Sodium channels contribute to microglia/macrophage activation and function in EAE and MS

Loss of axons is a major contributor to nonremitting deficits in the inflammatory demyelinating disease multiple sclerosis (MS). Based on biophysical studies showing that activity of axonal sodium channels can trigger axonal degeneration, recent studies have tested sodium channel-blocking drugs in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and have demonstrated a protective effect on axons. However, it is possible that, in addition to a direct effect on axons, sodium channel blockers may also interfere with inflammatory mechanisms. We therefore examined the novel hypothesis that sodium channels contribute to activation of microglia and macrophages in EAE and acute MS lesions. In this study, we demonstrate a robust increase of sodium channel Nav1.6 expression in activated microglia and macrophages in EAE and MS. We further demonstrate that treatment with the sodium channel blocker phenytoin ameliorates the inflammatory cell infiltrate in EAE by 75%. Supporting a role for sodium channels in microglial activation, we show that tetrodotoxin, a specific sodium channel blocker, reduces the phagocytic function of activated rat microglia by 40%. To further confirm a role of Nav1.6 in microglial activation, we examined the phagocytic capacity of microglia from med mice, which lack Nav1.6 channels, and show a 65% reduction in phagocytic capacity compared with microglia from wildtype mice. Our findings indicate that sodium channels are important for activation and phagocytosis of microglia and macrophages in EAE and MS and suggest that, in addition to a direct neuroprotective effect on axons, sodium channel blockade may ameliorate neuroinflammatory disorders via anti-inflammatory mechanisms.     

Human umbilical cord blood cell therapy blocks the morphological change and recruitment of CD11b‐expressing,isolectin‐binding proinflammatory cells after middle cerebral artery occlusion

Secondary neurodegeneration resulting from stroke is mediated by delayed proinflammatory signaling and immune cell activation. Although it remains unknown which cell surface markers signify a proinflammatory phenotype, increased isolectin binding occurs on CD11b‐expressing immune cells within injured brain tissue. Several reports have confirmed the efficacy of human umbilical cord blood (HUCB) cell therapy in reducing ischemic injury in rat after middle cerebral artery occlusion (MCAO), and these effects were attributed in part to dampened neuroinflammation. The present study examined the time course of lectin binding to cells of microglia/macrophage lineage within 96 hr after MCAO and whether delayed HUCB cell treatment alters the migration and/or morphological characteristics of these cells throughout the period of infarct expansion. Isolectin binding was up‐regulated in response to injury, was maximal at 96 hr, and colocalized with cells that expressed the putative proinflammatory markers MMP‐9 and nitric oxide. Isolectin‐tagged fluorescence was also significantly increased at 72 hr and localized to greater numbers of amoeboid, CD11b‐expressing cells relative to 51 hr. Treatment with 1 × 10⁶ HUCB cells significantly reduced total lectin binding at 72 hr, as well as the total area occupied by lectin‐tagged fluorescence at both 51 and 72 hr, relative to vehicle‐treated controls. This effect was accompanied by a shift in the morphology of CD11b‐positive cells from amoeboid to ramified shape. These data indicate that HUCB cell therapy suppressed the recruitment of proinflammatory, isolectin‐binding cells during the period of infarct expansion, thus offering a potential mechanism for the protective effects of HUCB cell therapy. © 2009 Wiley‐Liss, Inc.     

The relative number of macrophages/microglia expressing macrophage colony-stimulating factor and its receptor decreases in multiple sclerosis lesions

The activation of macrophages/microglia in multiple sclerosis (MS) lesions plays a central role in the effector phase of myelin breakdown. The precise patterns of macrophage/microglia activation during demyelination have not yet been defined. The growth and activating factor macrophage-colony stimulating factor (M-CSF) and its specific receptor (M-CSFR) may be involved in this process. The present study investigated the expression of M-CSF and M-CSFR mRNA by in situ hybridization in 60 lesions from 32 MS patients. In the control and periplaque white matter, microglia was almost completely M-CSFR positive. Irrespective of the demyelinating activity, an increased number of cells expressed M-CSF or M-CSFR mRNA within the lesions. However, despite the tremendous increase in macrophages/microglia within the lesions, the relative number of these cells expressing M-CSF or M-CSFR decreased. There was no correlation of M-CSF or M-CSFR expression with active myelin breakdown. The correlation between the clinical course and the expression of M-CSF or M-CSFR mRNA revealed significant differences with the lowest expression in primary progressive MS. These results suggest a downregulation of M-CSF and M-CSFR inside the MS plaque probably due to the high amount of macrophage-derived cytokines or mediators. Nevertheless, the differences in the relative number of cells expressing the M-CSF/M-CSFR pathway implicate that this pathway may be an important contributory factor in different forms of MS pathology.     

