Neurotoxic Mediators Research Articles

Human astrocytes and microglia-like cells constitutively express the novel cytosolic viral DNA sensor cGAS

Abstract Evidence is accumulating that the innate immune response to HIV-1 within the central nervous system (CNS) plays a critical role in neuroAIDS disease pathology. Microglia and astrocytes are uniquely positioned to respond to CNS pathogens, and these cells produce key inflammatory, neurotoxic, and antiviral mediators following exposure to retroviruses such as HIV-1. This virus infects glia but the mechanisms by which these cells perceive intracellular replicative HIV-1 have not been defined. However, it has become apparent that innate immune sensing of nucleic acids underpins many physiological and pathological responses to viral pathogens. Cyclic GMP-AMP synthase (cGAS) has been identified as a cytosolic DNA sensor and this molecule has recently been demonstrated to have a key role in sensing replicative HIV-1 DNA intermediates and eliciting responses in human monocytic cell lines. Furthermore, cGAS and its downstream adaptor molecule STING have been found to be expressed by murine microglia and astrocytes. Here, we have characterized cGAS mRNA and protein expression by human glial cells. Microglial and astrocytic cell lines and primary human astrocytes were found to constitutively express robust mRNA and protein levels of cGAS and such expression could not be elevated further by transfection with viral nucleic acid motifs. Importantly, cGAS mRNA and protein expression was maintained despite the successful establishment of pseudotyped HIV-1 infection. As such, the identification of robust levels of cGAS in both microglia and astrocytes that are maintained even in the face of HIV-1 challenge may indicate an important mechanism by which human glial cells can respond to this pathogen.

CX3CR1 ablation ameliorates motor and respiratory dysfunctions and improves survival of a Rett syndrome mouse model

Rett syndrome (RTT) is a neurodevelopmental disorder caused by loss-of-function mutations in the gene encoding MeCP2, an epigenetic modulator that binds the methyl CpG dinucleotide in target genes to regulate transcription. Previously we and others reported a role of microglia in the pathophysiology of RTT. Because microglia in the Mecp2 knockout (Mecp2KO) mouse model of RTT over-produce neurotoxic mediators glutamate and reactive oxygen species, we hypothesize that blocking neuron–microglia interaction by ablation of CX3CR1, a chemokine receptor expressed in microglia/myeloid cells mediating such interaction by pairing with its neuronal ligand CX3CL1, would ameliorate the RTT-like phenotype in Mecp2KO mice. Here we report that CX3CR1 ablation prolonged the lifespan of Mecp2KO mice from a median survival of 54.5–74days, and significantly improved the body weight gain, symptomatic scores, major respiratory parameters, and motor coordination and performance. CX3CR1 ablation rectified previously identified histological abnormalities in the Mecp2KO brain such as neuronal soma size in hippocampal CA2, and the number, soma size, and process complexity of microglia. Moreover, CX3CR1 ablation enhanced the neurotrophic action of microglia in Mecp2KO mice by producing higher amount of insulin-like growth factor 1. Our data support a role of myeloid cells/microglia in RTT and suggest a novel therapeutic approach for RTT by targeting CX3CR1 with specific antagonists or genetic downregulation.

Antioxidant potential of Thymoquinone against Arsenic mediated neurotoxicity

Introduction: Arsenic, an established poisonous metalloid has widespread occurrence in the environment which is posing a constant threat to health and survival of all living organisms. In view of arsenic induced oxidative stress and toxicity, this study focuses on the mitigatory role of thymoquinone, a major active component in the volatile oil of Nigella sativa (commonly known as black cumin); as its nutraceutical value can fulfil the demand of such dietary supplements in high risk population areas. Methods: In the current study, brain preparations of male wistar rats were used to assess different biochemical markers of oxidative stress and genotoxicity. Results: Significant and dose-dependent alterations in the level of enzymatic and biochemical biomarkers of oxidative stress were observed in the As-treated system. However, pre-treatment with thymoquinone brought about significant reduction in the As-induced neurotoxicity. Also, a significant decline in arsenic-induced DNA damage was recorded on pre-treatment with thymoquinone in comet assay. Conclusion: This study proves usefulness of antioxidant potential of thymoquione in mitigating the arsenic induced neurotoxicity.

