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PubMed2001

Ischemia-induced neuronal expression of the microglia attracting chemokine Secondary Lymphoid-tissue Chemokine (SLC).

Recently, it has been demonstrated that Secondary Lymphoid-tissue Chemokine (SLC) is constitutively expressed in secondary lymphoid organs and controls the homing of naive T-cells and mature dendritic cells. By screening cDNA isolated from ischemic mouse brain, we found expression of SLC mRNA 6 h up to 4 days after the onset of ischemia. In situ hybridization combined with immunohistochemistry showed neurons expressing SLC mRNA in the ischemic area of the cortex. SLC mRNA expression was also found in cultured neurones after various treatments known to induce neuronal death, but not in cultured glial cells. Stimulation with SLC induced intracellular calcium transients and chemotaxis in cultured microglia. Since mRNA encoding CXCR3, an alternative receptor for SLC, but no CCR7 mRNA was found in microglia, we suggest that the effects of SLC on microglia are mediated by CXCR3. This assumption was corroborated by cross-desensitization experiments using IP-10 as a ligand for CXCR3. The inducible expression of SLC in neurones acting on microglia suggests a new and important role of SLC in the neuroimmune system. We propose that SLC is part of a neurone-microglia signaling system which is related to pathological conditions of the brain like ischemia.

Animals↗
PubMed2004

Gangliosides activate microglia via protein kinase C and NADPH oxidase.

Microglia, the major immune effector cells in the central nervous system, are activated when the brain suffers injury. A number of studies indicate that gangliosides activate microglia. However, the signaling mechanisms involved in microglial activation are not yet to be elucidated. Our results show that gangliosides induce the expression of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) in rat brain microglia and BV2 murine microglia via protein kinase C (PKC) and NADPH oxidase. Expression of IL-1beta, TNF-alpha, and iNOS in ganglioside-treated cells was significantly reduced in the presence of inhibitors of PKC (GF109203X, Go6976, Ro31-8220, and rottlerin) and NADPH oxidase (diphenyleneiodonium chloride [DPI]). In response to gangliosides, PKC-alpha, betaII, and delta and NADPH oxidase p67(phox) translocated from the cytosol to the membrane. ROS generation was also activated within 5 min of ganglioside treatment. Ganglioside-induced ROS generation was blocked by PKC inhibitors. Furthermore, ganglioside-induced activation of NF-kappaB, an essential transcription factor that mediates the expression of IL-1beta, TNF-alpha, and iNOS, was reduced in the presence of GF109203X and DPI. Our results collectively suggest that gangliosides activate microglia via PKC and NADPH oxidase, which regulate activation of NF-kappaB.

Animals↗
PubMed2005

Retinoic acid inhibits expression of TNF-alpha and iNOS in activated rat microglia.

The release of proinflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha) and nitric oxide by microglia has been implicated in neurotoxicity in chronic neurodegenerative diseases such as Alzheimer's disease. As all-trans-retinoic acid (RA) has been reported to exert anti-inflammatory actions in various cell types, we have examined its effects on the expression of TNF-alpha and inducible nitric oxide synthase (iNOS) in microglia activated by beta-amyloid peptide (Abeta) and lipopolysaccharide (LPS). Exposure of primary cultures of rat microglial cells to Abeta or LPS stimulated the mRNA expression level of TNF-alpha (6-116-fold) and iNOS (8-500-fold) significantly. RA acted in a dose-dependent manner (0.1-10 microM) by attenuating both TNF-alpha (29-97%) and iNOS (61-96%) mRNA expression in microglia exposed to Abeta or LPS. RA-induced inhibition of TNF-alpha and iNOS mRNA expression in activated microglia was accompanied by the concomitant reduction in release of iNOS and TNF-alpha proteins as revealed by nitrite assay and ELISA, respectively. The anti-inflammatory effects of RA were correlated with the enhanced expression of retinoic acid receptor-beta, and transforming growth factor-beta1 as well as the inhibition of NF-kappaB translocation. These results suggest that RA may inhibit the neurotoxic effect of activated microglia by suppressing the production of inflammatory cytokines and cytotoxic molecules.