Proteomic fingerprints distinguish microglia, bone marrow, and spleen macrophage populations

Mononuclear phagocytes (MP; dendritic cells, monocytes, tissue macrophages, and microglia) maintain tissue homeostasis and provide a first line of defense against invading pathogens. In specific circumstances, MPs also induce inflammatory responses and as such affect disease onset and progression. Despite intensive research into MP biology, little is known of the functional and molecular properties of individual MP subtypes. Using a novel proteomics platform, unique protein patterns and protein identities were observed among populations of spleen and bone marrow macrophages and microglia. Cells were obtained from C57BL/6 mice and were cultivated in macrophage colony-stimulating factor. MP subtypes were indistinguishable by morphological or antigenic criteria. Protein profiling by Surface Enhanced Laser Desorption Ionization-Time of Flight (SELDI-TOF) ProteinChip assays with weak cationic exchange chips showed unique MP spectral profiles. Corresponding protein fractions were recovered by high performance liquid chromatography and identified by liquid chromatography tandem mass spectrometry. The results provide a unique means to distinguish microglia from other MP subtypes.     

Expression of growth differentiation factor-15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1) in the perinatal, adult, and injured rat brain

We and others have recently cloned a new member of the transforming growth factor-beta superfamily, growth differentiation factor-15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1). Using in situ hybridization and immunohistochemistry, we determined the distribution of GDF-15/MIC-1 mRNA and protein in the perinatal and cryolesioned adult rat brain. The choroid plexus epithelium of all ventricles represents the site of strongest and almost exclusive mRNA expression in the normal perinatal and adult brain. The newborn rat brain reveals GDF-15/MIC-1 immunoreactivity (ir) in ependymal cells lining the ventricles, in the striatal subventricular zone, and in populations of nonneural cells of the thalamic/hippocampal lamina affixa, in addition to that in the choroid plexus. Unilateral cryogenic cortical lesioning induced a significant increase of GDF-15/MIC-1 mRNA expression and ir at the lesion site and expression in presumed neurons within the dorsal thalamic area. At the lesion site, GDF-15/MIC-1-producing cells showed immuncytochemical features of neurons, macrophages, and activated microglial cells. Fluorescent microscopy revealed both intra- and extracellular GDF-15/MIC-1 ir. Up-regulation of GDF-15/MIC-1 in activated macrophages (Mstraight phi) is also supported by RT-PCR, ICC, and Western blot experiments showing pronounced induction of GDF-15/MIC-1 expression (mRNA and protein) in retinoic acid/phorbol ester-stimulated human M phi. Our data suggest that 1) GDF-15/MIC-1 is secreted into the cerebrospinal fluid and 2) in the newborn brain may penetrate through the ependymal lining and act on developing neurons and/or glial cells. As a constituent of cells in the lamina affixa, the protein might be involved in the regulation of mesenchyme-epithelial interactions. Finally, GDF-15/MIC-1 may also act within the antiinflammatory cytokine network activated in CNS lesions.     

Response of amoeboid microglia/brain macrophages in fetal rat brain exposed to a teratogen

This study examined the time course response of amoeboid microglia/brain macrophages in the rat fetus induced by a single intraperitoneal injection of cyclophosphamide, a teratogen, into the mother rat at 13 days of gestation. Compared to the normal fetal brain, a marked increase in amoeboid microglia was observed in the telencephalon and diencephalon of experimental fetuses, especially in those killed at embryonic day 15. Conglomerations of microglia occurred in the dorsal and superior neuroepithelium of diencephalon, basal telencephalon, cortical neuroepithelium, and hippocampal formation as identified with OX-42, OX-18, and ED-1 by immunohistochemistry. Rhodamine isothiocynate (RhIc) as a tracer was injected via the tail vein into the pregnant rat to assess the phagocytic capability of these cells. Following the tracer injection, none of microglial cells in normal fetal brain was detectable. RhIc, however, was readily taken up by amoeboid microglia in fetal brain with injury insult. Double labeling has shown that the RhIc-labeled cells were immunoreactive with ED-1, OX-42, OX-18, and OX-6, confirming their microglial nature. Microglial proliferation was assessed by immunohistochemistry using bromodeoxyuridine, which showed a marked increase in mitotic activity. Confocal microscopic analysis revealed that a varying number of microglia coexpressed iNOS, macrophage colony-stimulating factor (M-CSF), and ICAM-1. RT-PCR analysis showed increased expression of M-CSF mRNA. Furthermore, colony-stimulating factor-1 receptor mRNA was localized in microglia by in situ hybridization. The present results suggest that NO along with M-CSF and ICAM-1 is involved in microglial proliferation in prenatal brain injury.     