High-Glucose-Derived Oxidative Stress-Dependent Heme Oxygenase-1 Expression from Astrocytes Contributes to the Neuronal Apoptosis.

An elevated level of glucose has been found in the blood of hyperglycemia and diabetes patients associated with several central nervous system (CNS) complications. These disorders may be due to the up-regulation of many neurotoxic mediators by host cells triggered by high glucose (HG). Moreover, heme oxygenase-1 (HO-1) plays a crucial role in tissue pathological changes such as brain injuries. However, the molecular mechanisms underlying HG-induced HO-1 expression in brain cells remain poorly defined. Thus, we use the rat brain astrocytes (RBA-1) as a model to investigate the signaling mechanisms of HO-1 induction by HG and its effects on neuronal cells. We demonstrated that HG induced HO-1 expression via a reactive oxygen species (ROS)-dependent signaling pathway. NADPH oxidase (Nox)- and mitochondrion-dependent ROS generation led to activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun-N-terminal kinase (JNK) and then activated the downstream transcriptional factors nuclear factor-kappaB (NF-κB) and c-Fos/activator protein 1 (AP-1), respectively. Subsequently, the activated NF-κB and AP-1 turned on transcription of HO-1 gene. These results indicated that in brain astrocytes, activation of MAPK-mediated NF-κB and c-Fos/AP-1 cascades by Nox/ROS and mitoROS-dependent events is essential for HO-1 up-regulation induced by HG. Moreover, we found that HG-induced extracellular ROS increase and HO-1 expression from astrocytes resulted in neuronal apoptosis. These results offers new insights into the mechanisms and effects of the action of HG, supporting that HG may cause brain disorders in the development of diabetes- and hyperglycemia-induced CNS complications such as neurodegenerative diseases.

CB2 receptor activation prevents glial-derived neurotoxic mediator production, BBB leakage and peripheral immune cell infiltration and rescues dopamine neurons in the MPTP model of Parkinson's disease.

The cannabinoid (CB2) receptor type 2 has been proposed to prevent the degeneration of dopamine neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. However, the mechanisms underlying CB2 receptor-mediated neuroprotection in MPTP mice have not been elucidated. The mechanisms underlying CB2 receptor-mediated neuroprotection of dopamine neurons in the substantia nigra (SN) were evaluated in the MPTP mouse model of Parkinson's disease (PD) by immunohistochemical staining (tyrosine hydroxylase, macrophage Ag complex-1, glial fibrillary acidic protein, myeloperoxidase (MPO), and CD3 and CD68), real-time PCR and a fluorescein isothiocyanate-labeled albumin assay. Treatment with the selective CB2 receptor agonist JWH-133 (10 μg kg−1, intraperitoneal (i.p.)) prevented MPTP-induced degeneration of dopamine neurons in the SN and of their fibers in the striatum. This JWH-133-mediated neuroprotection was associated with the suppression of blood–brain barrier (BBB) damage, astroglial MPO expression, infiltration of peripheral immune cells and production of inducible nitric oxide synthase, proinflammatory cytokines and chemokines by activated microglia. The effects of JWH-133 were mimicked by the non-selective cannabinoid receptor WIN55,212 (10 μg kg−1, i.p.). The observed neuroprotection and inhibition of glial-mediated neurotoxic events were reversed upon treatment with the selective CB2 receptor antagonist AM630, confirming the involvement of the CB2 receptor. Our results suggest that targeting the cannabinoid system may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with glial activation, BBB disruption and peripheral immune cell infiltration.

Beyond Jugular and Subclavian: The Axillary Vein, First Option for Centrally Inserted Central Lines

