Daphnetin inhibits spinal glial activation via Nrf2/HO-1/NF-κB signaling pathway and attenuates CFA-induced inflammatory pain.

Daphnetin (7, 8-dihydroxycoumarin, DAPH), a coumarin derivative isolated from Daphne odora var., recently draws much more attention as a promising drug candidate to treat neuroinflammatory diseases due to its protective effects against neuroinflammation. However, itscontribution to chronic inflammatory pain is largely unknown. In the current work, we investigated the effects of DAPH in a murine model of inflammatory pain induced by complete Freund's adjuvant (CFA) and its possible underlying mechanisms. Our results showed that DAPH treatment significantly attenuated mechanical allodynia provoked by CFA. A profound inhibition of spinal glial activation, followed by attenuated expression levels of spinal pro-inflammatory cytokines, was observed in DAPH-treated inflammatory pain mice. Further study demonstrated that DAPH mediated negative regulation of spinal NF-κB pathway, as well as its preferential activation of Nrf2/HO-1 signaling pathway in inflammatory pain mice. This study, for the first time, indicated that DAPH might preventthe development of mechanical allodynia in mice with inflammatory pain. And more importantly, these data provide evidence for the potential application of DAPH in the treatment of chronic inflammatory pain.

The Role of Neuroglial Crosstalk and Synaptic Plasticity-Mediated Central Sensitization in Acupuncture Analgesia.

Although pain is regarded as a global public health priority, analgesic therapy remains a significant challenge. Pain is a hypersensitivity state caused by peripheral and central sensitization, with the latter considered the culprit for chronic pain. This study summarizes the pathogenesis of central sensitization from the perspective of neuroglial crosstalk and synaptic plasticity and underlines the related analgesic mechanisms of acupuncture. Central sensitization is modulated by the neurotransmitters and neuropeptides involved in the ascending excitatory pathway and the descending pain modulatory system. Acupuncture analgesia is associated with downregulating glutamate in the ascending excitatory pathway and upregulating opioids, 𝛾-aminobutyric acid, norepinephrine, and 5-hydroxytryptamine in the descending pain modulatory system. Furthermore, it is increasingly appreciated that neurotransmitters, cytokines, and chemokines are implicated in neuroglial crosstalk and associated plasticity, thus contributing to central sensitization. Acupuncture produces its analgesic action by inhibiting cytokines, such as interleukin-1ß, interleukin-6, and tumor necrosis factor-α, and upregulating interleukin-10, as well as modulating chemokines and their receptors such as CX3CL1/CX3CR1, CXCL12/CXCR4, CCL2/CCR2, and CXCL1/CXCR2. These factors are regulated by acupuncture through the activation of multiple signaling pathways, including mitogen-activated protein kinase signaling (e.g., the p38, extracellular signal-regulated kinases, and c-Jun-N-terminal kinase pathways), which contribute to the activation of nociceptive neurons. However, the responses of chemokines to acupuncture vary among the types of pain models, acupuncture methods, and stimulation parameters. Thus, the exact mechanisms require future clarification. Taken together, inhibition of central sensitization modulated by neuroglial plasticity is central in acupuncture analgesia, providing a novel insight for the clinical application of acupuncture analgesia.

Neuroprotective Effect of Alpha-Linolenic Acid against Aß-Mediated Inflammatory Responses in C6 Glial Cell.

