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At least 379 records · Page 21Linked to original sources

PubMed1998

Stereological estimation of total microglia number in mouse hippocampus.

Microglia are brain cells of considerable interest because of their role in CNS inflammatory responses and strong association with neuritic plaques in Alzheimer's disease (AD). In the present study, immunocytochemistry was combined with unbiased stereology to estimate the mean total number of microglia in dentate gyrus and CA1 regions of the mouse hippocampus. Systematic-uniform-random (SUR) sections were cut through the hippocampal formation of male C57BL/6J mice (n = 7, 4-5 months) and immunostained with Mac-1, an antibody to the complement subunit 3 receptor (CR3). The total number of Mac-1 immunopositive cells was determined using the optical fractionator method. The mean total number of microglia in the mouse dentate gyrus was estimated to be 20,300 (CV = 0.21) with a mean coefficient of error (CE) = 0.09. The mean total number of microglia in the mouse CA1 was estimated to be 43,200 (CV = 0.24) with a CE = 0.11. Comparison of total number estimates, derived from fraction- or volume-based methods, supported stereological theory regarding the equivalence of the two techniques. The time required to determine total microglia number in both hippocampal sub-regions was approximately 6 h per mouse from stained sections. The combination of immunocytochemistry and stereology provides a reliable means to assess microglia number that can easily be adopted for studies of transgenic and lesion-based models of aging and neurodegenerative diseases.

Animals↗

PubMed2002

Differential expression of MHC class II and B7 costimulatory molecules by microglia in rodent gliomas.

To assess the immune function of microglia and macrophages in brain tumors, the expression of MHC class II and B7 costimulatory molecules in three rodent glioma models was examined. Microglia and macrophages, which accounted for 5-12% of total cells, expressed B7.1 and MHC class II molecules in the C6 and 9L tumors, but not RG2 gliomas. Interestingly, the expression of B7.1 and MHC class II molecules by microglia and macrophage was associated with an increase in the number of tumor-infiltrating lymphocytes in C6 and 9L tumors. B7.2 expression, which was present at low levels on microglia and macrophages in normal brain, did not significantly change in tumors. Interestingly, the expression of all three surface antigens increased after microglia were isolated from intracranial C6 tumors and cultured for a short period of time. We conclude that microglia immune activity may be suppressed in gliomas and directly correlates to the immunogenecity of experimental brain tumors.

Animals↗

PubMed2003

Regulation of chemokine receptor expression in human microglia and astrocytes.

It has been proposed that the positioning of mobile cells within a tissue is determined by their overall profile of chemokine receptors. This study examines the profiles of chemokine receptors expressed on resting and activated adult human microglial cells, astrocytes and a microglial cell line, CHME3. Microglia express highest levels of CXCR1, CXCR3 and CCR3. Astrocytes also have moderate levels of CXCR1 and CXCR3, and some CCR3, while both cell types also expressed CCR4, CCR5, CCR6, CXCR2, CXCR4 and CXCR5 at lower levels. Activation of the cells with the inflammatory cytokine tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) increased the expression of some but not all receptors over a period of 24 h. Microglia showed moderate enhancement of receptor expression, while astrocytes responded particularly strongly to TNFalpha with enhanced CXCR3, CCR3 and CXCR1. However, the migratory and proliferative responses of the microglia and astrocytes to the same chemokine were different, with microglia migrating and astrocytes proliferating in response to CXCL10. The data indicates a mechanism by which activated microglia and astrocytes become selectively more sensitive to inflammatory chemokines during CNS disease, and the paper discusses which of the many chemokines present in CNS would have priority of action on microglia and astrocytes.

Astrocytes↗

PubMed1997

Activation of nuclear factor-kappa B by beta-amyloid peptides and interferon-gamma in murine microglia.