Animals↗
PubMed2006

Potentiation of the NMDA receptor-mediated responses through the activation of the glycine site by microglia secreting soluble factors.

We have previously reported that both transferred microglia and microglia-conditioned medium (MCM) potentiated the N-methyl-D-aspatate (NMDA) receptor-mediated synaptic responses in cortical neurons. To elucidate the mechanism underlying the potentiation of NMDA receptor-mediated responses by microglia, we examined the effects of MCM on NMDA-induced inward currents in mechanically dissociated hippocampal CA1 neurons under whole-cell patch recordings. MCM potentiated the amplitude of NMDA-induced currents up to 10-fold in a dose-dependent manner, and this effect of MCM remained even after boiling or cutting off molecules with a molecular mass more than 3 kDa. In the presence of glycine with a concentration sufficient to saturate the NMDA receptor glycine site, MCM failed to further potentiate the NMDA-induced currents. The glycine site antagonist 5, 7-dichrolokynurenic acid, significantly inhibited the effects of MCM. The effect of MCM was still observed even after treatment with D-amino acid oxidase, a D-serine degrading enzyme. On the other hand, MCM had no significant effect on the voltage-dependent Mg(2+) blockade of NMDA receptors. Furthermore, MCM enhanced the formation of the long-term potentiation in the Schaffer collateral pathway-CA1 pyramidal cell synapses. Using a high performance liquid chromatography system, we found the levels of both glycine and L-serine in MCM to be significantly higher than those in the control medium. It was also noted that an increased glycine productivity of microglia was observed in the hippocampus in the acute phase of neuronal injury. These observations strongly suggest that glycine is a major causative molecule released from microglia that potentiates the NMDA-induced currents.

Animals↗
PubMed2006

Challenge with innate and protein antigens induces CCR7 expression by microglia in vitro and in vivo.

Since activated microglia are able to phagocytose damaged cells and subsequently express major histocompatibility complex class II (MHC-II) and co-stimulatory proteins, they are considered to function as antigen presenting cells (APCs) in the central nervous system. The maturation and migratory potential of professional APCs is associated with the expression of chemokine receptor CCR7. We therefore investigated whether the immunological activation of microglia induces CCR7 expression. We here present that activation of cultured microglia by both the innate antigen lipopolysaccharide and protein antigen ovalbumin rapidly induces CCR7 expression, accompanied by increased MHC-II expression. Moreover, it is shown that CCR7 expression in IBA-1 positive cells is induced during the symptom onset and progression of experimental autoimmune encephalomyelitis, a rodent model for multiple sclerosis. These results suggest that microglia express CCR7 under specific inflammatory conditions, corroborating the idea that microglia develop into APCs with migratory potential toward lymphoid chemokines.

Animals↗
PubMed2001

Microglia and prion disease.

Gliosis is one of the hallmarks of the prion diseases. Prion diseases are fatal neurodegenerative conditions of low incidence made famous by both the hypothesis that a protein acts as the infectious agent without involvement of nucleic acid and the speculative idea that a disease of cattle, BSE, has spread to humans from the ingestion of prion-infected beef. Despite these unproved hypotheses, the aetiology of the prion diseases remains unsolved. The rapid degenerative course of the disease is preceded by a long incubation period with little or no symptoms. The rapid neurodegeneration in the disease follows from increased deposition of an abnormal isoform of a normal neuronal protein. Co-incident with the appearance of this abnormal protein is the activation of large numbers of microglia. Studies in cell culture with both the abnormal prion protein and a peptide-mimic suggest that neuronal degeneration occurs because of two concurrent effects. First, there is a reduction in neuronal resistance to toxic insults and, second, there is an increase in the production of toxic substances such as reactive oxygen species by microglia and a decrease in glutamate clearance by astrocytes. Microglia activated by the abnormal form of the prion protein also release cytokines, which stimulate changes in astrocytes such as proliferation. The implication of this is that microglia may play a major role in initiating the pathological changes in prion disease. This review discusses the role of microglia in these changes.

Animals↗
PubMed2001

Phagocytic properties of microglia in vitro: implications for a role in multiple sclerosis and EAE.

