Primary Microglial Cultures Research Articles

Abstract TMP55: TGF-β1 Induces Microglial BDNF Production and Improves Functional Outcome After Intracerebral Hemorrhage

Introduction: Intracerebral hemorrhage (ICH) results in the activation of microglia. Microglia can become classically activated, promoting neurotoxicity or alternatively activated, promoting tissue repair. Our lab has previously shown that microglia become alternatively activated between 3 and 14 days after ICH. This transition is critical to recovery, yet the mechanism is unknown. The anti-inflammatory cytokine TGF-β1 has a critical role in microglial development and homeostasis. We hypothesize that TGF-β1 is the mediator of microglia alternative activation after ICH and that TGF-β1 promotes microglial secretion of BDNF to promote tissue repair. Methods: Primary microglial cultures from C57BL/6 (WT) P0-P2 pups were used to study the mechanism of alternative activation. Microglia were activated with thrombin with or without TGF-β1 for 8 and 24 hours. BDNF was measured by intracellular cytokine staining (ICS) (flow cytometry) and ELISA from cell supernatants. ICH was induced by injecting 25ul of whole blood into the right striatum of male WT mice. Mice were treated with either 10ng TGF-β1 or PBS intracerebrally immediately prior to blood injection to target microglia. Behavioral outcomes were measured by cylinder test and beam walking. Brains were harvested at 7 days for immunohistochemistry (IHC). Results: Primary microglia that were activated with thrombin and TGF-β1 had more BDNF+ cells by ICS at 8 hours and secreted more BDNF by ELISA at 24 hours compared to thrombin alone in vitro (fig 1). Mice treated with TGF-β1 had better functional outcomes by cylinder test and beam walking than PBS treated mice 24 hours after ICH. TGF-β1 treated mice had significantly more BDNF+ CD11b+ cells (microglia/macrophages) than PBS treated 7 days after ICH by IHC. Conclusions: TGF-β1 treatment induces microglial BDNF production and improves functional outcomes 24 hours after ICH. Ongoing work will determine whether microglial BDNF production is the link to improved outcomes.

Blue Light Modulates Murine Microglial Gene Expression in the Absence of Optogenetic Protein Expression.

Neural optogenetic applications over the past decade have steadily increased; however the effects of commonly used blue light paradigms on surrounding, non-optogenetic protein-expressing CNS cells are rarely considered, despite their simultaneous exposure. Here we report that blue light (450 nm) repetitively delivered in both long-duration boluses and rapid optogenetic bursts gene-specifically altered basal expression of inflammatory and neurotrophic genes in immortalized and primary murine wild type microglial cultures. In addition, blue light reduced pro-inflammatory gene expression in microglia activated with lipopolysaccharide. These results demonstrate previously unreported, off-target effects of blue light in cells not expressing optogenetic constructs. The unexpected gene modulatory effects of blue light on wild type CNS resident immune cells have novel and important implications for the neuro-optogenetic field. Further studies are needed to elucidate the molecular mechanisms and potential therapeutic utility of blue light modulation of the wild type CNS.

Aconitum-Derived Bulleyaconitine A Exhibits Antihypersensitivity Through Direct Stimulating Dynorphin A Expression in Spinal Microglia.

Aconitine and its structurally-related diterpenoid alkaloids have been shown to interact differentially with neuronal voltage-dependent sodium channels, which was suggested to be responsible for their analgesia and toxicity. Bulleyaconitine A (BAA) is an aconitine analogue and has been prescribed for the management of pain. The present study aimed to evaluate the inhibitory effects of BAA on pain hypersensitivity and morphine antinociceptive tolerance, and explore whether the expression of dynorphin A in spinal microglia was responsible for its actions. Single intrathecal or subcutaneous (but not intraventricular or local) injection of BAA blocked spinal nerve ligation-induced painful neuropathy, bone cancer-induced pain, and formalin-induced tonic pain by 60 to 100% with the median effective dose values of 94 to 126ng per rat (intrathecal) and 42 to 59μg/kg (subcutaneous), respectively. After chronic treatment, BAA did not induce either self-tolerance to antinociception or cross-tolerance to morphine antinociception, and completely inhibited morphine tolerance. The microglial inhibitor minocycline entirely blocked spinal BAA (but not exogenous dynorphin A) antinociception, but failed to attenuate spinal BAA neurotoxicity. In a minocycline-sensitive and lidocaine- or ropivacaine-insensitive manner, BAA stimulated the expression of dynorphin A in the spinal cord, and primary cultures of microglia but not of neurons or astrocytes. The blockade effects of BAA on nociception and morphine tolerance were totally eliminated by the specific dynorphin A antiserum and/or κ-opioid receptor antagonist. Our results suggest that BAA eliminates pain hypersensitivity and morphine tolerance through directly stimulating dynorphin A expression in spinal microglia, which is not dependent on the interactions with sodium channels. The newly illustrated mechanisms underlying BAA antinociception help us to better understand and develop novel dynorphin A expression-based painkillers to treat chronic pain.