An increasing body of evidence suggests that amyloid-beta (A beta) peptides and microglia are crucially involved in the pathogenesis of Alzheimer's disease. In an effort to further elucidate the biological effects of A beta towards microglia, we investigated the ability of A beta peptides to activate nuclear factor (NF)-kappa B in the N9 murine microglial cell line. Co-stimulation of microglia with suboptimal concentrations of A beta(25-35) and 100 U/ml IFN gamma resulted in the detection of a specific NF-kappa B DNA-binding activity in nuclear extracts, as determined in gel mobility shift assays. This response required at least 120 min to be evident and supershift experiments revealed that the NF-kappa B complex contains both RelA and p50. Accordingly, immunoblot experiments showed that amongst NF-kappa B/Rel proteins, RelA and p50 are mobilized to the nucleus following microglial cell stimulation with A beta(25-35) plus IFN gamma. Higher concentrations of A beta(25-35) were effective by themselves in inducing NF-kappa B activation, both in the N9 microglial cell line and in rat primary microglia, as well as in human monocytes. For purposes of comparison, microglia were also stimulated with bacterial LPS, a known NF-kappa B inducer. As expected, LPS strongly induced the formation of two NF-kappa B DNA-binding activities, one of which was identified as RelA/p50. The LPS response was also more rapid, as it was already evident by 40 min and remained sustained for up to 3 h. Collectively, these findings indicate that NF-kappa B activation might constitute one of the mechanisms underlying the inducible expression of kappa B-dependent genes in microglia stimulated by A beta peptides and IFN gamma, or by LPS.

Amyloid beta-Peptides↗

PubMed1999

Cultures of astrocytes and microglia express interleukin 18.

Interleukin 18 (IL-18 or interferon-gamma inducing factor) is a recently discovered pro-inflammatory cytokine and powerful stimulator of the cell-mediated immune response. IL-18 is produced by several sources including monocytes/macrophages, keratinocytes and the zona reticularis and zona fasciculata of the adrenal cortex. IL-18 occurs in brain but its cellular source in the CNS has never been investigated. The presence of IL-18 and its response to stimulation in the brain was tested with primary cultures of microglia, astrocytes and hippocampal neurons. IL-18 mRNA was present in astrocytes and microglia, but not in neurons. The endotoxin lipopolysaccharide (LPS) did not affect IL-18 in astrocytes, but LPS robustly increased IL-18 mRNA in microglia. IL-18 protein was constitutively expressed in astrocytes and induced in microglia by LPS. The levels of interleukin-1beta converting enzyme (ICE), an activating enzyme, and caspase 3 (CPP32), an inactivating enzyme, were assessed to investigate the presence of the appropriate processing enzymes in the cultured cells. ICE was present at constitutive levels in microglia and astrocytes suggesting that these cell types may produce and secrete matured IL-18. Active forms of CPP32 were not detectable in either cell type indicating the absence of a degradative pathway of IL-18. The present results demonstrate that microglia and astrocytes are sources of brain IL-18 and add a new member to the family of cytokines produced in the brain.

Animals↗

PubMedDate not supplied

3D-Reconstruction of microglia and amyloid in APP23 transgenic mice: no evidence of intracellular amyloid.

Microglia cells are closely associated with compact amyloid plaques in Alzheimer's disease (AD) brains. Although activated microglia seem to play a central role in the pathogenesis of AD, mechanisms of microglial activation by beta-amyloid as well as the nature of interaction between amyloid and microglia remain poorly understood. We previously reported a close morphological association between activated microglia and congophilic amyloid plaques in the brains of APP23 transgenic mice at both the light and electron microscopic levels [25]. In the present study, we have further examined the structural relationship between microglia and amyloid deposits by using postembedding immunogold labeling, serial ultrathin sectioning, and 3-dimensional reconstruction. Although bundles of immunogold-labeled amyloid fibrils were completely engulfed by microglial cytoplasm on single sections, serial ultrathin sectioning and three-dimensional reconstruction revealed that these amyloid fibrils are connected to extracellular amyloid deposits. These data demonstrate that extracellular amyloid fibrils form a myriad of finger-like channels with the widely branched microglial cytoplasm. We conclude that in APP23 mice a role of microglia in amyloid phagocytosis and intracellular production of amyloid is unlikely.