The microglial cell, after many years of neglect, has become recognized as the sole representative cell of the immune system that resides in the normal central nervous system. While normally dormant, microglia can be activated by secretory substances or signals associated with disease or injury, and becomes a phagocytic cell, which also produces its own injurious molecules. In the activating process, its morphology is changed from a resting process-bearing cell, into a rounded amoebic form, and displays new or increased amounts of functional markers, such as receptors and Class I and Class II MHC molecules. Microglia prepared from newborn mice or rats for tissue culture are already activated, and can be used for studies of their phagocytic properties. Although they can phagocytize foreign substances, their uptake and metabolism of myelin are emphasized here, in keeping with their role in demyelinating diseases. A number of receptors have been implicated and appear to be important in the attachment to, and ingestion of, myelin particles in vitro, including the Fc, complement, macrophage scavenger, and the Galectin-3/MAC-2 receptors, although the alpha2-macroglobulin/low-density lipoprotein receptor and mannose receptors have also been suggested as participants in myelin phagocytosis. Certain cytokines and adhesion molecules also regulate the phagocytic activity of microglia. Comparative in vitro studies of phagocytosis by peritoneal macrophages and microglia have shown that the two kinds of cells respond differently to regulatory molecules, and it is concluded that they have different innate properties. The role of microglia in the demyelinative diseases experimental autoimmune encephalomyelitis and multiple sclerosis is emphasized here, and the possible means of intervention in the process leading to myelin destruction is discussed. Published 2001 Wiley-Liss, Inc.

Animals↗
PubMed1998

Melanocortin peptides inhibit production of proinflammatory cytokines and nitric oxide by activated microglia.

Inflammatory processes contribute to neurodegenerative disease, stroke, encephalitis, and other central nervous system (CNS) disorders. Activated microglia are a source of cytokines and other inflammatory agents within the CNS and it is therefore important to control glial function in order to preserve neural cells. Melanocortin peptides are pro-opiomelanocortin-derived amino acid sequences that include alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH). These peptides have potent and broad anti-inflammatory effects. We tested effects of alpha-MSH (1-13), alpha-MSH (11-13), and ACTH (1-24) on production of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO) in a cultured murine microglial cell line (N9) stimulated with lipopolysaccharide (LPS) plus interferon gamma (IFN-gamma). Melanocortin peptides inhibited production of these cytokines and NO in a concentration-related fashion, probably by increasing intracellular cAMP. When stimulated with LPS + IFN-gamma, microglia increased release of alpha-MSH. Production of TNF-alpha, IL-6, and NO was greater in activated microglia after innmunoneutralization of endogenous alpha-MSH. The results suggest that alpha-MSH is an autocrine factor in microglia. Because melanocortin peptides inhibit production of pro-inflammatory mediators by activated microglia they might be useful in treatment of inflammatory/degenerative brain disorders.

Adrenocorticotropic Hormone↗
PubMed2002

Astrocytic and microglia cells reactivity induced by neonatal administration of glutamate in cerebral cortex of the adult rats.

Recent studies confirm that astrocytes and neurons are associated with the synaptic transmission, particularly with the regulation of glutamate (Glu) levels. Therefore, they have the capacity to modulate the Glu released from neurons into the extracellular space. It has also been demonstrated an intense astrocytic and microglia response to physical or chemical lesions of the central nervous system. However, the persistence of the response of the glial cells in adult brain had not been previously reported, after the excitotoxic damage caused by neonatal dosage of monosodium glutamate (MSG) to newborn rats. In this study, 4 mg/g body weight of MSG were administered to newborn rats at 1, 3, 5, and 7 days after birth, at the age of 60 days the astrocytes and the microglia cells were analyzed with immunohistochemical methods in the fronto-parietal cortex. Double labeling to glial fibrillary acidic protein (GFAP) and BrdU, or isolectin-B(4) and BrdU identified astrocytes or microglia cells that proliferated; immunoblotting and immunoreactivity to vimentin served for assess immaturity of astrocytic intermediate filaments. The results show that the neonatal administration of MSG-induced reactivity of astrocytes and microglia cells in the fronto-parietal cortex, which was characterized by hyperplasia; an increased number of astrocytes and microglia cells that proliferated, hypertrophy; increased complexity of the cytoplasm extension of both glial cells and expression of RNAm to vimentin, with the presence of vimentin-positive astrocytes. This glial response to neuroexcitotoxic stimulus of Glu on the immature brain, which persisted to adulthood, suggests that the neurotransmitter Glu could trigger neuro-degenerative illnesses.

