less efficient antioxidative mechanisms available to counter ROS‐mediated cellular stress. Furthermore, we find that confocal microscopy is of limited utility in monitoring the presence of silver nanoparticles in tissues of exposed crayfish.

Aroclor 1254 selectively inhibits expression of glial GLT-1 glutamate transporter in the forebrain of chronically exposed adult rat.

Aroclor 1254, a commercially produced mixture of polychlorinated biphenyls, is known to cause many adverse conditions, including neurotoxicity. It has been recently postulated that upregulation of N-methyl-d-aspartate receptors (NMDARs) and enhanced glutamate signalling which leads to excitotoxicity, is the mechanism of Aroclor-induced neurotoxicity. To obtain insights into the mechanisms underlying glutamatergic overstimulation, we investigated the function and expression of sodium-dependent glutamate transporters which are known to regulate extracellular glutamate concentrations in the brain. Exposure to Aroclor 1254 was found to significantly lower the uptake of radioactive glutamate into gliosomal fractions obtained from adult rat brains. It also markedly decreased the expression of both protein and mRNA of GLT-1, the main glial glutamate transporter. This indicates that downregulation of GLT-1 may potentially lead to disturbances in glutamate clearance. The expression of GLAST, another astroglial glutamate transporter, was unchanged under conditions of Aroclor toxicity. Conversely, we observed enhanced glutamate uptake into nerve-endings fractions paralleled by increased EAAC1 protein expression. This may reflect the induction of protective mechanisms.

Blockade of the kinin B1 receptor affects the cytokine/chemokine profile in rat brain subjected to autoimmune encephalomyelitis

Kinins are bioactive peptides which provide multiple functions, including critical regulation of the inflammatory response. Released during tissue injury, kinins potentiate the inflammation which represents a hallmark of numerous neurological disorders, including those of autoimmune origin such as multiple sclerosis (MS). In the present work, we assess the expression of B1 receptor (B1R) in rat brain during the course of experimental autoimmune encephalomyelitis (EAE) which is an animal model of MS. We apply pharmacological inhibition to investigate the role of this receptor in the development of neurological deficits and in shaping the cytokine/chemokine profile during the course of the disease. Overexpression of B1R is observed in brain tissue of rats subjected to EAE, beginning at the very early asymptomatic phase of the disease. This overexpression is suppressed by a specific antagonist known as DALBK. The involvement of B1R in the progression of neurological symptoms in immunized rats is confirmed. Analysis of an array of cytokines/chemokines identified a sub-group as being B1R-dependent. Increase of the protein levels for the proinflammatory cytokines (Il-6, TNF-α but not IL-1β), chemokines attracting immune cells into nervous tissue (MCP-1, MIP-3α, LIX), and protein levels of fractalkine and vascular endothelial growth factor observed in EAE rats, were significantly diminished after DALBK administration. This may indicate the protective potential of pharmacological inhibition of B1R. However, simultaneously reduced protein levels of anti-inflammatory and neuroprotective factors (IL-10, IL-4, and CNTF) was noticed. The results show that B1R-mediated signaling regulates the cellular response profile following neuroinflammation in EAE.

Ultrastructural and biochemical features of cerebral microvessels of adult rat subjected to a low dose of silver nanoparticles.

The widespread use of silver nanoparticles (AgNPs) in medicine and in multiple commercial products has motivated researchers to investigate their potentially hazardous effects in organisms. Since AgNPs may easily enter the brain through the blood-brain barrier (BBB), characterization of their interactions with cellular components of the neurovascular unit (NVU) is of particular importance. Therefore, in an animal model of prolonged low-dose exposure, we investigate the extent and mechanisms of influence of AgNPs on cerebral microvessels. Adult rats were treated orally with small (10 nm) AgNPs in a dose of 0.2 mg/kg b.w. over a 2-week period. A silver citrate-exposed group was established as a positive control of ionic silver effects. Alterations in the expression of tight junction proteins claudin-5, ZO-1, and occludin, were observed. These effects are accompanied by ultrastructural features indicating enhanced permeability of microvessels such as focal edema of perivascular astrocytic processes and surrounding neuropil. We did not identify changes in the expression of PDGFβR which is a marker of pericytes. Ultrastructural alterations in these cells were not identified. The results show that altered integrity of cerebral vessels under a low-dose of AgNP-exposure may be the consequence of dysfunction of endothelial cells caused by disruption of tight junction proteins.

Administration of an antagonist of P2X7 receptor to EAE rats prevents a decrease of expression of claudin-5 in cerebral capillaries

Purinergic P2X receptors, when activated under pathological conditions, participate in induction of the inflammatory response and/or cell death. Both neuroinflammation and neurodegeneration represent hallmarks of multiple sclerosis (MS), an autoimmune disease of the central nervous system. In the current study, we examined whether P2X7R is expressed in brain microvasculature of rats subjected to experimental autoimmune encephalomyelitis (EAE) and explore possible relationships with blood-brain barrier (BBB) protein—claudin-5 after administration of P2X7R antagonist—Brilliant Blue G (BBG). Capillary fraction isolated from control and EAE rat brains was subjected to immunohistochemical and Western blot analyses. We document the presence of P2X7R in brain capillaries isolated from brain tissue of EAE rats. P2X7R is found to be localized on the abluminal surface of the microvessels and is co-expressed with PDGFβR, a marker of pericytes. We also show over-expression of this receptor in isolated capillaries during the course of EAE, which is temporally correlated with a lower protein level of PDGFβR, as well as claudin-5, a tight junction-building protein. Administration of a P2X7R antagonist to the immunized rats significantly reduced clinical signs of EAE and enhances protein expression of both claudin-5 and PDGFβR. These results indicate that P2X7 receptor located on pericytes may contribute to pathological mechanisms operated during EAE in cerebral microvessels influencing the BBB integrity.