Human herpesvirus (HHV)-6 is a β-herpesvirus that infects most infants by 2 years of age and persists in a variety of host cells after primary infection, with intermittent reactivation typically during periods of immunosuppression. HHV-6 has two closely related species, HHV-6A and B, which are both neurotropic and can be detected in up to 85% of brain samples at autopsy. This pleiotropic virus has been implicated in many central nervous system (CNS) diseases, including febrile seizures (FS), epilepsy due to medial temporal lobe sclerosis, multiple sclerosis, encephalomyelitis, progressive multifocal leukoencephalopathy, chronic fatigue syndrome, and cognitive dysfunction. The significance of HHV-6 infection is often controversial due to the challenge implicit in attributing disease to a commensal virus of the brain. Studies have used a variety of unstandardized techniques on an array of sample sources to detect HHV-6, making direct comparisons problematic. In addition, many detection methods do not distinguish between latent and active infection. However, there is accumulating evidence implicating HHV-6 as a cause of CNS pathology, especially in FS and encephalitis. Treatment options are limited, fraught with side-effects, and poorly studied. Whether HHV-6 is a commensal pathogen of the brain, marker of immune dysregulation, trigger of autoimmunity, or directly neurotoxic mediator of CNS disease will remain uncertain for many CNS diseases until more refined studies are performed.

Anti-inflammatory effects and antioxidant activity of dihydroasparagusic acid in lipopolysaccharide-activated microglial cells.

The activation of microglia and subsequent release of toxic pro-inflammatory factors are crucially associated with neurodegenerative disease, characterized by increased oxidative stress and neuroinflammation, including Alzheimer and Parkinson diseases and multiple sclerosis. Dihydroasparagusic acid is the reduced form of asparagusic acid, a sulfur-containing flavor component produced by Asparagus plants. It has two thiolic functions able to coordinate the metal ions, and a carboxylic moiety, a polar function, which may enhance excretion of the complexes. Thiol functions are also present in several biomolecules with important physiological antioxidant role as glutathione. The aim of this study is to evaluate the anti-inflammatory and antioxidant potential effect of dihydroasparagusic acid on microglial activation in an in vitro model of neuroinflammation. We have used lipopolysaccharide to induce an inflammatory response in primary rat microglial cultures. Our results suggest that dihydroasparagusic acid significantly prevented lipopolysaccharide-induced production of pro-inflammatory and neurotoxic mediators such as nitric oxide, tumor necrosis factor-α, prostaglandin E2, as well as inducible nitric oxide synthase and cyclooxygenase-2 protein expression and lipoxygenase activity in microglia cells. Moreover it effectively suppressed the level of reactive oxygen species and affected lipopolysaccharide-stimulated activation of mitogen activated protein kinase, including p38, and nuclear factor-kB pathway. These results suggest that dihydroasparagusic acid's neuroprotective properties may be due to its ability to dampen induction of microglial activation. It is a compound that can effectively inhibit inflammatory and oxidative processes that are important factors of the etiopathogenesis of neurodegenerative diseases.

Matrix-derived inflammatory mediator N-acetyl proline-glycine-proline is neurotoxic and upregulated in brain after ischemic stroke.

BackgroundN-acetyl proline-glycine-proline (ac-PGP) is a matrix-derived chemokine produced through the proteolytic destruction of collagen by matrix metalloproteinases (MMPs). While upregulation and activation of MMPs and concomitant degradation of the extracellular matrix are known to be associated with neurological injury in ischemic stroke, the production of ac-PGP in stroke brain and its effects on neurons have not been investigated.FindingsWe examined the effects of ac-PGP on primary cortical neurons and found that it binds neuronal CXCR2 receptors, activates extracellular signal-regulated kinase 1/2 (ERK1/2), and induces apoptosis associated with caspase-3 cleavage in a dose-dependent manner. After transient ischemic stroke in rats, ac-PGP was significantly upregulated in infarcted brain tissue.ConclusionsThe production of ac-PGP in brain in ischemia/reperfusion injury and its propensity to induce apoptosis in neurons may link MMP-mediated destruction of the extracellular matrix and opening of the blood-brain barrier to progressive neurodegeneration associated with the initiation and propagation of inflammation. Ac-PGP may be a novel neurotoxic inflammatory mediator involved in sustained inflammation and neurodegeneration in stroke and other neurological disorders associated with activation of MMPs.