The novel Nrf2 activator CDDO-EA attenuates cerebral ischemic injury by promoting microglia/macrophage polarization toward M2 phenotype in mice

The aim of present study was to explore whether 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid (CDDO)-ethylamide (CDDO-EA) attenuates cerebral ischemic injury and its possible mechanisms using a middle cerebral artery occlusion (MCAO) model in C57BL/6 mice. Our results showed that intraperitoneal injection (i.p.) of CDDO-EA (2 and 4 mg/kg) augmented NFE2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in ischemic cortex after MCAO. Moreover, CDDO-EA (2 mg/kg, i.p.) significantly enhanced Nrf2 nuclear accumulation, associated with increased cytosolic HO-1 expression, reduced neurological deficit and infarct volume as well as neural apoptosis, and shifted polarization of microglia/macrophages toward an antiinflammatory M2 phenotype in ischemic cortex after MCAO. Using an in vitro model, we confirmed that CDDO-EA (100 μg/mL) increased HO-1 expression and primed microglial polarization toward M2 phenotype under inflammatory stimulation in BV2 microglial cells. These findings suggest that a novel Nrf2 activator CDDO-EA confers neuroprotection against ischemic injury.     

Membrane‐type 1 metalloproteinase is upregulated in microglia/brain macrophages in neurodegenerative and neuroinflammatory diseases

We previously reported that glioma cells induce the expression of membrane‐type 1 metalloproteinase (MT1‐MMP or MMP‐14) in tumor‐associated microglia/macrophages and promote tumor growth, whereas MMP‐14 expression in microglia under physiological conditions is very low. Here, we show that the increase in MMP‐14 expression is also found in microglia/macrophages associated with neurodegenerative and neuroinflammatory pathologies in mouse models as well as in human biopsies or post‐mortem tissue. We found that microglial/macrophage MMP‐14 expression was upregulated in Alzheimer's disease tissue, in active lesions of multiple sclerosis, and in tissue from stage II stroke as well as in the corresponding mouse models for the human diseases. In contrast, we observed no upregulation for MMP‐14 in microglia/macrophages in the early phase of stroke or in the corresponding mouse model, in human amyotrophic lateral sclerosis (ALS) tissue or in a mouse model of ALS as well as in human cases of acute brain trauma. These data indicate that MMP‐14 expression is not a general marker for activated microglia/macrophages but is upregulated in defined stages of neuroinflammatory and neurodegenerative diseases and that there is generally a good match between mouse models and human brain pathologies. © 2013 Wiley Periodicals, Inc.     

Downregulation of miR‐181b in mouse brain following ischemic stroke induces neuroprotection against ischemic injury through targeting heat shock protein A5 and ubiquitin carboxyl‐terminal hydrolase isozyme L1

Understanding the molecular mechanism of cerebral hypoxic preconditioning (HPC)‐induced endogenous neuroprotection may provide potential therapeutic targets for ischemic stroke. By using bioinformatics analysis, we found that miR‐181b, one of 19 differentially expressed miRNAs, may target aconitate hydratase (ACO2), heat shock protein A5 (HSPA5), and ubiquitin carboxyl‐terminal hydrolase isozyme L1 (UCHL1) among 26 changed protein kinase C isoform‐specific interacting proteins in HPC mouse brain. In this study, the role of miR‐181b in oxygen–glucose deprivation (OGD)‐induced N2A cell ischemic injury in vitro and mouse middle cerebral artery occlusion (MCAO)‐induced cerebral ischemic injury in vivo, and its regulation of ACO2, HSPA5, and UCHL1 were further determined. We found that miR‐181b expression levels significantly decreased in mouse brain following MCAO and in OGD‐treated N2A cells. Up‐ and downregulation of miR‐181b by transfection of pre‐ or anti‐miR‐181b could negatively regulate HSPA5 and UCHL1 (but not ACO2) protein levels as well as N2A cell death and programmed cell death in OGD‐treated N2A cells. By using a T7 promoter‐driven control dual luciferase assay, we confirmed that miR‐181b could bind to the 3′‐untranslated rergions of HSPA5 and UCHL1 mRNAs and repress their translations. miR‐181b antagomir reduced caspase‐3 cleavage and neural cell loss in cerebral ischemic cortex and improved neurological deficit of mice after MCAO. In addition, HSPA5 and UCHL1 short interfering RNAs (siRNAs) blocked anti‐miR‐181b‐mediated neuroprotection against OGD‐induced N2A cell injury in vitro. These results suggest that the downregulated miR‐181b induces neuroprotection against ischemic injury through negatively regulating HSPA5 and UCHL1 protein levels, providing a potential therapeutic target for ischemic stroke. © 2013 Wiley Periodicals, Inc.     

