Nadezda Nedeljkovic

Up-regulation of ectonucleotidase activity after cortical stab injury in rats

The objective of this study was to examine the changes in the activity and expression of ectonucleotidase enzymes in the model of unilateral cortical stab injury (CSI) in rat. The activities of ecto‐nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1) and ecto 5′‐nucleotidase were assessed by measuring the levels of ATP, ADP and AMP hydrolysis in the crude membrane preparations obtained from injured left cortex, right cortex, left and right caudate nucleus, whole hippocampus and cerebellum. Significant increase in NTPDase and ecto 5′‐nucleotidase activities was observed in the injured cortex following CSI, whereas in other brain areas only an increase in ecto 5′‐nucleotidase activity was seen. Immunohistochemical analysis performed using antibodies specific to NTPDase 1 and ecto 5′‐nucleotidase demonstrated that CSI induced significant changes in enzyme expression around the injury site. Immunoreactivity patterns obtained for NTPDase 1 and ecto 5′‐nucleotidase were compared with those obtained for glial fibrillary acidic protein, as a marker of astrocytes and complement receptor type 3 (OX42), as a marker of microglia. Results suggest that up‐regulation of ectonucleotidase after CSI is catalyzed by cells that activate in response to injury, i.e. cells immunopositive for NTPDase 1 were predominantly microglial cells, whereas cells immunopositive for ecto 5′‐nucleotidase were predominantly astrocytes.

Benfotiamine Attenuates Inflammatory Response in LPS Stimulated BV-2 Microglia

Microglial cells are resident immune cells of the central nervous system (CNS), recognized as key elements in the regulation of neural homeostasis and the response to injury and repair. As excessive activation of microglia may lead to neurodegeneration, therapeutic strategies targeting its inhibition were shown to improve treatment of most neurodegenerative diseases. Benfotiamine is a synthetic vitamin B1 (thiamine) derivate exerting potentially anti-inflammatory effects. Despite the encouraging results regarding benfotiamine potential to alleviate diabetic microangiopathy, neuropathy and other oxidative stress-induced pathological conditions, its activities and cellular mechanisms during microglial activation have yet to be elucidated. In the present study, the anti-inflammatory effects of benfotiamine were investigated in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. We determined that benfotiamine remodels activated microglia to acquire the shape that is characteristic of non-stimulated BV-2 cells. In addition, benfotiamine significantly decreased production of pro-inflammatory mediators such as inducible form of nitric oxide synthase (iNOS) and NO; cyclooxygenase-2 (COX-2), heat-shock protein 70 (Hsp70), tumor necrosis factor alpha α (TNF-α), interleukin-6 (IL-6), whereas it increased anti-inflammatory interleukin-10 (IL-10) production in LPS stimulated BV-2 microglia. Moreover, benfotiamine suppressed the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and protein kinase B Akt/PKB. Treatment with specific inhibitors revealed that benfotiamine-mediated suppression of NO production was via JNK1/2 and Akt pathway, while the cytokine suppression includes ERK1/2, JNK1/2 and Akt pathways. Finally, the potentially protective effect is mediated by the suppression of translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus. Therefore, benfotiamine may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and enhancing anti-inflammatory factor production in activated microglia.

Developmental profile of NTPDase activity in synaptic plasma membranes isolated from rat cerebral cortex

In the present study the developmental profile of ATP‐hydrolyzing activity promoted by NTPDase 1, its kinetic properties and the enzyme protein abundance associated with synaptic plasma membrane from rat cerebral cortex were characterized. NTPDase 1 activity increased from birth to day 30; afterwards it decreased and remained unchanged from adulthood (90 days) to senescence (365 days). Kinetic analysis revealed that enzyme exhibited the highest specific activity at day 30 and highest apparent affinity for ATP at day 365; however, Vmax/Km values remained unchanged for each age studied. Immunoblot analysis demonstrated that relative abundance of NTPDase 1 is highest at day 15 during ontogeny. The discrepancy between maximum enzyme activity and maximum enzyme protein abundance indicates that NTPDase 1 may have an additional role during development.