Nutrition, Neuroinflammation and Cognition

Activation of microglia and astrocytes leads to the production of cytokines and other inflammatory mediators which may contribute to the apoptotic cell death of neurons observed in many neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Vulnerability of the central nervous system (CNS) to oxidative and inflammatory stress increases with age and has been postulated to be a leading contributing factor to the cognitive impairment and thereby development of neurodegenerative diseases. Suppression of microglial production of neurotoxic mediators may result in neuroprotection. This heightens the interest in the development of neuroinflammation-targeted therapeutics. Nutrition is involved in the pathogenesis of age-related cognitive decline and also neurodegenerative diseases. Certain nutrients facilitate human brain function with their immediate and long term effects. On the other hand, malnutrition influences the brain throughout life, with profound implications on cognitive decline and dementia. Several phytochemicals with potent antioxidant and anti-inflammatory activities, have been shown to repress microglial activation and exert neuroprotective effects. Thus this review highlights the role of foods, nutrients and phytochemicals in suppressing neuro-inflammation and also enhancing cognition.

Minocycline attenuates Aβ oligomers-induced pro-inflammatory phenotype in primary microglia while enhancing Aβ fibrils phagocytosis

Microglia, the brain innate immune cells, are activated in response to amyloid beta (Aβ) resulting in neuroinflammation in AD brains. Recently, two phenotypes have been described for microglia: the pro-inflammatory classical and the anti-inflammatory alternative. Changes in microglia phenotype that control their phagocytic function are yet to be determined. The highly neurotoxic Aβ oligomers (oAβ) formed at an early disease stage induce pro-inflammatory microglia activation releasing neurotoxic mediators and contributing to neurodegeneration. A novel strategy for AD treatment is to attenuate microglia-induced inflammation while maintaining efficient Aβ clearance. Minocycline effectively crosses the blood-brain barrier and has widely reported neuroprotective effects. Yet, its exact mechanism of neuroprotection and its effects on microglia are still unknown. The aim of this study is to investigate the effect of minocycline on the phagocytic uptake of fAβ by primary microglia in relation to their activation state in an inflammatory milieu generated by oAβ or LPS. The study shows that minocycline is able to attenuate oAβ-induced neuroinflammatory response of microglia by inhibiting their pro-inflammatory phenotype activation. In addition, a significant enhancement of fAβ phagocytosis by minocycline- treated microglia is reported for the first time, providing novel insight into its neuroprotective role in AD.

Resveratrol attenuates hypoxia-induced neurotoxicity through inhibiting microglial activation

Resveratrol is a natural polyphenol enriched in Polygonum cuspidatum and has been found to afford neuroprotective effects against neuroinflammation in the brain. Activated microglia can secrete various pro-inflammatory cytokines and neurotoxic mediators, which may contribute to hypoxic brain injuries. The aim of this study is to investigate the potential role of resveratrol in attenuating hypoxia-induced neurotoxicity via its anti-inflammatory actions through in vitro models of the BV-2 microglial cell line and primary microglia. We found that resveratrol significantly inhibited hypoxia-induced microglial activation and reduced subsequent release of pro-inflammatory factors. In addition, resveratrol inhibited the hypoxia-induced degradation of IκB-alpha and phosphorylation of p65 NF-κB protein. Hypoxia-induced ERK1/2 and JNK phosphorylation was also strongly inhibited by resveratrol, whereas resveratrol had no effect on hypoxia-stimulated p38 MAPK phosphorylation. Importantly, treating primary cortical neurons with conditioned medium (CM) from hypoxia-stimulated microglia induced neuronal apoptosis, which was reversed by CM co-treated with resveratrol. Taken together, resveratrol exerts neuroprotection against hypoxia-induced neurotoxicity through its anti-inflammatory effects in microglia. These effects were mediated, at least in part, by suppressing the activation of NF-ĸB, ERK and JNK MAPK signaling pathways.