Therapeutic approaches for neurodegeneration, such as Alzheimer's disease (AD), have been widely studied. One of the critical hallmarks of AD is accumulation of amyloid beta (Aß). Aß induces neurotoxicity and releases inflammatory mediators or cytokines through activation of glial cell, and these pathological features are observed in AD patient's brain. The purpose of this study is to investigate the protective effect of alpha-linolenic acid (ALA) on Aß25-35-induced neurotoxicity in C6 glial cells. Exposure of C6 glial cells to 50 µM Aß25-35 caused cell death, overproduction of nitric oxide (NO), and pro-inflammatory cytokines release [interleukin (IL)-6 and tumor necrosis factor-α], while treatment of ALA increased cell viability and markedly attenuated Aß25-35-induced excessive production of NO and those inflammatory cytokines. Inhibitory effect of ALA on generation of NO and cytokines was mediated by down-regulation of inducible nitric oxide synthase and cyclooxygenase-2 protein and mRNA expressions. In addition, ALA treatment inhibited reactive oxygen species generation induced by Aß25-35 through the enhancement of the nuclear factor-erythroid 2-related factor-2 (Nrf-2) protein levels and subsequent induction of heme-oxygenase-1 (HO-1) expression in C6 glial cells dose- and time-dependently. Furthermore, the levels of neprilysin and insulin-degrading enzyme protein expressions, which contribute to degradation of Aß, were also increased by treatment of ALA compared to Aß25-35-treated control group. In conclusion, effects of ALA on Aß degradation were shown to be mediated through inhibition of inflammatory responses and activation of antioxidative system, Nrf-2/HO-1 signaling pathway, in C6 glial cells. Our findings suggest that ALA might have the potential for therapeutics of AD.

Anti­inflammatory effect of Migri­Heal® in an in vitro inflammatory model of primary mixed glial cells.

Migri­Heal®, is a novel herbal remedy that was introduced for the treatment of migraine headaches. Previous studies revealed that this drug may reduce nitric oxide (NO) in an in vitro inflammatory model. The aim of the present study was to investigate the anti­inflammatory effect of Migri­Heal® on primary mix glial cells stimulated with LPS. In the current study, neonatal rat primary mix glial cells were isolated from the mixed glial cultures via shaking, and cultured in Dulbecco's' modified Eagle's medium supplemented with 10% fetal bovine serum. Following pretreatment with Migri­Heal® (25, 75, 100, 150, 200 and 300 µg/ml) and cells were treated with LPS (10 µg/ml) for 1 h, and incubated for 48 h. The present study determined that 150 µg/ml Migri­Heal® significantly reduced the production of NO in rat mix glial cells stimulated with 10 µg/ml LPS. Migri­Heal® also suppressed mRNA expression level of LPS­induced inducible nitric oxide synthase and tumor necrosis factor α, which was accompanied by inhibition of the transcription factor nuclear factor­κB. Additionally, MTT assay determined that Migri­Heal® was not cytotoxic, suggesting that the anti­inflammatory effects of Migri­Heal® observed were not due to cell death. In conclusion, the findings of the present study demonstrated that Migri­Heal® may be useful as a potential anti­inflammatory agent in inflammatory diseases. However, additional studies are required to confirm these findings.

IL-17A and Multiple Sclerosis: Signaling Pathways, Producing Cells and Target Cells in the Central Nervous System.

Multiple sclerosis (MS) is an immune mediated demyelinating disease of the central nervous system (CNS). The importance of immune cells to MS pathology is supported by clinical data linking the depletion of T and B cells, or the prevention of their migration into the brain with significant reduction in relapses and development of new lesions. In vitro studies, preclinical animal models and encouraging data with the anti-IL-17A antibody secukinumab in a small proof of concept study in man, indicate that IL-17A, a key interleukin associated with many inflammatory and autoimmune diseases, may be involved in MS. Not only cells involved in adaptive immune responses such as Th17 cells and cytotoxic T cells, or innate immune responses such as mucosa-associated invariant T (MAIT) cells and γδT cells, but also CNS resident cells such as astrocytes and oligodendrocytes might contribute to the local production of IL-17A. IL-17A synergizes with other proinflammatory cytokines, by inducing the release of additional cytokines, mediators of tissue damage and chemokines, that recruit new inflammatory cells. IL-17A adversely affects the functions of microglia, astrocytes, oligodendrocytes, neurons, neural precursor cells and endothelial cells. Blockade of IL-17A might be beneficial to MS patients not only by inhibiting inflammation and tissue destruction, but also by enhancing repair processes.