Amitriptyline induces brain-derived neurotrophic factor (BDNF) mRNA expression through ERK-dependent modulation of multiple BDNF mRNA variants in primary cultured rat cortical astrocytes and microglia

A significant role of brain-derived neurotrophic factor (BDNF) has been previously implicated in the therapeutic effect of antidepressants. To ascertain the contribution of specific cell types in the brain that produce BDNF following antidepressant treatment, the effects of the tricyclic antidepressant amitriptyline on rat primary neuronal, astrocytic and microglial cortical cultures were examined. Amitriptyline increased the expression of BDNF mRNA in astrocytic and microglial cultures but not neuronal cultures. Antidepressants with distinct mechanisms of action, such as clomipramine, duloxetine and fluvoxamine, also increased BDNF mRNA expression in astrocytic and microglial cultures. There are multiple BDNF mRNA variants (exon I, IIA, IV and VI) expressed in astrocytes and microglia and the variant induced by antidepressants has yet to be elaborated. Treatment with antidepressants increased the expression of exon I, IV and VI in astrocyte and microglia. Clomipramine alone significantly upregulated expression of exon IIA. The amitriptyline-induced expression of both total and individual BDNF mRNA variants (exon I, IV and VI) were blocked by MEK inhibitor U0126, indicating MEK/ERK signaling is required in the expression of BDNF. These findings indicate that non-neural cells are a significant target of antidepressants and further support the contention that glial production of BDNF is crucial role in the therapeutic effect of antidepressants. The current data suggest that targeting of glial function could lead to the development of antidepressants with a truly novel mechanism of action.

Membrane protein remodeling in microglia exposed to amyloid peptides

Infection, neurodegeneration, and other conditions associated with loss of brain homeostasis, induce changes in microglial morphology, gene expression and function, generally referred to as “activation”. Alzheimer’s disease (AD) is the most common dementia and is characterized by neuroinflammatory changes, including alterations in the morphology and distribution of microglia and astrocytes, and deposition of complement and other inflammatory mediators. Our previous observations show that microglial cells challenged in vitro with amyloid peptides clustered and rounded up, dramatically changing their morphology. Besides, in these cells we observed the early acetylation and then the phosphorylation of STAT3 which is required for the expression of the epsilon isoform of 14-3-3, a marker of Abeta-activated microglia (1, 2). We applied affinity partitioning approach combined with high throughput mass spectrometric analysis in order to identify variation of proteins on plasma membrane of BV2 immortalized microglia upon treatment with amyloid peptides. By this method several proteins up- or down-regulated by amyloid treatment were identified in microglial plasma membrane. Among them annexins (5 and 7), IFITM3 and MARK3. These data have been confirmed in primary microglial cultures.

A new approach to follow a single extracellular vesicle-cell interaction using optical tweezers.

Extracellular vesicles (EVs) are spherical membrane structures released by most cells. These highly conserved mediators of intercellular communication carry proteins, lipids, and nucleic acids, and transfer these cellular components between cells by different mechanisms, such as endocytosis, macropinocytosis, or fusion. However, the temporal and spatial dynamics of vesicle-cell interactions still remain largely unexplored. Here we used optical tweezers to drive single EVs produced by microglial cells onto the surface of astrocytes or microglia in primary culture. By visualizing single EV-cell contacts, we observed that microglial vesicles displayed different motilities on the surface of astrocytes compared with microglia. After contact, EVs positioned on astrocytes displayed some minor oscillatory motion around the point of adhesion, while vesicles dragged to microglia displayed quite regular directional movement on the plasma membrane. Both the adhesion and motion of vesicles on glial cells were strongly reduced by cloaking phosphatidylserine (PS) residues, which are externalized on the vesicle membrane and act as determinants for vesicle recognition by target cells. These data identify optical manipulation as a powerful tool to monitor in vitro vesicle-cell dynamics with high temporal and spatial resolution and to determine in a quantitative manner the contribution of surface receptors/extracellular protein ligands to the contact.