Immunohistological Determination of Ecto-nucleoside Triphosphate Diphosphohydrolase1 (NTPDase1) and 5′-nucleotidase in Rat Hippocampus Reveals Overlapping Distribution

Distribution of two enzymes involved in the ectonucleotidase enzyme chain, ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1) and ecto-5′-nucleotidase, was assessed by immunohistochemistry in the rat hippocampus. Obtained results have shown co-expression of the enzymes in the hippocampal region, as well as wide and strikingly similar cellular distribution. Both enzymes were expressed at the surface of pyramidal neurons in the CA1 and CA2 sections, while cells in the CA3 section were faintly stained. The granule cell layer of the dentate gyrus was moderately stained for NTPDase1, as well as for ecto-5′-nucleotidase. Glial association for ecto-5′-nucleotidase was also observed, and fiber tracts were intensively stained for both enzymes. This is the first comparative study of NTPDase1 and ecto-5′-nucleotidase distribution in the rat hippocampus. Obtained results suggest that the broad overlapping distribution of these enzymes in neurons and glial cells reflects the functional importance of ectonucleotidase actions in the nervous system.

Dynamic changes in the expression pattern of ecto‐5′‐nucleotidase in the rat model of cortical stab injury

Traumatic injury induces massive release of ATP in the extracellular space, where it influences numerous aspects of neuronal, astrocytic, and microglial responses to injury by activating P2X and P2Y receptors. The extracellular ATP actions are controlled by the ectonucleotidase enzyme pathway, which hydrolyses ATP to adenosine at all neuronal and nonneuronal cell types. Adenosine activates its P1 receptors, which have important neuroprotective roles. The rate‐limiting enzyme in the ectonucleotidase pathway is ecto‐5′‐nucleotidase (e‐5NT), which catalyzes the final step of dephosphorylation of AMP to adenosine. The aim of the present study was to characterize the expression pattern and cellular distribution of e‐5NT in the perilesioned cortex at 4 hr and 1, 2, 7, and 15 days after unilateral cortical stab injury (CSI). Immunoblot and immunohistochemical studies showed that overall e‐5NT expression was lower 4 hr and 1 day postinjury and then gradually increased above the control levels. Double‐immunofluorescence studies further showed in control tissue the presence of the enzyme in the membranes surrounding neuronal somata and apical dendrites and less frequently in astrocytes. CSI caused a rapid (after 4 hr) and irreversible loss of the enzyme from neurons, accounting for a decrease in the overall enzyme expression. This was accompanied with a gradual increase in e‐5NT‐positive astrocytes, accounting for up‐regulation of the enzyme levels in the injured area. Thus, CSI induced dynamic changes in the expression pattern of e‐5NT that modify the ATP/adenosine ratio and the extent of P1 and P2 receptors activation and, therefore, outcome of the pathological processes after CSI.

Ribavirin ameliorates experimental autoimmune encephalomyelitis in rats and modulates cytokine production.

To determine the mechanism underlying ribavirin induced amelioration of experimental autoimmune encephalomyelitis (EAE), cytokine profiles were evaluated in draining lymph node (DLN) cell culture supernatants and spinal cord obtained from EAE and/or ribavirin-treated EAE Dark Agouti rats. Administration of ribavirin to EAE rats markedly affected the production of pro-inflammatory cytokines IFN-gamma, IL-1beta and TNF-alpha in DLN and spinal cord, thus shifting the balance towards the anti-inflammatory cytokines IL-10 and TGF-beta. These findings suggest that ribavirin attenuates EAE by limiting cytokine-mediated immunoinflammatory events leading to CNS destruction. The conducted experiments provide rationale for ribavirin to be considered as a candidate drug in the development of new therapeutic strategies for the treatment of autoimmune diseases in humans, such as multiple sclerosis.

Immunolocalization of ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) in the rat forebrain

Immunohistochemical study was performed to determine distribution of ecto-nucleotide pyrophosphatase/phosphodiesterase1 (NPP1) in adult rat forebrain. The study revealed widespread distribution of NPP1 in rat forebrain, yet with regional differences in the expression pattern and abundance. Strong NPP1 immunoreaction was detected in pyramidal cell layer of cerebral cortex and hippocampus, and in the midline regions of hypothalamus and thalamus. In many immunopositive forebrain areas, NPP1 was mainly localized at neuronal cell bodies. However, prominent immunoreaction was also detected at ependymal cells, tanycytes, endothelial cells of the capillaries and cells of the choroid plexus, suggesting that NPP1 could be involved in some highly specialized transport process.