Aging↗
PubMed2001

Lipopolysaccharide-induced switch between retinoid receptor (RXR) alpha and glucocorticoid attenuated response gene (GARG)-16 messenger RNAs in cultured rat microglia.

Glucocorticoid-attenuated response genes (GARG) belong to a recently described family of genes responsive to the action of dexamethasone. Full-length cDNA of one member of this family, GARG16, has been cloned from rat microglia and regulation of its mRNA expression has been studied. Moreover, regulation of retinoid/retinoic acid activated transcription factor (RXR/RAR) mRNAs in mixed astrocyte and in purified microglia cultures has been investigated. RARbeta mRNA was undetectable in microglia by RT-PCR, whereas clearly present in the mixed cultures. RXRalpha, RARgamma, and GARG16 mRNAs were found in both culture systems. RXRalpha mRNA was strongly expressed in control microglia but rapidly declined upon treatment with LPS. Conversely, GARG16 mRNA was almost untraceable in control microglia but rapidly increased by LPS. Time-course studies revealed an oscillating behavior of expression of both mRNAs during the first 6 hr, which receded to control levels (RXRalpha high, GARG16 low) at 72 hr of LPS-treatment. Additionally, p38 MAPK and SEK phosphorylations peaked at 1 hr followed by steady declines, whereas MEK and c-Jun showed double peaks at 1+4 hr and 1+6 hr, respectively, before subsiding to control levels. This behavior was not observed in comparative studies with TNF-alpha, interleukin-10 (IL-10), or interferon-gamma inducible protein 10 (IP-10). Finally, inhibitors of p38 MAPK, p42/p44 ERK, and PKCalpha as well as the use of dexamethasone revealed major influences of the p38 MAPK-c-Jun-AP-1 signaling pathway on RXRalpha and GARG16 mRNA expressions. The counter regulatory control of GARG16 and RXRalpha mRNA expression is believed to be an example of a fine-tuned cellular mechanism to react to inflammatory stimuli.

Animals↗
PubMed2001

Transformation of donor-derived bone marrow precursors into host microglia during autoimmune CNS inflammation and during the retrograde response to axotomy.

Macrophages in the brain can have a triple source. They may originate from recently blood-derived precursors, from the largely resident perivascular cell population (perivascular macrophages and related cells), and from intrinsic parenchymal as well as perivascular microglia. Although continuous exchange of part of the perivascular cell population with bone marrow-derived precursors is now accepted, the turnover of adult parenchymal microglia has remained enigmatic. Using bone-marrow chimeras carrying an unexpressed marker gene and carbon labeling of peripheral monocyte/macrophages in a combined model of facial nerve axotomy and transfer experimental autoimmune encephalitis, we demonstrate for the first time that there is an easy to induce exchange between parenchymal central nervous system (CNS) microglia and the macrophage precursor cell pool of the bone marrow. Furthermore, very low level infiltration of the CNS parenchyma by recently bone marrow-derived microglia could be observed after simple peripheral nerve axotomy that is followed by neuronal regeneration. Thus, microglial cells can be considered wanderers between the peripheral immune system and the CNS where they may act as a "Trojan horse" in infections. The fact that recently bone marrow-derived parenchymal microglia fully integrate into a regenerating brain nucleus' architecture encourages entirely new approaches for delivering genes into the adult CNS.

Animals↗
PubMed2005

Purinergic mediated changes in Ca2+ mobilization and functional responses in microglia: effects of low levels of ATP.