Lithium Distinctly Modulates the Secretion of Pro- and Anti- Inflammatory Interleukins in Co-Cultures of Neurons and Glial Cells at Therapeutic and Sub-Therapeutic Concentrations.

Lithium is associated with various effects on immune functions, some of which are still poorly understood. The roles of many cytokines have been characterized in a variety of neurodevelopmental processes including neurogenesis, neuronal and glial cell migration, proliferation, differentiation, synaptic maturation and synaptic pruning. This work aims to evaluate the effects of different doses of lithium (0.02; 0.2 and 2mM) on the secretion of cytokines in co-cultures of cortical and hippocampal neurons with glial cells. Our results indicate that chronic treatment with lithium chloride at subtherapeutic concentrations are able to modify the secretion of pro- and anti-inflammatory interleukins in co-cultures of cortical and hippocampal neurons with glial cells.

Hypothalamic innate immune reaction in obesity.

Findings from rodent and human studies show that the presence of inflammatory factors is positively correlated with obesity and the metabolic syndrome. Obesity-associated inflammatory responses take place not only in the periphery but also in the brain. The hypothalamus contains a range of resident glial cells including microglia, macrophages and astrocytes, which are embedded in highly heterogenic groups of neurons that control metabolic homeostasis. This complex neural-glia network can receive information directly from blood-borne factors, positioning it as a metabolic sensor. Following hypercaloric challenge, mediobasal hypothalamic microglia and astrocytes enter a reactive state, which persists during diet-induced obesity. In established mouse models of diet-induced obesity, the hypothalamic vasculature displays angiogenic alterations. Moreover, proopiomelanocortin neurons, which regulate food intake and energy expenditure, are impaired in the arcuate nucleus, where there is an increase in local inflammatory signals. The sum total of these events is a hypothalamic innate immune reactivity, which includes temporal and spatial changes to each cell population. Although the exact role of each participant of the neural-glial-vascular network is still under exploration, therapeutic targets for treating obesity should probably be linked to individual cell types and their specific signalling pathways to address each dysfunction with cell-selective compounds.

Dual orexin receptor antagonist 12 inhibits expression of proteins in neurons and glia implicated in peripheral and central sensitization.

Sensitization and activation of trigeminal nociceptors is implicated in prevalent and debilitating orofacial pain conditions including temporomandibular joint (TMJ) disorders. Orexins are excitatory neuropeptides that function to regulate many physiological processes and are reported to modulate nociception. To determine the role of orexins in an inflammatory model of trigeminal activation, the effects of a dual orexin receptor antagonist (DORA-12) on levels of proteins that promote peripheral and central sensitization and changes in nocifensive responses were investigated. In adult male Sprague-Dawley rats, mRNA for orexin receptor 1 (OX1R) and receptor 2 (OX2R) were detected in trigeminal ganglia and spinal trigeminal nucleus (STN). OX1R immunoreactivity was localized primarily in neuronal cell bodies in the V3 region of the ganglion and in laminas I-II of the STN. Animals injected bilaterally with complete Freund's adjuvant (CFA) in the TMJ capsule exhibited increased expression of P-p38, P-ERK, and lba1 in trigeminal ganglia and P-ERK and lba1 in the STN at 2 days post injection. However, levels of each of these proteins in rats receiving daily oral DORA-12 were inhibited to near basal levels. Similarly, administration of DORA-12 on days 3 and 4 post CFA injection in the TMJ effectively inhibited the prolonged stimulated expression of protein kinase A, NFkB, and Iba1 in the STN on day 5 post injection. While injection of CFA mediated a nocifensive response to mechanical stimulation of the orofacial region at 2h and 3 and 5 days post injection, treatment with DORA-12 suppressed the nocifensive response on day 5. Somewhat surprisingly, nocifensive responses were again observed on day 10 post CFA stimulation in the absence of daily DORA-12 administration. Our results provide evidence that DORA-12 can inhibit CFA-induced stimulation of trigeminal sensory neurons by inhibiting expression of proteins associated with sensitization of peripheral and central neurons and nociception.