A novel nicotinic mechanism underlies β-amyloid-induced neurotoxicity

Loss of basal forebrain cholinergic neurons (BFCN) correlates with cognitive deficits in Alzheimer disease (AD). Our recent evidence suggests that chronic exposure to Aβ up-regulated neuronal α7-nAChRs and increased neuronal excitability in cultured hippocampal neurons. However, the impact of the up-regulated α7-nAChRs on Aβ-induced neurotoxicity remains unclear. In this study, we investigated the role of α7-nAChRs in the mediation of Aβ-induced neurotoxicity. The effects of Aβ exposure on α7-nAChRs and cytotoxicity were examined using whole-cell patch clamp recordings, atomic force microscope (AFM) imaging, immunoprecipitation, and lactate dehydrogenase (LDH) release assay in primary cultured hippocampal neurons as well as differentiated human neuroblastoma (SH-SY5Y) cells with cholinergic characteristics. We found that α7-nAChRs are necessary for Aβ-induced neurotoxicity in hippocampal neurons because chronic Aβ significantly increased LDH level in hippocampal cultures, which was prevented by either α7-nAChR antagonist methyllycaconitine (MLA) or by α7 subunit gene deletion (cultures prepared from nAChR α7 subunit KO mice), whereas β2-containing nAChR antagonist (dihydro-β-erythroidine, DhβE) or the genetic deletion of nAChR β2 subunit (cultures prepared from β2 KO mice) failed to prevent Aβ-induced toxicity. In SH-SY5Y cells, larger aggregates of Aβ preferentially up-regulated α7-nAChR expression and function accompanied by a significant decrease in cell viability. Co-treatment MLA, but not mecamylamine (MEC), prevented Aβ exposure-induced neurotoxicity. Our results suggest a detrimental role of upregulated α7-nAChRs in the mediation of Aβ-induced neurotoxicity.

Mast cells, brain inflammation and autism

Increasing evidence indicates that brain inflammation is involved in the pathogenesis of neuropsychiatric diseases. Mast cells (MCs) are located perivascularly close to neurons and microglia, primarily in the leptomeninges, thalamus, hypothalamus and especially the median eminence. Corticotropin-releasing factor (CRF) is secreted from the hypothalamus under stress and, together with neurotensin (NT), can stimulate brain MCs to release inflammatory and neurotoxic mediators that disrupt the blood–brain barrier (BBB), stimulate microglia and cause focal inflammation. CRF and NT synergistically stimulate MCs and increase vascular permeability; these peptides can also induce each other׳s surface receptors on MCs leading to autocrine and paracrine effects. As a result, brain MCs may be involved in the pathogenesis of “brain fog,” headaches, and autism spectrum disorders (ASDs), which worsen with stress. CRF and NT are significantly increased in serum of ASD children compared to normotypic controls further strengthening their role in the pathogenesis of autism. There are no clinically affective treatments for the core symptoms of ASDs, but pilot clinical trials using natural-antioxidant and anti-inflammatory molecules reported statistically significant benefit.

Proteolytic cleavage of ataxin-7 promotes SCA7 retinal degeneration and neurological dysfunction.

The neurodegenerative disorder spinocerebellar ataxia type 7 (SCA7) is caused by a polyglutamine (polyQ) expansion in the ataxin-7 protein, categorizing SCA7 as one member of a large class of heritable neurodegenerative proteinopathies. Cleavage of ataxin-7 by the protease caspase-7 has been demonstrated in vitro, and the accumulation of proteolytic cleavage products in SCA7 patients and mouse models has been identified as an early pathological change. However, it remains unknown whether a causal relationship exists between ataxin-7 proteolysis and in vivo SCA7 disease progression. To determine whether caspase cleavage is a critical event in SCA7 disease pathogenesis, we generated transgenic mice expressing polyQ-expanded ataxin-7 with a second-site mutation (D266N) to prevent caspase-7 proteolysis. When we compared SCA7-D266N mice with SCA7 mice lacking the D266N mutation, we found that SCA7-D266N mice exhibited improved motor performance, reduced neurodegeneration and substantial lifespan extension. Our findings indicate that proteolysis at the D266 caspase-7 cleavage site is an important mediator of ataxin-7 neurotoxicity, suggesting that inhibition of caspase-7 cleavage of polyQ-ataxin-7 may be a promising therapeutic strategy for this untreatable disorder.