Microglia, the immune effector cells of the brain, are stimulated by a diversity of agents to transiently increase levels of intracellular calcium ([Ca2+]i). Changes in [Ca2+]i induced by compounds such as adenosine triphosphate (ATP) serve important roles in cellular signal transduction linking stimuli with cellular functional responses. Purinergic responses in microglia, like that in other cells, are mediated by two families of receptors classified as P2Y and P2X. Activation of metabotropic receptors (P2YR) leads to increased [Ca2+]i due to depletion of intracellular stores, a process that can trigger activation of Ca2+ entry through plasmalemmal store-operated channels (SOC). Activation of ionotropic receptors (P2XR) is associated with influx of Na+ and Ca2+ and efflux of K+ through nonselective cationic channels, leading to cellular depolarization. An intriguing property of purinergic stimulation of microglia is the dependence of cellular responses on agonist concentration. As one example, activation of the subtype P2X7R by higher levels of ATP (millimolar range), leads to a marked enhancement in microglial secretion of inflammatory mediators. Other members of the ionotropic P2XR family sensitive to lower levels of ATP, however, are also important in mediating microglial inflammatory responses in brain. At lower concentrations of ATP (100 microM), activation of SOC in human microglia is not only coupled to P2YR-dependent depletion of internal stores, but is also modulated by ATP binding to a P2XR (not P2X7R). The modulation is consistent with a P2XR-mediated influx of Na+ and inhibition of SOC by depolarization. In this review, a primary focus is placed on the effects of low concentrations of ATP (< or =100 microM) to induce changes in [Ca2+]i and modify functional processes in microglia. In essence, responses mediated by purinergic receptors other than P2X7R are considered.

Adenosine Triphosphate↗
PubMed2005

Effects of axotomy on telomere length, telomerase activity, and protein in activated microglia.

The adult central nervous system (CNS) is generally thought of as a postmitotic organ. However, DNA labeling studies have shown that one major population of nonneuronal cells, called microglia, retain significant mitotic potential. Microglial cell division is prominent during acute CNS injury involving neuronal damage or death. Prior work from this laboratory has shown that purified microglia maintained in vitro with continual mitogenic stimulation exhibit telomere shortening before entering senescence. In the current study, we sought to investigate whether telomere shortening occurs in dividing microglia in vivo. For this purpose, we used a nerve injury model that is known to trigger localized microglial proliferation in a well-defined CNS region, the facial motor nucleus. Adult Sprague-Dawley rats underwent facial nerve axotomy, and facial motor nuclei were microdissected after 1, 4, 7, and 10 days. Whole tissue samples were subjected to measurements of telomere length, telomerase activity, and telomerase protein. Results revealed a tendency for all of these parameters to be increased in lesioned samples. In addition, microglial cells isolated directly from axotomized facial nuclei with fluorescence-activated cell sorting (FACS) showed increased telomerase activity relative to unoperated controls, suggesting that microglia are the primary cell type responsible for the increases observed in whole tissue samples. Overall, the results show that microglia activated by injury are capable of maintaining telomere length via telomerase during periods of high proliferation in vivo. We conclude that molecular mechanisms pertaining to telomere maintenance are active in the injured CNS.

Animals↗
PubMed2006

CCL5 evokes calcium signals in microglia through a kinase-, phosphoinositide-, and nucleotide-dependent mechanism.

Microglia, the resident macrophages of the CNS, are responsible for the innate immune response in the brain and participate in the pathogenesis of certain neurodegenerative disorders. Chemokines initiate activation and migration of microglia. The beta-chemokine CCL5 induces an elevation in intracellular calcium concentration ([Ca(2+)](i)) in human microglia. Here, we examined the signal transduction pathway linking activation of chemokine receptor CCR5 to an elevation in [Ca(2+)](i) in cultured microglia by using pharmacological approaches in combination with Fura-2-based digital imaging. The CCL5-induced response required Janus kinase (Jak) activity and the stimulation of an inhibitory G protein. Multiple downstream signaling pathways were involved, including phosphatidylinositol 3-kinase (PI3K), Bruton's tyrosine kinase (Btk), and phospholipase C (PLC)-mediated release of Ca(2+) from inositol 1,4,5-trisphosphate (IP(3))-sensitive stores. Activation of both the kinase and the lipase pathways was required for eliciting the Ca(2+) response. However, the majority of the [Ca(2+)](i) increase was derived from sources activated by NAD metabolites. Cyclic ADP-ribose (cADPR) evoked Ca(2+) release from intracellular stores, and ADPR evoked Ca(2+) influx via a nimodipine-sensitive channel. Thus, a multistep cascade couples CCR5 activation to Ca(2+) increases in human microglia. Because changes in [Ca(2+)](i) affect chemotaxis, secretion, and gene expression, pharmacologic modulation of this pathway may alter inflammatory and degenerative processes in the CNS.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2006

Kainate induces rapid redistribution of the actin cytoskeleton in ameboid microglia.