[Basic mechanisms of action of fingolimod in relation to multiple sclerosis]. / Mecanismos basicos de accion del fingolimod en relacion con la esclerosis multiple.

INTRODUCTION: Fingolimod has recently been approved for the therapy of relapsing multiple sclerosis. This drug binds to different sphingosine-1-phosphate receptors. AIM: To analyze basic mechanisms of action that can account for the efficacy of this drug in multiple sclerosis. DEVELOPMENT: Fingolimod acts as an inverse agonist on sphingosine-1-phosphate receptors, inducing degradation of receptors. On lymphoid circulation, this effect causes retention in lymph nodes of naive and central memory T cells, including Th17 T lymphocytes, bearing CCR7 and CD62L receptors. As a result, the level of circulating T cells is markedly decreased. B ell circulation is impaired and complex effects on other immune cells are also induced. Fingolimod enters the central nervous system and binds to receptors on glial cells and neurons. In experimental autoimmune encephalomyelitis, the therapeutic efficacy of fingolimod is not only associated with a reduced entry of inflammatory cells into the nervous system, but also with a direct effect mostly on astroglial cells. CONCLUSIONS: In multiple sclerosis patients, the available evidence indicates that fingolimod efficacy is directly associated with impairment of circulation of several T cell subsets and possibly B cells. Animal studies raise the possibility that an additional effect on glial cells might also contribute to the clinical efficacy.

Immunomodulation of experimental allergic encephalomyelitis by cinnamon metabolite sodium benzoate.

Experimental allergic encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), the most common human demyelinating disease of the central nervous system. Sodium benzoate (NaB), a metabolite of cinnamon and a FDA-approved drug against urea cycle disorders in children, is a widely used food additive, which is long known for its microbicidal effect. However, recent studies reveal that apart from its microbicidal effects, NaB can also regulate many immune signaling pathways responsible for inflammation, glial cell activation, switching of T-helper cells, modulation of regulatory T cells, cell-to-cell contact, and migration. As a result, NaB alters the neuroimmunology of EAE and ameliorates the disease process of EAE. In this review, we have made an honest attempt to analyze these newly-discovered immunomodulatory activities of NaB and associated mechanisms that may help in considering this drug for various inflammatory human disorders including MS as primary or adjunct therapy.

Neuron-immune interactions in the sensitized thalamus induced by mustard oil application to rat molar pulp.

We have reported that mustard oil application to the rat dental pulp induces neuronal activation in the thalamus. To address the mechanisms involved in the thalamic changes, we performed neuronal responsiveness recording, immunohistochemistry, and molecular biological analysis. After mustard oil application, neuronal responsiveness was increased in the mediodorsal nucleus. When MK801 (an N-methyl-D-aspartate receptor antagonist) was applied to the mediodorsal nucleus, the enhanced responsiveness was decreased. N-methyl-D-aspartate receptor 2D, glial fibrillary acidic protein, and antigen-presenting cell-related gene mRNAs in the contralateral thalamus were up-regulated at 10 minutes after mustard oil application, but were down-regulated within 10 minutes after the antagonist application. OX6-expressing microglia and glial fibrillary acidic protein-expressing astrocytes did not increase until 60 minutes after mustard oil application. These results suggested that the thalamic neurons play some roles in regulating the glial cell activation in the mediodorsal nucleus via N-methyl-D-aspartate receptor 2D during pulp inflammation-induced central sensitization.

Polyethylene glycol-conjugated immunoliposomes specific for olfactory ensheathing glial cells.

A novel immunoliposome delivery system was developed for directed transport into cultured olfactory epithelium cells. Monoclonal antibodies against glial fibrillary acidic protein (GFAP) served as a vector. Fluorescence microscopy showed that the target cells are specifically stained with Dil dye incorporated into liposomal membranes. This transport system holds promise for the delivery of bioactive substances to olfactory epithelial cells and modulation of their capacity to stimulate axonal regeneration.