Reactive cell proliferation and microglia following injury to the rat brain

The non–astrocytic cells which proliferate in the rat brain after the induction of an area of necrosis have been characterized and counted by means of combined in vivo bromodeoxyuridine (BrdU) administration and immuno–histochemical demonstration of glial fibrillary acid protein (GFAP), vimentin, Ricinus communis agglutinin 120 (RCA–1), Griffonia simplicifolia B4 isolectin (GSI–B4), keratan sulphate (KS), carbonic anhydrase C (CA.C), transferrin (TF) and ferritin. Two days after the injury, 7.5% of the proliferating cells were GFAP–positive reactive astrocytes, 5.7% were RCA–1–positive cells and 17.4% were GSI–B4–positive cells. Lectin–binding cells had the microscopic and ultrastructural aspects of microglia; they proliferated around the needle track and in the corpus callosum. Microglia represented a large fraction of the proliferating cells. Evidence is presented for the origin of at least a proportion of perilesional astrocytes and microglia from the periventricular matrix, and of microglia from blood precursors. Other non–proliferating microglia cells transiently appeared in the normal brain around the wound, in agreement with the existence of two different microglia cell populations reacting with different modalities to an area of necrosis.     

In vitro proliferation of axotomized rat facial nucleus-derived activated microglia in an autocrine fashion

Transection of rat adult facial nerve leads to an increase in the number of activated microglia in the facial nucleus (FN), with a peak in proliferation 3 days after transection. To investigate the characteristics of these activated microglia, we isolated the cells with high purity from axotomized FN (axFN) 3 days after transection according to the previously reported procedure for explant culture. The isolated microglia exhibited immunocytochemical properties similar to those in vivo, and their numbers increased approximately five- to sevenfold over a period of 10 days without the addition of any mitogens, suggesting that self-reproduction was occurring. Actually, the microglia actively incorporated bromodeoxyuridine (BrdU) and strongly expressed an S-phase-specific protein marker, proliferating cell nuclear antigen (PCNA). To examine the mechanism underlying this proliferation, the expression of the mitogens and specific receptors of the microglia were analyzed in conditioned medium (CM) and cells. Macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-CSF (GM-CSF) were detected in the CM as well as in the cells. Their specific receptor proteins, c-Fms and GMCSFRalpha, were also detected in the cell homogenate. These proliferating microglia were not found to produce deleterious factors for neurons. In summary, the microglia isolated from the axFN were found to be proliferative in an autocrine fashion and to have some cellular properties in common with those observed in vivo.     

Characterization of microglial reaction after middle cerebral artery occlusion in rat brain

We have studied the microglial reaction that accompanies cortical infarction induced by middle cerebral artery occlusion (MCAO). Lectin histochemistry with the B₄-isolectin from Griffonia simplicifoliaas well as immunocytochemistry with a panel of monoclonal antibodies directed against major histocompatibility complex (MHC) and lymphocytic antigens were performed. Principal attention was focused on neocortical and thalamic regions, representative of primary and secondary ischemic damage, respectively. With the lectin procedure, activated microglial cells were abundant in the neocortex 24 hours after MCAO. In contrast, microglial activation in the thalamus was not apparent until day 2 after MCAO. On day 5, MHC class II antigen was expressed by reactive microglia in fiber tracts traversing the striatum, but was absent from activated microglia in the primary cortical infarction area. MHC class I and lymphocytic antigens were expressed differentially on microglia with class I antigens appearing early and lymphocytic antigens appearing late in the time course after focal ischemia. The findings are compatible with previous studies during global ischemia and confirm the early activation and the progressive nature of immunomolecule expression on activated microglia after an ischemic insult. In addition to neocortical and thalamic sites, our results showed an early microglial activation to be present also in forebrain regions outside of the middle cerebral artery (MCA) territory, such as the contralateral cortex and hippocampus. A unilateral microglial reaction was also detectable after long-term survival (≥4 weeks) in the pyramidal tracts, as well as in the corticospinal tracts at cervical but not lumbar spinal cord levels. Ischemia-induced neuronal damage, as evaluated by Nissl staining, was found only in cortical and thalamic regions. We conclude that the demonstration of reactive microglia indicates not only imminent ischemic neuronal damage within MCA territory but can also delineate extra-focal disturbances, possibly reflecting subtle and transitory changes in neuronal activity. © 1993 Wiley-Liss, Inc.