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Ion channels in cultured microglia.

Inward and, depending on activation state, outward potassium currents are the dominant ion channels in microglial cells in culture. During transition between resting and activated phases, there is also an upregulated expression of stretch/swelling-activated chloride currents. Pharmacological blockade of the specific potassium channels does not prevent the transition, whereas blockade of chloride channels does, suggesting that this current may be involved in phase changes. Interestingly, this chloride current is far less studied than the potassium currents with regard to the different microglial phases. One puzzling finding when studying microglial state is that despite changes in current densities and membrane oscillations during transition, there is no evidence of an accompanying change in membrane potential. In other cells of the immune system, membrane oscillations and alterations in membrane potential are correlated with transitions in cellular phases. This discrepancy in microglia may be a result of the fact that almost all ion channel and membrane potential studies in culture are undertaken with concomitant dialysis of cytoplasm with pipette solution. Further complicating matters is that the few studies that use microglia in situ, find fundamental differences in ion channel current patterns of "resting" microglia as well as different temporal changes to pathological events or stimuli.

Calcium Channels↗

Interferon-gamma acutely induces calcium influx in human microglia.

The acute actions of the cytokine, interferon-gamma (IFN-gamma), on intracellular calcium [Ca(2+)](i) levels in human microglia were investigated. In the presence of a calcium-containing physiological solution (Ca(2+)-PSS), IFN-gamma caused a progressive increase in [Ca(2+)](i) to a plateau level with a mean rate of increase of 0.81 +/- 0.17 nM/s and mean amplitude of 102 +/- 12 nM (n = 67 cells). Washout of the cytokine did not alter the plateau established with IFN-gamma in Ca(2+)-PSS; however, introduction of a Ca(2+)-free PSS diminished [Ca(2+)](i) to baseline levels. The decrease in [Ca(2+)](i) with Ca(2+)-free PSS would indicate that the response to IFN-gamma was mediated by an influx pathway. This result was confirmed in separate experiments showing the lack of an induced change in [Ca(2+)](i) with IFN-gamma applied in Ca(2+)-free PSS. The increase in [Ca(2+)](i) induced in Ca(2+)-PSS was reduced to near baseline levels when the external solution contained low Cl(-) in the maintained presence of IFN-gamma suggesting that cellular depolarization inhibited the cytokine mediated entry pathway. The compound SKF96365, which blocks store operated influx of Ca(2+) in human microglia, was ineffective in altering the increase in [Ca(2+)](i), however, La(3+) completely inhibited the Ca(2+) response induced by IFN-gamma. Whole-cell patch clamp studies showed no effect of IFN-gamma to alter outward currents and inward rectifier K(+) currents. The influx of Ca(2+) may serve a signaling role in microglia linking IFN-gamma to functional responses of the cells to infiltrating T lymphocytes into the central nervous system (CNS) during inflammatory processes.

Calcium↗

Microglia and multiple sclerosis.

Microglia participate in all phases of the multiple sclerosis (MS) disease process. As members of the innate immune system, these cells have evolved to respond to stranger/danger signals; such a response within the central nervous system (CNS) environment has the potential to induce an acute inflammatory response. Engagement of Toll-like receptors (TLRs), a major family of pattern-recognition receptors (PRRs), provides an important mechanism whereby microglia can interact with both exogenous and endogenous ligands within the CNS. Such interactions modulate the capacity of microglia to present antigens to cells of the adaptive immune system and thus contribute to the initiation and propagation of the more sophisticated antigen-directed responses. This inflammatory response introduces the potential for bidirectional feedback between CNS resident and infiltrating systemic cells. Such interactions acquire particular relevance in the era of therapeutics for MS because the infiltrating cells can be subjected to systemic immunomodulatory therapies known to change their functional properties. Phagocytosis by microglia/macrophages is a hallmark of the MS lesion; however, the extent of tissue damage and the type of cell death will dictate subsequent innate responses. Microglia/macrophages are armed with a battery of effector molecules, such as reactive nitrogen species, that may contribute to CNS tissue injury, specifically to the injury of oligodendrocytes that is associated with MS. A therapeutic challenge is to modulate the dynamic properties of microglia/macrophages so as to limit potentially damaging innate responses, to protect the CNS from injury, and to promote local recovery.