Age and neuroinflammation: a lifetime of psychoneuroimmune consequences.

This article reviews the literature indicating that the innate immune cells of the brain become more reactive with age. Although it is unclear how glia reactivity increases, emerging evidence suggests these alterations allow exacerbated neuroinflammation and sickness behavior following peripheral immune activation. This amplified or prolonged exposure to inflammatory cytokines in the brain may impair neuronal plasticity and underlie a heightened neuroinflammatory response in the aged that also may lead to other neurobehavioral impairments such as delirium, depression, and, potentially, the onset of neurologic disease. Therefore pharmacologic strategies to decrease neuroinflammation associated with infection may be important for improving recovery from sickness and reducing neurobehavioral deficits in the elderly.

Immunity, neuroglia and neuroinflammation in autism.

Autism is a complex neurodevelopmental disorder of early onset that is highly variable in its clinical presentation. Although the causes of autism in most patients remain unknown, several lines of research support the view that both genetic and environmental factors influence the development of abnormal cortical circuitry that underlies autistic cognitive processes and behaviors. The role of the immune system in the development of autism is controversial. Several studies showing peripheral immune abnormalities support immune hypotheses, however until recently there have been no immune findings in the CNS. We recently demonstrated the presence of neuroglial and innate neuroimmune system activation in brain tissue and cerebrospinal fluid of patients with autism, findings that support the view that neuroimmune abnormalities occur in the brain of autistic patients and may contribute to the diversity of the autistic phenotypes. The role of neuroglial activation and neuroinflammation are still uncertain but could be critical in maintaining, if not also in initiating, some of the CNS abnormalities present in autism. A better understanding of the role of neuroinflammation in the pathogenesis of autism may have important clinical and therapeutic implications.

Lesional RhoA+ cell numbers are suppressed by anti-inflammatory, cyclooxygenase-inhibiting treatment following subacute spinal cord injury.

Inhibition of the small GTPase RhoA or its downstream target Rho-associated coiled kinase (ROCK) has been shown to promote axon regeneration and to improve functional recovery following spinal cord injury (SCI) in the adult rat. RhoA has also been implicated in delayed secondary injury pathophysiology, such as free radical formation and loss of endothelial integrity leading to edema formation. In the present report, we have analyzed the effect of the central nervous system (CNS) permissive, putatively neuroprotective, anti-inflammatory cyclooxygenase-1/-2 (COX-1/-2) inhibitor indomethacin in CNS effective dosage (2 mg/kg/day) on lesional RhoA expression following subacute spinal cord injury. In control rats receiving vehicle alone, RhoA+ cells accumulate at the lesion site (Th8). At day 3 following SCI, the RhoA+ cellular composition is composed prevailingly of microglia/macrophages and polymononuclear granulocytes, but few reactive astrocytes. In contrast, in the verum group, lesional numbers of RhoA cells were reduced by indomethacin treatment by more than 60% (P < 0.0001). Inflammation-dependent RhoA expression accessible by cyclooxygenase inhibition proposes an immune-related mechanism. Our results identify COX blockers as candidates for a safe, synergistic, adjuvant treatment option in combination with cell-specific approaches to Rho inactivation, effectively minimizing the pool of RhoA+ cells at the lesion site following SCI.

Calpain expression and infiltration of activated T cells in experimental allergic encephalomyelitis over time: increased calpain activity begins with onset of disease.