Soluble Sugar-Based Quinoline Derivatives as New Antioxidant Modulators of Metal-Induced Amyloid Aggregation

Oxidative stress and protein aggregation have been demonstrated to be the major factors involved in neurodegenerative diseases. Metal ions play a pivotal role, acting as mediators of neurotoxicity either by favoring or redox cycling. Thus, they represent a promising and suitable therapeutic target for the treatment of neurodegenerative disorders. In particular, the development of bifunctional or multifunctional molecules, which have antiaggregant and metal-chelating/antioxidant properties, may be considered as a valuable strategy for the treatment of neurodegeneration considering its multifactorial nature. Herein, we report the design and the characterization of four new multifunctional sugar-appended 8-hydroxyquinolines focusing on the effects of the conjugation with trehalose, a nonreducing disaccharide involved in the protection of proteins and cells against environmental stresses. These glycoconjugates do not exhibit any antiproliferative activity against three human cell lines of different histological origin, unlike 8-hydroxyquinolines. The multiple properties of the new derivatives are highlighted, reporting their Cu(2+) and Zn(2+) binding ability, and antioxidant and antiaggregant capacities. In particular, these latter were determined by different assays, including the evaluation of their ability to modulate or even suppress the aggregation of Aβ1-40 and Aβ1-42 peptides induced by copper or zinc ions.

Endocrine dysfunction in sepsis: a beneficial or deleterious host response?

Sepsis is a systemic, deleterious inflammatory host response triggered by an infective agent leading to severe sepsis, septic shock and multi-organ failure. The host response to infection involves a complex, organized and coherent interaction between immune, autonomic, neuroendocrine and behavioral systems. Recent data have confirmed that disturbances of the autonomic nervous and neuroendocrine systems could contribute to sepsis-induced organ dysfunction. Through this review, we aimed to summarize the current knowledge about the endocrine dysfunction as response to sepsis, specifically addressed to vasopressin, copeptin, cortisol, insulin and leptin. We searched the following readily accessible, clinically relevant databases: PubMed, UpToDate, BioMed Central. The immune system could be regarded as a "diffuse sensory organ" that signals the presence of pathogens to the brain through different pathways, such as the vagus nerve, endothelial activation/dysfunction, cytokines and neurotoxic mediators and the circumventricular organs, especially the neurohypophysis. The hormonal profile changes substantially as a consequence of inflammatory mediators and microorganism products leading to inappropriately low levels of vasopressin, sick euthyroid syndrome, reduced adrenal responsiveness to ACTH, insulin resistance, hyperglycemia as well as hyperleptinemia. In conclusion, clinical diagnosis of this "pan-endocrine illness" is frequently challenging due to the many limiting factors. The most important benefits of endocrine markers in the management of sepsis may be reflected by their potential to be used as biomarkers in different scoring systems to estimate the severity of the disease and the risk of death.

Fluoxetine and S-citalopram inhibit M1 activation and promote M2 activation of microglia in vitro

Increasing evidence has suggested that microglia dysfunction plays an important role in the pathogenesis of depression. Both classical activation (M1 activation) and alternative activation (M2 activation) may be involved in the process. M1-activated microglia secrete various pro-inflammatory cytokines and neurotoxic mediators, which may contribute to the development of depression, while M2-activated microglia promote tissue reconstruction by releasing anti-inflammatory cytokines involved in the process of depression. Selective serotonin reuptake inhibitors (SSRIs) are first-line treatments for depression, and their effects on immune system modulation have recently gained attention. Several studies have suggested that SSRIs affect the M1 activation of microglia, but results have varied. In addition, little is known about the effect of SSRIs on M2 activation in depression. The aim of this study was to investigate the effects of fluoxetine and S-citalopram, two widely used SSRIs in clinical, on both the M1 and M2 activation of microglia (the murine BV2 cell line and mouse primary microglia cell). The indexes of activation were measured by real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and Western blot. The present results showed that both fluoxetine and S-citalopram significantly down-regulated the indexes of M1 activation and up-regulated the M2 activation indexes on mRNA and protein levels either in cell line or primary cells. Taken together, the results suggested that fluoxetine and S-citalopram modulated the immune system by inhibiting M1 activation and by improving M2 activation of microglia and that the immune system modulation may partially mediate the therapeutic effects of antidepressant drugs-SSRIs.

Downregulation of microglial activation by achillolide A.