Animals↗

Microglia-derived tumor necrosis factor-alpha exaggerates death of newborn hippocampal progenitor cells in vitro.

Production of new hippocampal neurons continues in adult mammals and different brain insults can significantly increase this process. However, many hippocampal progenitor cells (HPC) die shortly after birth. Here we investigated the possibility that increased release of cytokines by activated microglia contributes to the death of HPC. We showed that addition of tumor necrosis factor-alpha (TNFalpha) to the medium of a cultured HPC line (HiB5) shortly after the cells stopped division causes significant apoptotic cell death. Conditioned medium from an activated microglial cell line (BV-2) had a similar effect, though conditioned medium from nonactivated microglia increased the survival of HPC. Reverse transcription-PCR indicated that HPC and microglial cells express both TNF receptors, TNF-R1 and TNF-R2. Coculturing of HPC with activated microglial cells aggravated death of hippocampal progenitors and also caused death of microglial cells themselves. Our data indicate that activated microglia-released TNFalpha might be an important contributor in inflammation-induced exaggeration of death of newly formed HPC in the adult brain after an insult.

Animals↗

Effects of statins on microglia.

High serum cholesterol level has been shown as one of the risk factors for Alzheimer's disease (AD), and epidemiological studies indicate that treatment with cholesterol-lowering substances, statins, may provide protection against AD. An acute-phase reaction and inflammation, with increased levels of proinflammatory cytokines, are well known in the AD brain. Notably, there is evidence for antiinflammatory activities of statins, such as reduction in proinflammatory cytokines. Consequently, it is of interest to analyze the effects of statins on microglia, the main source of inflammatory factors in the brain, such as in AD. The aims of this study were to determine the effects of statins (atorvastatin and simvastatin) on microglial cells with regard to the secretion of the inflammatory cytokine interleukin-6 (IL-6) and cell viability after activation of the cells with bacterial lipopolysaccharides (LPS) or beta-amyloid1-40 (Abeta1-40) and in unstimulated cells. Cells of the human microglial cell line CHME-3 and primary cultures of rat neonatal cortical microglia were used. Incubation with LPS or Abeta1-40 induced secretion of IL-6, and Abeta1-40, but not LPS, reduced cell viability. Both atorvastatin and simvastatin reduced the basal secretion of IL-6 and the cell viability of the microglia, but only atorvastatin reduced LPS- and Abeta1-40-induced IL-6 secretion. Both statins potentiated the Abeta1-40-induced reduction in cell viability. The data indicate the importance of also considering the microglial responses to statins in evaluation of their effects in AD and other neurodegenerative disorders with an inflammatory component.

Amyloid beta-Peptides↗

Responses of microglia in vitro to the gram-positive bacterial component, lipoteichoic acid.

An increase in incidence and severity of gram-positive infections has emerged in the past decade. In this regard, attention has been focused recently on immune responses of microglial cells in the central nervous system to gram-positive bacteria. The underlying immunological and cellular events in microglial activation induced by specific bacterial toxin of gram-positive bacteria, however, have not yet been clarified fully. This study reports that a simple cell wall product, lipoteichoic acid (LTA), derived from gram-positive bacteria (Staphylococcus aureus) could trigger microglial activation in vitro. Microglia challenged with LTA showed intense ruffling of plasma membrane in the form of lamellipodia or rounded up forming cell aggregates. MTT assay and Western blot analysis with anti-proliferating cell nuclear antigen antibody showed a significant microglial proliferation that may be induced at the later phases of LTA treatment with low doses but at the early period with a high dose. Concentrated LTA also caused apoptotic death of cultured microglia showing fragmented nuclei and increased expression of annexin V or caspase 3. In response to LTA, isolated microglia increased the expression of inducible nitric oxide synthase and major histocompatibility complex class II antigen. Microglial LTA receptors such as CD14 molecule, complement receptor type 3, and macrophage scavenger receptor were upregulated concurrently. In conclusion, staphylococcal LTA can exert an immunomodulatory effect on microglial morphology, cell cycle, and immunomolecules, including its receptors.