Microglial Phagocytosis Assay.

Phagocytosis is essential for microglial clearance of apoptotic cells, extracellular protein aggregates, and infectious bacteria in the central nervous system (CNS). While the preparation of primary microglial culture has been described elsewhere, this protocol describes the microglial phagocytosis experimental procedure and the subsequent measurement of microglial phagocytic ability using fluorescent latex beads or fluorescent amyloid beta 42 (Aβ42) peptides.

Protocol for Primary Microglial Culture Preparation.

Primary microglia, in either mono-culture or co-culture with neurons or astrocytes, are a powerful tool for studying mechanisms underlying microglial inflammatory responses and cell type-specific interactions in the central nervous system (CNS). This protocol provides the details of how to prepare high purity primary microglia from newborn mouse pups. The overall steps include brain cell dissociation, mixed glial cell culture, and microglia isolation.

Intracellular degradation of chemically functionalized carbon nanotubes using a long-term primary microglial culture model.

Chemically functionalized carbon nanotubes (f-CNTs) have been used in proof-of-concept studies to alleviate debilitating neurological conditions. Previous in vivo observations in brain tissue have suggested that microglia - acting as resident macrophages of the brain - play a critical role in the internalization of f-CNTs and their partial in situ biodegradation following a stereotactic administration in the cortex. At the same time, several reports have indicated that immune cells such as neutrophils, eosinophils and even macrophages could participate in the processing of carbon nanomaterials via oxidation processes leading to degradation, with surface properties acting as modulators of CNT biodegradability. In this study we questioned whether degradability of f-CNTs within microglia could be modulated depending on the type of surface functionalization used. We investigated the kinetics of degradation of multi-walled carbon nanotubes (MWNTs) functionalized via different chemical strategies that were internalized within isolated primary microglia over three months. A cellular model of rat primary microglia that can be maintained in cell culture for a long period of time was first developed. The Raman structural signature of the internalized f-CNTs was then studied directly in cells over a period of up to three months, following a single exposure to a non-cytotoxic concentration of three different f-CNTs (carboxylated, aminated and both carboxylated and aminated). Structural modifications suggesting partial but continuous degradation were observed for all nanotubes irrespective of their surface functionalization. Carboxylation was shown to promote more pronounced structural changes inside microglia over the first two weeks of the study.

Selective A2A receptor antagonist prevents microglia-mediated neuroinflammation and protects retinal ganglion cells from high intraocular pressure–induced transient ischemic injury

Glaucoma is a leading cause of vision loss and blindness worldwide, characterized by chronic and progressive neuronal loss. Reactive microglial cells have been recognized as a neuropathologic feature, contributing to local inflammation and retinal neurodegeneration. In a recent in vitro work (organotypic cultures), we demonstrated that blockade of adenosine A2A receptor (A2AR) prevents the neuroinflammatory response and affords protection to retinal ganglion cells (RGCs) against exposure to elevated hydrostatic pressure (EHP), to mimic elevated intraocular pressure (IOP), the main risk factor for glaucoma development. Herein, we investigated whether a selective A2AR antagonist (SCH 58261) could modulate retinal microglia reactivity and their inflammatory response. Furthermore, we took advantage of the high IOP-induced transient ischemia (ischemia-reperfusion, I-R) animal model to evaluate the protective role of A2AR blockade in the control of retinal neuroinflammation and neurodegeneration. Primary microglial cell cultures were challenged either with lipopolysaccharide or with EHP, in the presence or absence of A2AR antagonist SCH 58261 (50 nM). In addition, I-R injury was induced in adult Wistar rats after intravitreal administration of SCH 58261 (100 nM, 5 μL). Our results showed that SCH 58261 attenuated microglia reactivity and the increased expression and release of proinflammatory cytokines. Moreover, intravitreal administration of SCH 58261 prevented I-R-induced cell death and RGC loss, by controlling microglial-mediated neuroinflammatory response. These results prompt the proposal that A2AR blockade may have great potential in the management of retinal neurodegenerative diseases characterized by microglia reactivity and RGC death, such as glaucoma and ischemic diseases.