Microglia are key mediators of the immune response in the central nervous system (CNS). They are closely related to macrophages and undergo dramatic morphological and functional changes after CNS trauma or excitotoxic lesions. Microglia can be directly stimulated by excitatory neurotransmitters and are known to express many neurotransmitter receptors. The role of these receptors, however, is not clear. This study describes the microglial response to the glutamate receptor agonist kainate (KA) and shows via immunochemistry that the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptor subunit GluR1 is present on cultured microglia. In the presence of 100 microM or 1 mM KA, cultured microglia underwent dramatic morphological and cytoskeletal changes as observed by time-lapse photography and quantitative confocal analysis of phalloidin labeling. KA-stimulated microglia showed condensation of cytoplasmic actin filaments, rapid de- and repolymerization, and cytoplasmic redistribution of condensed actin bundles. Rearrangement of actin filaments-thought to be involved in locomotion and phagocytosis and to indicate an increased level of activation (for reviews see Greenberg [ 1995] Trends Cell Biol. 5:93-99; Imai and Kohsaka [ 2002] Glia 40:164-174)-was significantly increased in treated vs. control cultures. Morphological plasticity and membrane ruffling were also seen. These findings suggest direct microglial excitation via glutamate receptor pathways. Thus, neurotransmitter release after brain or spinal cord injury might directly modulate the inflammatory response.

Actins↗
PubMed2006

IL-1beta, an immediate early protein secreted by activated microglia, induces iNOS/NO in C6 astrocytoma cells through p38 MAPK and NF-kappaB pathways.

In the present study we sought to examine cell-cell interactions by investigating the effects of factors released by stimulated microglia on inducible nitric oxide (NO) synthase (iNOS) induction in astrocytoma cells. After examining the temporal profiles of proinflammatory molecules induced by lipopolysaccharide (LPS) stimulation in BV2 microglial cells, iNOS and IL-1beta were observed to be the first immediate-response molecules. Removal of LPS after 3 hr stimulation abrogated NO release, whereas a full induction of IL-1beta was retained in BV2 cells. We observed consistently that conditioned medium (CM) from activated microglia resulted in the induction of iNOS in C6 cells, and IL-1beta was shown to be a key regulator of iNOS induction. An IL-1beta-neutralizing antibody diminished NO induction. Incubation with recombinant IL-1beta stimulated NO release to a lesser extent compared to microglial CM; co-treatment of LPS and IL-1beta had a potent, synergistic effect on NO release from C6 cells. Transient transfection with MEK kinase 1 (MEKK1) or nuclear factor-kappa B (NF-kappaB) expression plasmids induced iNOS, and IL-1beta further enhanced the MEKK1 response. Furthermore, IL-1beta-mediated NO release from C6 cells was significantly suppressed by inhibition of p38 mitogen activated protein kinase (MAPK) or NF-kappaB by specific chemical inhibitors. Both IL-1beta and MEKK1 stimulated p38 and JNK MAPKs, as well as the NF-kappaB pathway, to induce iNOS in C6 cells. Microglia may represent an anti-tumor response in the central nervous system, which is potentiated by the local secretion of immunomodulatory factors that in turn affects astrocytoma (glioma) cells. A better understanding of microglia-glioma or microglia-astrocyte interactions will help in the design of novel immune-based therapies for brain tumors or neuronal diseases.

Analysis of Variance↗
PubMed1995

Effect of bacterial wall lipopolysaccharide (LPS) on morphology, motility, and cytoskeletal organization of microglia in cultures.