Chronic inflammation has been implicated in the pathogenesis of various neurodegenerative diseases. During the neuroinflammatory process, microglial cells release neurotoxic and proinflammatory mediators. In the present study, using activity-guided fractionation, we have purified an anti-inflammatory compound determined by spectroscopic methods to be a sesquiterpene lactone named achillolide A from Achillea fragrantissima (Forsk.) Sch. Bip. In primary cultures of lipopolysaccharide-activated microglial cells, achillolide A inhibited the lipopolysaccharide-induced levels of proinflammatory and toxic mediators including glutamate, nitric oxide, matrix metalloproteinase-9, cyclooxygenase-2, induced nitric oxide synthase, interleukin-1β, and tumor necrosis factor-α. Achillolide A also exhibited an antioxidant capacity, as was shown in a cell free system as well as by its ability to reduce intracellular reactive oxygen species levels in microglial cells. Thus, achillolide A might have therapeutic potential for treatment of neurodegenerative diseases and deserves further studies.

Abstract W P251: Novel Platelet-Activating Factor Receptor Antagonists Attenuate Brain Injury after Experimental Stroke

INTRODUCTION: Platelet-activating factor (PAF) is a bioactive phospholipid that regulates synaptic activity and when overproduced is a potent mediator of leukocyte functions, including platelet aggregation and inflammation. PAF concentration increases during ischemia-reperfusion and is a mediator of neurotoxicity. The inhibition of this process plays a critical role in neuronal survival. We synthesized various analogues and identified them as PAF receptor antagonists, which were then selected for in vivo experiments to evaluate anti-ischemic activity. METHODS: Physiologically controlled Sprague-Dawley rats received 2 h MCAo by poly-L-lysine-coated intraluminal suture. LAU compounds (LAU-0901, LAU-09015, LAU-09018, LAU-09019, LAU-09020 or LAU-09023; 30 mg/kg) or vehicle (45% cyclodextran) was administered IP at 2 h from onset of MCAo. Neurological status was evaluated during occlusion (at 60 min) and on day 1, 2, 3 and 7. Histopathology was carried out on day 7. RESULTS: Physiological variables were stable and showed no significant differences between groups. All LAU compounds significantly reduced the neurobehavioral deficit compared to the vehicle group, beginning from day 1 and persisting through the 7 day survival period. Histologically, the vehicle group showed large hemispheric infarcts with pan-necrosis. By contrast, LAU-treated rats showed preserved hemispheric structure and small subcortical infarcts. Treatment with LAU (LAU-0901, LAU-09015, LAU-09018, LAU-09019, or LAU-09023) significantly reduced total infarct volume compared to the vehicle group by 68%, 52%, 40%, 51% and 54%, respectively. CONCLUSION: Our results clearly indicate that novel LAU PAF receptor antagonists are highly neuroprotective on behavior and reduction of brain infarction in experimental stroke. These compounds may provide the basis for future therapeutics in patients suffering ischemic stroke.

CCR2 antagonism alters brain macrophage polarization and ameliorates cognitive dysfunction induced by traumatic brain injury.

Traumatic brain injury (TBI) is a major risk factor for the development of multiple neurodegenerative diseases. With respect to the increasing prevalence of TBI, new therapeutic strategies are urgently needed that will prevent secondary damage to primarily unaffected tissue. Consistently, neuroinflammation has been implicated as a key mediator of secondary damage following the initial mechanical insult. Following injury, there is uncertainty regarding the role that accumulating CCR2(+) macrophages play in the injury-induced neuroinflammatory sequelae and cognitive dysfunction. Using CX3CR1(GFP/+)CCR2(RFP/+) reporter mice, we show that TBI initiated a temporally restricted accumulation of peripherally derived CCR2(+) macrophages, which were concentrated in the hippocampal formation, a region necessary for learning and memory. Multivariate analysis delineated CCR2(+) macrophages' neuroinflammatory response while identifying a novel therapeutic treatment window. As a proof of concept, targeting CCR2(+) macrophages with CCX872, a novel Phase I CCR2 selective antagonist, significantly reduced TBI-induced inflammatory macrophage accumulation. Concomitantly, there was a significant reduction in multiple proinflammatory and neurotoxic mediators with this treatment paradigm. Importantly, CCR2 antagonism resulted in a sparing of TBI-induced hippocampal-dependent cognitive dysfunction and reduced proinflammatory activation profile 1 month after injury. Thus, therapeutically targeting the CCR2(+) subset of monocytes/macrophages may provide a new avenue of clinical intervention following TBI.