Animals↗

Astroglia growth retardation and increased microglia proliferation by lithium and ornithine decarboxylase inhibitor in rat cerebellar cultures: Cytotoxicity by combined lithium and polyamine inhibition.

Lithium, the most prevalent treatment for manic-depressive illness, might have a neuroprotective effect after brain injury. In culture, lithium can exert neurotoxic effects associated with reduction in polyamine synthesis but neuroprotective effects as cultured neurons mature. Cumulative evidence suggests that lithium may exert some of its effects on neurons indirectly, by initially acting on glial cells. We used rat cerebellar cultures to ascertain the effects of lithium on ornithine decarboxylase (ODC) activity, the enzyme catalyzing the first step in polyamine synthesis, and to compare effects of lithium with those of the ODC inhibitor alpha-difluoromethylornithine (DFMO) on neuron survival and glial growth. Switching cultures from high (25 mM) to low (5 mM) KCl concentrations served as the traumatic neuronal insult. The results indicate the following. 1) Whereas high depolarizing KCl concentration enhances neuron survival, it inhibits astroglial growth. 2) Lithium (LiCl; 1-5 mM) enhances neuronal survival but inhibits astroglial growth. 3) Lithium treatment leads to reduced ODC activity. 4) DFMO enhances neuron survival but inhibits astroglial growth. 5) Lithium and DFMO lead to transformation of astroglia from epithelioid (flat) to process-bearing morphology and to increased numbers of microglia. 6) Combined lithium plus DFMO treatment is cytolethal to both neurons and glia in culture. In conclusion, lithium treatment results in growth retardation and altered cell morphology of cultured astroglia and increased microglia proliferation, and these effects may be associated with inhibition of polyamine synthesis. This implies that direct effects on astrocytes and microglia may contribute to the effects of lithium on neurons.

Animals↗

Lipocortin 1 immunoreactivity identifies microglia in adult rat brain.

Immunohistochemical localization of two Ca(++)-binding proteins, Lipocortin 1 (LC1) and S100-beta, demonstrates two distinct classes of primitive glia in the floor plate of rat embryos. With proper fixation (formalin-lysine-periodate-acetic acid), dendritic glia in the CNS of adult rats also apparently stain for either LC1 or S100-beta in the ratio of 1:3. In order to further distinguish and identify these two glial classes, we have examined their population density, topography, and responses to localized neuron death. Neurons of the ipsilateral thalamus undergo apoptosis following cortical ablation; the contralateral thalamus serves as control. By eight days post-lesion, the number of LC1 cells in the ipsilateral thalamus has increased > 4-fold, the increase comprising primarily activated phagocytes adjacent to degenerating neurons. The S100-beta glia in the same region are virtual- ly indistinguishable from control; but background staining (apparently representing extra-cellular S100-beta) is increased. Thus, the responses of dendritic LC1 glia resemble these previously described for microglia and are quite different from the astrocytes identified by S100-beta immunoreactivity. Both dendritic and activated forms of LC1 glia stain with the microglial marker, Griffonia simplicifolia iso-lectin B4. However, before the correspondence of LC1 glia and microglia can be confirmed, two anomalies require resolution: (1) the LC1 glia are greater in number and more evenly distributed than microglia marked with other methods; (2) the dendritic LC1 glia apparently are progeny of primitive glia that form the midline raphe of the embryonic floor plate. The participation of LC1 glia in the removal of CNS debris supports the hypothesis that LC1 plays anti-inflammatory and/or immunosuppressive roles in phagocytes.

Animals↗

Ion channels in rat microglia and their different sensitivity to lipopolysaccharide and interferon-gamma.