Alzheimer Disease↗

PubMedDate not supplied

Age-related activation of microglia and astrocytes: in vitro studies show persistent phenotypes of aging, increased proliferation, and resistance to down-regulation.

Astrocytes and microglia from cerebral cortex of 3-, 6-, 12-, and 24-month-old F344 male rat donors showed progressively greater proliferation during primary culture. Microglia from aging donor brains exhibited an amoeboid-like morphology and express antigens characteristic of an activated state (e.g., major histocompatibility complex class II). Moreover, microglia from aging donors were less sensitive to several types of regulators. Granulocyte-macrophage colony stimulating factor stimulated proliferation in microglia from young, but not aging brains. Transforming growth factor (TGF)-beta1 inhibited astrocytic and microglial proliferation in cultures from young, but not aging donors. Similarly, the inhibition of lipopolysaccharide-induced NO production by TGF-beta1 in microglia was impaired in cultures from 12-month (middle-age) brains. Another aging change detected by middle age, increased glial fibrillary acidic protein (GFAP) expression, also persisted in astrocytes from 12- to 24-month-old brains, as evaluated by increased activity of a 5'-upstream GFAP promoter construct. Thus, both microglia and astrocytes originated from aging cerebral cortex maintain in vitro at least some of the activated phenotypes of aging glia that are observed in vivo. This new in vitro cell model may allow efficient analysis of glial age changes.

Aging↗

PubMed2001

Voltage-gated proton channels in microglia.

Microglia, macrophages that reside in the brain, can express at least 12 different ion channels, including voltage-gated proton channels. The properties of H+ currents in microglia are similar to those in other phagocytes. Proton currents are elicited by depolarizing the membrane potential, but activation also depends strongly on both intracellular pH (pH(i)) and extracellular pH (pH(o)). Increasing pH(o) or lowering pH(i) promotes H+ channel opening by shifting the activation threshold to more negative potentials. H+ channels in microglia open only when the pH gradient is outward, so they carry only outward current in the steady state. Time-dependent activation of H+ currents is slow, with a time constant roughly 1 s at room temperature. Microglial H+ currents are inhibited by inorganic polyvalent cations, which reduce H+ current amplitude and shift the voltage dependence of activation to more positive potentials. Cytoskeletal disruptive agents modulate H+ currents in microglia. Cytochalasin D and colchicine decrease the current density and slow the activation of H+ currents. Similar changes of H+ currents, possibly due to cytoskeletal reorganization, occur in microglia during the transformation from ameboid to ramified morphology. Phagocytes, including microglia, undergo a respiratory burst, in which NADPH oxidase releases bactericidal superoxide anions into the phagosome and stoichiometrically releases protons into the cell, tending to depolarize and acidify the cell. H+ currents may help regulate both the membrane potential and pH(i) during the respiratory burst. By compensating for the efflux of electrons and counteracting intracellular acidification, H+ channels help maintain superoxide anion production.

Animals↗

PubMed2001

Ability of rat microglia to uptake extracellular glutamate.

Since it has been suggested that microglia in vivo act as glutamate scavengers, this possibility was investigated in primary cultured microglia. The microglia showed specific abilities to uptake (14)C-glutamate depending on incubation time and numbers of cells used. The activity was suppressed by a specific inhibitor for a glial cell-type transporter, glutamate transporter-1 (GLT-1) (EAAT2). However, that of cultured astrocytes was not affected. These results suggest that microglia uptake glutamate by means of GLT-1. Supporting these results, immunoblotting revealed the presence of GLT-1 in the microglia, while only glutamate-aspartate transporter (GLAST) (EAAT1: another glial cell-type transporter) was detected in the astrocytes. All together, these results indicate that microglia can act as glutamate scavengers in vivo by expressing the glutamate transporter GLT-1.