We examined the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the morphology, cell motility, cytoskeletal organization, and phagocytic activity of microglia in tissue cultures initiated from neopallia of newborn C3H/OuJ mice. Normally, the microglia in our cultures are non-migratory and Mac-1 positive, have ameboid cell morphology, no polarity, many short processes that extend into lamellipodia in opposing directions, and undulating cell membrane projections. When 1-5 micrograms/ml LPS is added to such cultures, some cells acquire polarity by forming a large lamellipodium and begin to migrate. Two hours later migration ceases; the membrane undulations stop; and the cells become non-polar, assume a large, round, flat shape, and gradually develop many microspikes all over the cell body. Those cells that do not transform into large, round, flat cells enlarge and extend numerous lamellipodia in opposing directions. We found that the cytoskeleton of microglia is composed of actin, vimentin-containing intermediate filaments (IF) and microtubules (MT). Vimentin-containing IF and MT form dense networks that radiate into the cell periphery, whereas F-actin is diffusely arranged throughout the cytoplasm. The LPS-treated cells show changes in the organization of the main components of the cytoskeleton. F-actin is reorganized by the formation of bundles underneath and parallel to the cell membrane and other bundles projecting into the cores of the microspikes. The vimentin-containing IF dense network reorganizes into two condensed rings, with fine strands of IF extended between the two rings and the MT networks become less dense and extend throughout the cytoplasm. The LPS treatment potentiates the phagocytic activity of the microglia. However, approximately 30% of microglia lose the expression of MHC class II antigens.

Actins↗
PubMed1995

192IgG-saporin immunotoxin-induced loss of cholinergic cells differentially activates microglia in rat basal forebrain nuclei.

To characterize the specificity of a novel cholinergic immunotoxin (conjugate of the monoclonal antibody 192IgG against the low-affinity nerve growth factor receptor with the cytotoxic protein saporin), coronal sections through the basal forebrain of adult rats, that received a single intracerebro-ventricular injection of 4 micrograms of 192IgG-saporin conjugate, were subjected to histochemical and immunocytochemical procedures to evaluate cholinergic (choline acetyltransferase (ChAT)-immunoreactive, acetylcholinesterase-positive, NADPH-diaphorase-positive) and GABAergic structures (parvalbumin-immunoreactive, labeling of perineuronal nets with Wisteria floribunda agglutinin) as well as microglia (visualized with Griffonia simplicifolia agglutinin) and astrocytes (immunostaining for glial fibrillary acidic protein). Seven days following injection of the immunotoxin, ChAT-immunoreactive cells nearly completely disappeared throughout the magnocellular basal forebrain complex, including globus pallidus, as compared to vehicle-injected controls. However, there was no significant difference in the number of ChAT-positive cells in the adjacent ventral pallidum and in the caudate-putamen of immunolesioned and control animals. NADPH-diaphorase-containing cells, including a significant subpopulation of cholinergic cells, also strikingly decreased in number by more than 90% in the magnocellular basal forebrain complex following immunolesion, and only a few noncholinergic diaphorase-positive cells survived in the medial septum, vertical and horizontal diagonal band, and nucleus basalis of Meynert. In contrast, the number of parvalbumin-containing GABAergic projection neurons in the septum-diagonal band of Broca complex and nucleus basalis of Meynert from immunolesioned rats was not different from that of vehicle-injected control animals. Immunolesioning also did not result in any change in either number or shape of cells surrounded by perineuronal nets, which are frequently associated with parvalbumin-containing GABAergic neurons. Seven days following injection of the immunotoxin, a very strong activation of microglia with an identical distribution pattern was observed in all experimental animals. Large numbers of activated microglia were found in all magnocellular basal forebrain nuclei, corresponding to the distribution of degenerating cholinergic cells. Additionally, immunolesioning also resulted in a dramatic activation of microglia in the lateral septal nuclei, which are known to be almost free of cholinergic cells, but not of penetrating cholinergic dendrites in adjacent zones, and in the ventral pallidum, where there was no observed loss of cholinergic cells. There was no significant increase in microglia activation in striatum and cortical areas, and no astrocytic response in any of the basal forebrain nuclei at this particular time point of survival. These results suggest that 192IgG-saporin specifically destroys basal forebrain cholinergic neurons and does not suppress their neuronal activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