Calpain activity and expression at the protein level were examined in inflammatory cells, activated microglia, and astrocytes prior to or at onset of symptomatic experimental allergic encephalomyelitis (EAE), an animal model for the human demyelinating disease multiple sclerosis (MS). EAE was induced in Lewis rats by injection of guinea pig spinal cord homogenate and myelin basic protein (MBP) emulsified with Complete Freund's Adjuvant (CFA). Calpain translational expression, determined by Western blot and immunocytochemistry, was correlated with calpain activity, infiltration of inflammatory cells, and myelin loss at 2-11 days following challenge with antigen. Controls (CFA only) did not show any changes over time in these parameters and very few changes (CD11+ microglia/mononuclear phagocytes) were seen in either group from days 2 to 8 post-induction. In contrast, from days 9 to 11, the animals that developed the disease (at least grade 1) demonstrated extensive cellular infiltration (CD4+, CD25+, and CD11+ as well as increased calpain expression (content) and activity. This study demonstrates that cell infiltration and increased calpain activity do not begin in the CNS until the onset of clinical signs.

Immunocytochemical study of an early microglial activation in ischemia.

Transient arrest of the cerebral blood circulation results in neuronal cell death in selectively vulnerable regions of the rat brain. To elucidate further the involvement of glial cells in this pathology, we have studied the temporal and spatial distribution pattern of activated microglial cells in several regions of the ischemic rat brain. Transient global ischemia was produced in rats by 30 min of a four-vessel occlusion. Survival times were 1, 3, and 7 days after the ischemic injury. The microglial reaction was studied immunocytochemically using several monoclonal antibodies, e.g., against CR3 complement receptor and major histocompatibility complex (MHC) antigens. Two recently produced monoclonal antibodies against rat microglial cells, designated MUC 101 and 102, were also used to identify microglial cells. Following ischemia, the microglial reaction was correlated with the development of neuronal damage. The earliest presence of activated microglial cells was observed in the dorsolateral striatum, the CA1 area, and the dentate hilus of the dorsal hippocampus. However, the microglial reaction was not confined to areas showing selective neuronal damage, but also occurred in regions that are rather resistant to ischemia, such as the CA3 area. Particularly in the frontoparietal cortex, the appearance of MHC class II-positive microglial cells provided an early indication of the subsequent distribution pattern of neuronal damage. The microglial reaction would thus seem to be an early, sensitive, and reliable marker for the occurrence of neuronal damage in ischemia.

[The immunological status indices of monkeys inoculated with a meningococcal B vaccine]. / Pokazateli immunologicheskogo statusa obez'ian, privitykh meningokokkovoi B-vaktsinoi.

The method for the determination of bacterial antibodies to group B meningococci was worked out. The method was used for the determination of antibodies to group B meningococcal vaccine produced in the USSR. The dynamic study of antibodies to protein, polysaccharide and lipopolysaccharide antigens of group B meningococci was made by the method of the enzyme immunoassay (EIA), and the safety of the vaccine was studied by the determination of autoantibodies active against brain tissue antigens. The data thus obtained were indicative of the immunological activity of group B protein-polysaccharide vaccines, manifested by the capacity for stimulating bactericidal antibodies whose level increased 8- to 10-fold after the immunization of monkeys in 2 and 3 injections. Similarity in the dynamics of the formation of bacteriolysins and antibodies to protein antigen, as determined in EIA, was noted. The vaccine was found to stimulate no cytotoxic anticerebral antibodies in the glia migration test, which was indicative of the safety of group B meningococcal vaccine.

Identification of the normal microglial population in human and rodent nervous tissue using lectin-histochemistry.

Mistletoe lectin-1 (ML-1) and Ricinus communis agglutinin-120 (RCA-1) both possess D-galactose-specific surface-binding sites. They were used to selectively identify microglial populations in aldehyde-fixed normal brain tissue by lectin immunohistochemistry on paraffin and frozen sections. Mistletoe lectin-1 was superior to RCA-1 in labelling microglia in the rat brain, whereas RCA-1 labelled human microglia better than ML-1. Thus, RCA-1 and ML-1 supplement each other for identifying microglial in human and rodent central nervous system tissues. The high reproducibility of the results and the applicability of the technique to routine histology, using formalin-fixed tissue, should facilitate study of the histogenesis and role of microglia in the CNS.