Expression of Ecto-Nucleoside Triphosphate Diphosphohydrolase1-3 (NTPDase1-3) by Cortical Astrocytes After Exposure to Pro-inflammatory Factors In Vitro

Nucleoside triphosphate diphosphohydrolases (NTPDases) are ecto-enzymes catalyzing the first step of sequential hydrolysis of extracellular ATP to adenosine, as the final product. Among eight members of NTPDase family, NTPDases1–3 have been shown to be expressed in the brain. Although altered NTPDase expression has been observed in relation to cell death and reactive gliosis in several experimentally induced neuropathologies, regulators of NTPDases expression and function are largely unknown. The present study explored the effects of several inflammatory factors (i.e., INF-γ, TNF-α, LPS, peroxide, and glutamate) on NTPDase1–3 activity and expression by cultured cortical astrocytes. We were able to demonstrate that INF-γ and TNF-α increased both ATP and ADP hydrolysis, while LPS specifically increased ATP hydrolysis. Consistent with the observed enhanced nucleotidase activity, INF-γ induced the upregulation of NTPDase1 at the mRNA and protein level. Furthermore, we were able to demonstrate that INF-γ and TNF-α decreased the relative abundance of dominant astrocytic NTPDase2 in favor of NTPDase1. In summary, these results suggest that INF-γ, TNF-α, and LPS may be relevant in vivo regulators of NTPDase expression in neuropathologies associated with neuroinflammation.

Therapeutic effects of combined treatment with ribavirin and tiazofurin on experimental autoimmune encephalomyelitis development : Clinical and histopathological evaluation

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS) and the helpful tool in preclinical testing of various substances considered for treatment of this human CNS disease. Ribavirin (R) and tiazofurin (T) are purine nucleoside analogues, with the broad spectrum of anti-viral, anti-tumoral and anti-inflammatory properties. We proposed that combined treatment with RT, administrated during the effector phase of EAE, would attenuate disease severity, both clinically and pathologically. Ribavirin was given daily at a dosage of 30 mg/kg and tiazofurin was given at a dosage of 10 mg/kg every other day for 15 days. We detected amelioration of clinical signs and faster recovery in the RT group compared to the control group. Immunohistochemical analyses revealed that RT treatment decrease the number of T cells, macrophages and microglia. In the controls, we detected reactive type of microglia, while in the RT group we noticed ramified/resting form. Demyelination areas and axonal damage were not recorded in the RT group, in contrast to the control group where multiple areas of demyelination zones and axonal loss were found. RT combination treatment suppresses ongoing EAE, prevents demyelination and axonal loss, and therefore may well be the potential therapy for the treatment of MS.

Benfotiamine upregulates antioxidative system in activated BV-2 microglia cells

Chronic microglial activation and resulting sustained neuroinflammatory reaction are generally associated with neurodegeneration. Activated microglia acquires proinflammatory cellular profile that generates oxidative burst. Their persistent activation exacerbates inflammation, which damages healthy neurons via cytotoxic mediators, such as superoxide radical anion and nitric oxide. In our recent study, we have shown that benfotiamine (S-benzoylthiamine O-monophosphate) possesses anti-inflammatory effects. Here, the effects of benfotiamine on the pro-oxidative component of activity of LPS-stimulated BV-2 cells were investigated. The activation of microglia was accompanied by upregulation of intracellular antioxidative defense, which was further promoted in the presence of benfotiamine. Namely, activated microglia exposed to non-cytotoxic doses of benfotiamine showed increased levels and activities of hydrogen peroxide- and superoxide-removing enzymes—catalase and glutathione system, and superoxide dismutase. In addition, benfotiamine showed the capacity to directly scavenge superoxide radical anion. As a consequence, benfotiamine suppressed the activation of microglia and provoked a decrease in NO and ·O−2 production and lipid peroxidation. In conclusion, benfotiamine might silence pro-oxidative activity of microglia to alleviate/prevent oxidative damage of neighboring CNS cells.