ATP-Binding Cassette Transporters↗

PubMed2001

Preservation of neurotrophin expression in microglia that migrate into the gerbil's brain across the blood-brain barrier.

Microglia isolated from a mixed glial culture drawn from neonatal Mongolian gerbils were demonstrated to produce high amounts of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The gerbil microglia retained the capability to migrate into the brain parenchyma after intra-arterial injection. We found that exogenously migrated microglia retained their BDNF and GDNF productive ability and expressed large amounts of BDNF and GDNF in damaged brain areas which suggests microglia's role as a protectant of damaged neurons. Since peripherally injected microglia exhibit specific affinity for areas of neural damage within the brain, we suggest that microglia are possible tools for cell therapy of brain damage.

Animals↗

PubMed2003

Suppression of lipopolysaccharide-dependent tumor necrosis factor alpha induction in rat microglia, in which protein kinase C alpha is down-regulated.

Microglia are thought to produce a cytotoxic cytokine, tumor necrosis factor alpha (TNF alpha), in the pathological brain. Thus, the mechanism that suppresses the induction of TNF alpha in microglia is of interest. We found that lipopolysaccharide (LPS)-induced TNF alpha is strongly suppressed by pretreatment with specific protein kinase C (PKC) inhibitors, Gö6976 and bisindolylmaleimide, suggesting that PKC alpha plays a role in the signaling cascade of TNF alpha induction. Therefore, the association of PKC alpha with TNF alpha induction was investigated in PKC alpha-deficient microglia prepared by pretreatment with phorbol-12-myristate-13-acetate (PMA). PMA-pretreated microglia strongly decreased the TNF alpha induction in the absence of PKC alpha when activated with LPS. The lack of PKC alpha in the PMA-treated microglia was confirmed by determining conventional PKC activity. Taken together, the present study demonstrated that PKC alpha is a requisite PKC isoform in the signaling cascade of TNF alpha induction in LPS-stimulated microglia.

Animals↗

PubMed1997

Transforming growth factor-beta1 induces apoptosis of rat microglia without relation to bcl-2 oncoprotein expression.

Transforming growth factor-beta (TGF-beta) plays a role as an immunosuppressive cytokine within the central nervous system (CNS). The CNS cells targeted by action of TGF-beta1 have not been defined. In this study, we tested the effect of TGF-beta1 on microglia, astrocytes and oligodendrocytes from newborn rats. TGF-beta1 selectively induced apoptosis of microglia, and not of astrocytes or oligodendrocytes. To study the apoptotic mechanism, bcl-2 oncoprotein expression in microglia, astrocytes and oligodendrocytes was measured. Bcl-2 was mainly expressed in microglia, indicating that microglial bcl-2 does not prevent TGF-beta1-mediated microglial apoptosis. The relative protein expression of bcl-2 in microglia was not related to frequency of microglial apoptosis. Thus, TGF-beta1-induced microglial apoptosis was regulated by a bcl-2-independent mechanism. Expression of cytokine (IL-1beta, IL-6, IL-12, IFN-gamma and TNF-alpha) mRNA on microglia was not influenced by treatment with TGF-beta1.

Animals↗

PubMed2002

Lysophospholipids induce membrane hyperpolarization in microglia by activation of IKCa1 Ca(2+)-dependent K(+) channels.