In order to study the voltage-dependent ion channels in microglia, and their possible modulation by pro-inflammatory substances like lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) we employed the patch-clamp technique on purified rat microglial cell subcultures grown for 1 - 5 days in control condition or after a 24 hour treatment with those agents. Regardless of the culture condition, almost 100% of the cells presented inward-rectifying (IR) K+ currents identified by the following features: (a) extracellular K(+)-dependence of Vrev and whole-cell conductance; (b) inward-rectifying property; (c) channel blocking mechanism by Cs+; and (d) single channel conductance of 27 pS. A 'n' type outward-rectifying (OR) K+ current was present in 30% of the cells during the first 2 days of subcultivation. Its occurrence was strongly dependent on the preparation, varying from 0% to almost 80%, and it decreased to 13% of the cells after three days in culture. It showed the following features: (i) threshold of activation close to -30 mV; (ii) sigmoid current onset; (iii) voltage-dependent kinetics; and (iv) sensitivity to 4-aminopyridine (4-AP) and tetraethylammonium (TEA). Furthermore, we detected two ion currents not previously described in microglia: (i) a slowly activating outward current which appeared at potentials more positive than +20 mV and with a reversal potential close to 0 mV, tentatively identified as a proton current; and (ii) a Cl- conductance identified in ion substitution experiments as the current sensitive to the Cl- channel blocker SITS. The two agents, LPS (20 - 2,000 ng/ml) and IFN-gamma (10 - 100 u/ml), shared the following effects: (a) enhancement of membrane capacitance, and (b) increase of OR current amplitude and frequency of occurrence. Moreover, IFN-gamma was also able to increase IR current density, especially in cells with ameboid morphology, while LPS was ineffective. We conclude that the voltage-dependent ion channel pattern of microglia is more complex than previously thought and that activating agents such as LPS and IFN-gamma share some electrophysiological effects, but differ in others.

Animals↗

Microglia and inflammation: impact on developmental brain injuries.

Inflammation during the perinatal period has become a recognized risk factor for developmental brain injuries over the past decade or more. To fully understand the relationship between inflammation and brain development, a comprehensive knowledge about the immune system within the brain is essential. Microglia are resident immune cells within the central nervous system and play a critical role in the development of an inflammatory response within the brain. Microglia are critically involved with both the innate and adaptive immune system, regulating inflammation and cell damage within the brain via activation of Toll-like receptors, production of cytokines, and a myriad of other intracellular and intercellular processes. In this article, microglial physiology is reviewed along with the role of microglia in developmental brain injuries in humans and animal models. Last, microglial functions within the innate and adaptive immune system will be summarized. Understanding the processes of inflammation and microglial activation is critical for formulating effective preventative and therapeutic strategies for developmental brain injuries.

Biomarkers↗

Methylene blue blocks cGMP production and disrupts directed migration of microglia to nerve lesions in the leech CNS.

Migration and accumulation of microglial cells at sites of injury are important for nerve repair. Recent studies on the leech central nervous system (CNS), in which synapse regeneration is successful, have shown that nitric oxide (NO) generated immediately after injury by endothelial nitric oxide synthase (eNOS) stops migrating microglia at the lesion. The present study obtained results indicating that NO may act earlier, on microglia migration, and aimed to determine mechanisms underlying NO's effects. Injury induced cGMP immunoreactivity at the lesion in a pattern similar to that of eNOS activity, immunoreactivity, and microglial cell accumulation, which were all focused there. The soluble guanylate cyclase (sGC) inhibitor methylene blue (MB) at 60 microM abolished cGMP immunoreactivity at lesions and blocked microglial cell migration and accumulation without interfering with axon conduction. Time-lapse video microscopy of microglia in living nerve cords showed MB did not reduce cell movement but reduced directed movement, with significantly more cells moving away from the lesion or reversing direction and fewer cells moving toward the lesion. The results indicate a new role for NO, directing the microglial cell migration as well as stopping it, and show that NO's action may be mediated by cGMP.

Animals↗

Inhibition of nitric oxide synthase expression in activated microglia and peroxynitrite scavenging activity by Opuntia ficus indica var. saboten.