Glycogen synthase kinase 3 is part of the molecular machinery regulating the adaptive response to LPS stimulation in microglial cells

Repeated stimulation of TLR4 signaling by lipopolysaccharide (LPS) in microglia induces a state of tolerance/sensitization consisting in the reprogramming of the expression of pro-inflammatory genes in favor of anti-inflammatory ones. The molecular mechanisms underlying this adaptive response are far to be elucidated. Glycogen synthase kinase 3 (GSK3) has emerged as crucial regulator of TLR signaling, mediating the balance between pro- and anti-inflammatory functions in both periphery and central nervous system. The present study extends this notion identifying GSK3 as part of the molecular machinery regulating the LPS-adaptive response in microglial cells, by using primary microglial cultures and organotypic hippocampal slices (OHSCs). We found that lithium chloride (LiCl), a widely used GSK3 inhibitor and the mainstay treatment for bipolar disorder, reinforced the LPS adaptive response by enhancing both downregulation of pro-inflammatory genes (inducible nitric oxide synthase, interleukin 1β, interleukin 6, tumor necrosis factor α), and upregulation of genes typically associated to anti-inflammatory functions (interleukin 10 and MRC1). The effects of GSK3 inhibition were mimicked by Wnt3a, added exogenously, and reversed by Inhibitor of Wnt-Response-1-endo, a pharmacological disruptor of the canonical Wnt/β-catenin pathway, and GW9662, a selective peroxisome proliferator activated receptor γ antagonist, suggesting that these two pathways are involved in the regulation of LPS-tolerance/sensitization by GSK. Finally, LiCl treatment of OHSCs enhanced the protective functional consequences of the microglial adaptive response to LPS on oligodendrocyte maturation, as indicated by MBP mRNA upregulation. These results further indicate GSK3 as key component in the orchestration of neuroinflammation and target for neuroprotective strategies.

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.

Peptidic exenatide and herbal catalpol mediate neuroprotection via the hippocampal GLP-1 receptor/β-endorphin pathway

Both peptidic agonist exenatide and herbal agonist catalpol of the glucagon-like peptide-1 receptor (GLP-1R) are neuroprotective. We have previously shown that activation of spinal GLP-1Rs expresses β-endorphin in microglia to produce antinociception. The aim of this study was to explore whether exenatide and catalpol exert neuroprotection via activation of the hippocampal GLP-1R/β-endorphin pathway. The rat middle cerebral artery occlusion model was employed, and the GLP-1R immunofluorescence staining and β-endorphin measurement were assayed in the hippocampus and primary cultures of microglia, neurons and astrocytes. The immunoreactivity of GLP-1Rs on microglia in the hippocampus was upregulated after ischemia reperfusion. Intracerebroventricular (i.c.v.) injection of exenatide and catalpol produced neuroprotection in the rat transient ischemia/reperfusion model, reflected by a marked reduction in brain infarction size and a mild recovery in neurobehavioral deficits. In addition, i.c.v. injection of exenatide and catalpol significantly stimulated β-endorphin expression in the hippocampus and cultured primary microglia (but not primary neurons or astrocytes). Furthermore, exenatide and catalpol neuroprotection was completely blocked by i.c.v. injection of the GLP-1R orthosteric antagonist exendin (9–39), specific β-endorphin antiserum, and selective opioid receptor antagonist naloxone. Our results indicate, for the first time, that the neuroprotective effects of catalpol and exenatide are GLP-1R-specific, and that these effects are mediated by β-endorphin expression probably in hippocampal microglia. We postulate that in contrast to the peripheral tissue, where the activation of GLP-1Rs in pancreas islet β-cells causes secretion of insulin to perform glucoregulation, it leads to β-endorphin expression in microglial cells to produce neuroprotection and analgesia in the central nervous system.

Non-Selective Cannabinoid Receptor Antagonists, Hinokiresinols Reduce Infiltration of Microglia/Macrophages into Ischemic Brain Lesions in Rat via Modulating 2-Arachidonolyglycerol-Induced Migration and Mitochondrial Activity.