Effects of the lysophospholipids sphingosine-1-phosphate and lysophosphatidic acid were studied in cultured murine microglia using the patch-clamp and video imaging techniques. Both lysophospholipids induced transient membrane hyperpolarization and K(+) current activation. The lysophospholipid-induced K(+) current was blocked by charybdotoxin or iberiotoxin, but was unaffected by apamin. In recordings with 1 microM intracellular free Ca(2+), Ca(2+)-dependent K(+) currents of microglia showed a similar pharmacological profile to lysophospholipid-induced currents. The Ca(2+)-dependent K(+) channels activated in microglia by lysophospholipids are most likely encoded by the IKCa1 channel gene. The presence of IKCa1 mRNA in microglia was demonstrated by reverse transcriptase-polymerase chain reaction studies. Ca(2+) imaging experiments revealed increases in the intracellular free Ca(2+) concentration of microglia to a mean value of about 400 nM after application of 1 microM sphingosine-1-phosphate or 1 microM lysophosphatidic acid. We suggest that the transient membrane hyperpolarization seen in microglia following exposure to sphingosine-1-phosphate or lysophosphatidic acid is caused by activation of IKCa1 Ca(2+)-dependent K(+) channels. Increases in the concentration of intracellular free Ca(2+) evoked by the lysophospholipids are sufficient to activate microglial Ca(2+)-dependent K(+) channels.

Animals↗

PubMedDate not supplied

The monoclonal antibody H386F labels microglia in the retinal nerve fiber layer of several mammals.

The antibody H386F revealed microglia in the retinae of several species: owl monkey, slow loris, galago, ferret, raccoon, and tree shrew. The shape, size, and density of labeled microglia were identical to those labeled by OX-42 and OX-41, two antibodies specific for microglia, in both galago and owl monkey. The labeled microglia varied little in retinal location. There was remarkably little variability in density, shape, number, and size of the labeled microglia between species. All labeled microglia were evenly distributed across, but restricted to, the nerve fiber layer. Possible reasons for this restriction in location are discussed.

Animals↗

PubMed2000

Effects of macrophage-colony-stimulating factor deficiency on the maturation of microglia and brain macrophages and on their expression of scavenger receptor.

Macrophage-colony-stimulating factor (M-CSF) regulates the survival, proliferation and differentiation of the mononuclear phagocyte lineage. Osteopetrotic (op/op) mice defective in producing functional M-CSF were used in order to investigate the role of M-CSF on the development of microglia and brain macrophages and the expression of scavenger receptor (SR). Adult op/op and littermate mice at 10-47 weeks of age were investigated by immunohistochemistry with a panel of monoclonal antibodies (F4/80, Mac-1, anti-major histocompatibility complex (MHC) class II and anti-SR), electron microscopy and reverse transcriptase-polymerase chain reaction (RT-PCR). Microglia were weakly immunolabeled with F4/80 and Mac-1 in op/op and littermate mice, but the number of microglia in op/op mice was reduced in the cerebrum, cerebellum and brainstem compared with that of normal littermates. The numbers of Mac-1-positive microglia in op/op mice was 39% (pons) and 30% (cerebellar cortex) lower than that in normal littermates (P<0.05). In addition, the microglia cell processes in op/op mice were often shorter than those in control mice. In op/op and littermate mice, both MHC class II and SR were present in perivascular cells and macrophages of the leptomeninx and choroid plexus. Ultrastructurally, perivascular cells appeared to be immature, since their cytoplasm was narrow and contained few inclusion bodies compared with those of control mice. Reverse transcriptase-polymerase chain reaction showed a weak expression for SR mRNA in the brains of op/op mice as well as littermate mice. These results indicate that microglia are partly dependent on M-CSF for their proliferation and differentiation and that M-CSF has no significant effect on the expression of SR in the physiological brain. The study also suggests that M-CSF affects the maturation of perivascular cells at the ultrastructural level.

Animals↗

PubMed2000

Extracellular ATP triggers tumor necrosis factor-alpha release from rat microglia.

Brain microglia are a major source of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), which have been implicated in the progression of neurodegenerative diseases. Recently, microglia were revealed to be highly responsive to ATP, which is released from nerve terminals, activated immune cells, or damaged cells. It is not clear, however, whether released ATP can regulate TNF-alpha secretion from microglia. Here we demonstrate that ATP potently stimulates TNF-alpha release, resulting from TNF-alpha mRNA expression in rat cultured brain microglia. The TNF-alpha release was maximally elicited by 1 mM ATP and also induced by a P2X(7) receptor-selective agonist, 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, suggesting the involvement of P2X(7) receptor. ATP-induced TNF-alpha release was Ca(2+)-dependent, and a sustained Ca(2+) influx correlated with the TNF-alpha release in ATP-stimulated microglia. ATP-induced TNF-alpha release was inhibited by PD 098059, an inhibitor of extracellular signal-regulated protein kinase (ERK) kinase 1 (MEK1), which activates ERK, and also by SB 203580, an inhibitor of p38 mitogen-activated protein kinase. ATP rapidly activated both ERK and p38 even in the absence of extracellular Ca(2+). These results indicate that extracellular ATP triggers TNF-alpha release in rat microglia via a P2 receptor, likely to be the P2X(7) subtype, by a mechanism that is dependent on both the sustained Ca(2+) influx and ERK/p38 cascade, regulated independently of Ca(2+) influx.

Adenosine Triphosphate↗

PubMed2002

Heterogeneous expression of the triggering receptor expressed on myeloid cells-2 on adult murine microglia.

Microglial activation is an early and common feature of almost all neuropathologies, including multiple sclerosis, Alzheimer's disease and mechanical injury. To better understand the relative contributions microglia make toward neurodegeneration and neuroprotection, we used TOGA(R) to identify molecules expressed by microglia and regulated by inflammatory signals. Triggering receptor expressed on myeloid cells-2 (TREM-2) was among the mRNAs identified as being expressed by unactivated microglia, but down-regulated by lipopolysaccharide/interferon gamma. In the healthy CNS, not all microglia expressed TREM-2. Microglial expression of TREM-2 varied not only between brain regions but also within each brain region. Brain regions with an incomplete blood-brain barrier had the lowest percentages of TREM-2- expressing microglia, whereas the lateral entorhinal and cingulate cortex had the highest percentages. A novel form of TREM-2b that lacked a transmembrane domain was detected, perhaps indicating a soluble form of the protein. Taken together, these data suggest that (1) subsets of microglia are specialized to respond to defined extracellular signals; and (2) regional variations in TREM-2 expression may contribute to the varying sensitivities of different brain regions to similar pathological signals.

Amino Acid Sequence↗

PubMed2004

Role of reactive oxygen species in LPS-induced production of prostaglandin E2 in microglia.

We determined the roles of reactive oxygen species (ROS) in the expression of cyclooxygenase-2 (COX-2) and the production of prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-activated microglia. LPS treatment increased intracellular ROS in rat microglia dose-dependently. Pre-treatment with superoxide dismutase (SOD)/catalase, or SOD/catalase mimetics that can scavenge intracellular ROS, significantly attenuated LPS-induced release in PGE2. Diphenylene iodonium (DPI), a non-specific NADPH oxidase inhibitor, decreased LPS-induced PGE2 production. In addition, microglia from NADPH oxidase-deficient mice produced less PGE2 than those from wild-type mice following LPS treatment. Furthermore, LPS-stimulated expression of COX-2 (determined by RT-PCR analysis of COX-2 mRNA and western blot for its protein) was significantly reduced by pre-treatment with SOD/catalase or SOD/catalase mimetics. SOD/catalase mimetics were more potent than SOD/catalase in reducing COX-2 expression and PGE2 production. As a comparison, scavenging ROS had no effect on LPS-induced nitric oxide production in microglia. These results suggest that ROS play a regulatory role in the expression of COX-2 and the subsequent production of PGE2 during the activation process of microglia. Thus, inhibiting NADPH oxidase activity and subsequent ROS generation in microglia can reduce COX-2 expression and PGE2 production. These findings suggest a potential therapeutic intervention strategy for the treatment of inflammation-mediated neurodegenerative diseases.

Animals↗