Growing evidence suggests that therapeutic strategies to modulate the post-ischemic inflammatory responses are promising approaches to improve stroke outcome. Although the endocannabinoid system has been emerged as an endogenous therapeutic target to regulate inflammation after stroke insult, the downstream mechanisms and their potentials for therapeutic intervention remain controversial. Here we identified trans- and cis-hinokiresinols as novel non-selective antagonists for two G-protein-coupled cannabinoid receptors, cannabinoid receptor type 1 and type 2. The Electric Cell-substrate Impedance Sensing and Boyden chamber migration assays using primary microglial cultures revealed that both hinokiresinols significantly inhibited an endocannabinoid, 2-arachidonoylglycerol-induced migration. Hinokiresinols modulated 2-arachidonoylglycerol-induced mitochondrial bioenergetics in microglia as evidenced by inhibition of ATP turnover and reduction in respiratory capacity, thereby resulting in impaired migration activity. In rats subjected to transient middle cerebral artery occlusion (1.5-h) followed by 24-h reperfusion, post-ischemic treatment with hinokiresinols (2 and 7-h after the onset of ischemia, 10 mg/kg) significantly reduced cerebral infarct and infiltration of ED1-positive microglial/macrophage cells into cerebral ischemic lesions in vivo. Co-administration of exogenous 2-AG (1 mg/kg, i.v., single dose at 2 h after starting MCAO) abolished the protective effect of trans-hinokiresionol. These results suggest that hinokiresinols may serve as stroke treatment by targeting the endocannabinoid system. Alteration of mitochondrial bioenergetics and consequent inhibition of inflammatory cells migration may be a novel mechanism underlying anti-ischemic effects conferred by cannabinoid receptor antagonists.

Intranasal Delivery of RGD Motif-Containing Osteopontin Icosamer Confers Neuroprotection in the Postischemic Brain via αvβ3 Integrin Binding.

Osteopontin (OPN) is a phosphorylated glycoprotein possessing an arginine-glycine-aspartate (RGD)-motif, which binds to several cell surface integrins and mediates a wide range of cellular processes. Inductions of OPN have been reported in the postischemic brain, and the neuroprotective effects of OPN have been demonstrated in animal models of stroke. In the present study, we showed a robust neuroprotective effect of RGD-containing icosamer OPN peptide (OPNpt20) in a rat model of focal cerebral ischemia (middle cerebral artery occlusion, MCAO). Intranasally administered OPNpt20 reduced mean infarct volume by 79.7% compared to the treatment-naïve MCAO control animals and markedly ameliorated neurological deficits. In addition, OPNpt20 significantly suppressed the inductions of iNOS and of inflammatory markers in postischemic brains and in primary microglial cultures, demonstrating anti-inflammatory effects. Administration of a mutant peptide, in which RGD was replaced by arginine-alanine-alanine (RAA), failed to suppress infarct volumes in MCAO animals and co-administration of OPNpt20 with anti-αvβ3 integrin antibody failed to suppress iNOS induction in primary microglia culture, indicating that the RGD motif in OPNpt20 and endogenous αvβ3 integrin play critical roles. Furthermore, pull-down assay revealed a direct binding between OPNpt20 and αvβ3 integrin in primary microglia culture. Together, these results indicate that RGD-containing OPN icosamer has therapeutic potential in the postischemic brain and αvβ3 integrin-mediated anti-inflammatory effect might be an underlying mechanism.

The mitochondrial uncoupling protein-2 is a master regulator of both M1 and M2 microglial responses.

Microglial activation is a dynamic process, central to neuroinflammation, which can have beneficial or pathogenic effects to human health. Mitochondria are key players in neuroinflammatory and neurodegenerative processes, common to most brain diseases. To the best of our knowledge on the role of mitochondria in the modulation of neuroinflammation, we focused on the mitochondrial uncoupling protein-2 (UCP2), known to control mitochondrial functions and to be implicated in a variety of physiological and pathological processes. In primary microglial cultures, the M1 stimulus lipopolysaccharide induced an early and transitory decrease in UCP2 levels. The initial UCP2 down-regulation was paralleled by mitochondrial inner membrane potential (mMP) depolarization and increased mitochondrial reactive oxygen species production. The key role of UCP2 in controlling mMP and reactive oxygen species production was confirmed by both pharmacological inhibition and down-regulation by RNA interference. Additionally, UCP2-silenced microglia stimulated with lipopolysaccharide showed an enhanced inflammatory response, characterized by a greater production of nitric oxide and interleukin-6. UCP2 was differently regulated by M2 stimuli, as indicated by its persistent up-regulation by interleukin-4. In UCP2-silenced microglia, interleukin-4 failed to induce M2 genes (mannose receptor 1 and interleukin-10) and to reduce M1 genes (inducible nitric oxide synthase and tumour necrosis factor-α). Our findings indicate that UCP2 is central to the process of microglial activation, with opposite regulation of M1 and M2 responses, and point to UCP2 manipulation as a potential strategy for redirecting microglial response towards protective phenotypes in several brain diseases where neuroinflammation is recognized to contribute to neurodegeneration. We show that the mitochondrial uncoupling protein-2 (UCP2) is central to the process of microglial activation, with opposite regulation of M1 and M2 responses. In UCP2-silenced microglia, lipopolysaccharide (LPS) triggers an enhanced inflammatory response characterized by a greater expression of M1 genes, whereas interleukin-4 (IL-4) fails in inducing M2 genes and reducing M1 genes. We propose UCP2 manipulation as a potential strategy for redirecting microglial response towards protective phenotypes.

The effects of tiagabine on lipopolysaccharide (LPS)-induced proinflammatory cytokine release from primary rat microglial cell cultures

The effects of tiagabine on lipopolysaccharide (LPS)-induced proinflammatory cytokine release from primary rat microglial cell cultures

S100B inhibition reduces behavioral and pathologic changes in experimental traumatic brain injury.

Neuroinflammation following traumatic brain injury (TBI) is increasingly recognized to contribute to chronic tissue loss and neurologic dysfunction. Circulating levels of S100B increase after TBI and have been used as a biomarker. S100B is produced by activated astrocytes and can promote microglial activation; signaling by S100B through interaction with the multiligand advanced glycation end product-specific receptor (AGER) has been implicated in brain injury and microglial activation during chronic neurodegeneration. We examined the effects of S100B inhibition in a controlled cortical impact model, using S100B knockout mice or administration of neutralizing S100B antibody. Both interventions significantly reduced TBI-induced lesion volume, improved retention memory function, and attenuated microglial activation. The neutralizing antibody also significantly reduced sensorimotor deficits and improved neuronal survival in the cortex. However, S100B did not alter microglial activation in BV2 cells or primary microglial cultures stimulated by lipopolysaccharide or interferon gamma. Further, proximity ligation assays did not support direct interaction in the brain between S100B and AGER following TBI. Future studies are needed to elucidate specific pathways underlying S100B-mediated neuroinflammatory actions after TBI. Our results strongly implicate S100B in TBI-induced neuroinflammation, cell loss, and neurologic dysfunction, thereby indicating that it is a potential therapeutic target for TBI.

Dimethyl Fumarate Protects Brain From Damage Produced by Intracerebral Hemorrhage by Mechanism Involving Nrf2.

Intracerebral hemorrhage (ICH) represents a devastating form of stroke for which there is no effective treatment. This preclinical study was designed to evaluate dimethyl fumarate (DMF), a substance recently approved for the treatment of multiple sclerosis, as therapy for ICH. We hypothesized that DMF through activating the master regulator of cellular self-defense responses, transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), would act as effective treatment for ICH-mediated damage. Male rats and mice, including Nrf2 knockouts, were subjected to intracerebral injection of blood (to mimic ICH) and then treated with DMF. Neurological deficit, brain edema, gene induction profile and hematoma resolution were evaluated. Phagocytic functions of primary microglia in culture were used to study hematoma resolution. Treatment with DMF induced Nrf2-target genes, improved hematoma resolution, reduced brain edema, and ultimately enhanced neurological recovery in rats and wild-type, but not Nrf2 knockout, mice. Most importantly, the treatment of ICH with DMF showed a 24 h window of therapeutic opportunity. A clinically relevant dose of DMF demonstrates potent therapeutic efficacy and impressive 24 h therapeutic window of opportunity. This study merits further evaluation of this compound as potential treatment for ICH in humans.

The Receptor CMRF35-Like Molecule-1 (CLM-1) Enhances the Production of LPS-Induced Pro-Inflammatory Mediators during Microglial Activation.

CMRF35-like molecule-1 (CLM-1) belongs to a receptor family mainly expressed in myeloid cells that include activating and inhibitory receptors. CLM-1 contains two ITIMs and a single immunoreceptor tyrosine-based switch motif (ITSM), although also displays a binding site for p85α regulatory subunit of PI3K. By using murine primary microglial cultures, we show the presence of all CLM members in microglial cells and characterize the expression of CLM-1 both in basal conditions and during microglial activation. The TLR4 agonist lipopolysaccharide (LPS) and the TLR3 agonist polyinosinic–polycytidylic acid (Poly I:C) induce an increase in microglial CLM-1 mRNA levels in vitro, whereas the TLR2/6 heterodimer agonist peptidoglycan (PGN) produces a marked decrease. In this study we also describe a new soluble isoform of CLM-1 that is detected at mRNA and protein levels in basal conditions in primary microglial cultures. Interestingly, CLM-1 engagement enhances the transcription of the pro-inflammatory mediators TNFα, COX-2 and NOS-2 in microglial cells challenged with LPS. These results reveal that CLM-1 can acts as a co-activating receptor and suggest that this receptor could play a key role in the regulation of microglial activation.