Activated microglia by neuronal injury or inflammatory stimulation overproduce nitric oxide (NO) by inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS) such as superoxide anion, resulting in neurodegenerative diseases. The toxic peroxynitrite (ONOO-), the reaction product of NO and superoxide anion further contributes to oxidative neurotoxicity. A butanol fraction obtained from 50% ethanol extracts of Opuntia ficus indica var. saboten (Cactaceae) stem (SK OFB901) and its hydrolysis product (SK OFB901H) inhibited the production of NO in LPS-activated microglia in a dose dependent manner (IC50 15.9, 4.2 microg/mL, respectively). They also suppressed the expression of protein and mRNA of iNOS in LPS-activated microglial cells at higher than 30 microg/mL as observed by western blot analysis and RT-PCR experiment. They also inhibited the degradation of I-kappaB-alpha in activated microglia. Moreover, they showed strong activity of peroxynitrite scavenging in a cell free bioassay system. These results imply that Opuntia ficus indica may have neuroprotective activity through the inhibition of NO production by activated microglial cells and peroxynitrite scavenging activity.

Animals↗

Cytokine and free radical production by porcine microglia.

Swine have been used increasingly as an animal model for a variety of immunologic purposes. Because the functional activities of porcine microglia, the resident macrophages of the brain, have not been elucidated, highly enriched porcine microglial cell cultures were developed in the present study to assess cytokine and free radical production by these cells compared to microglia of human and murine origin. Porcine microglial cells were found to behave similarly to both human and murine cells in releasing tumor necrosis factor-alpha and interleukin-1 and in generating superoxide anion. In contrast to murine cells, porcine microglial cells, like human cells, failed to generate NO in response to cytokine stimulation. These findings suggest that swine will serve as an excellent model for investigations of central nervous system diseases in which microglia are involved in host defense or neuronal injury.

Animals↗

Phagocytosis of nonopsonized Cryptococcus neoformans by swine microglia involves CD14 receptors.

The interaction of the opportunistic fungus Cryptococcus neoformans with swine microglia was studied in vitro in the presence and absence of anti-CD14 monoclonal antibodies. In the absence of anti-CD14 antibodies, 36% of microglia had phagocytized nonopsonized, encapsulated cryptococci after 2 hr incubation (effector-to-target ratio, 1:50). Preincubation of microglia with anti-CD14 antibodies resulted in a 63% reduction of phagocytosis. These findings suggest that CD14 receptors facilitate uptake of nonopsonized C, neoformans by resident macrophages within the brain.

Animals↗

Contribution of peripheral macrophages and microglia to the cellular reaction after mechanical or neurotoxin-induced lesions of the rat brain.

Lesions of the central nervous system result in invasion of peripheral phagocytes and/or in situ activation and proliferation of microglia, depending on the direct or indirect nature of the injury. Neurotoxins which are widely used to induce neurodegeneration have been reported to elicit a pure microglial reaction when administered intraventricularly. However, the mechanical lesion at the injection site, although remote from the lesioned area, could give access to blood-derived cells. Therefore, this study compares the phagocytic reaction occurring after a traumatic lesion of the brain causing a breakdown of the blood-brain barrier (BBB), or after degeneration of the inferior olivary neurons induced by intraperitoneal administration of 3-acetylpyridine, a process that respects the integrity of the BBB as suggested by the results of intravascular injection of Evans blue. The identification of the macrophages at the lesion site used specific binding of the B4 isolectin from Griffonia simplicifolia, preloading of the peripheral monocytes/macrophages with trypan blue, and characteristic morphological features. In traumatically lesioned rats, the lectin-labeled macrophages were mainly large rounded peripheral cells recruited 1-3 days postlesion, whereas in chemically lesioned rats, the cellular reaction appeared 24-36 h postinjection and peaked between 3 and 12 days before undergoing a slow decline. Lectin binding and morphological characteristics indicated that these small cells were reactive microglia. These results confirm that a brain injury leaving the BBB intact involves essentially the recruitment and/or the proliferation of microglia.

Animals↗

Murine leukemia virus-induced neurodegeneration of rats: enhancement of neuropathogenicity correlates with enhanced viral tropism for macrophages, microglia, and brain vascular cells.

A highly neuropathogenic retrovirus, NT40, was generated by serially passaging an infectious molecular clone of Friend murine leukemia virus, FB29, through F344 Fisher rats. NT40 induced severe neurological signs such as reflex abnormalities and ataxia within 4-6 weeks following neonatal inoculation. FB29 led to only very mild neurological dysfunctions with longer incubation periods. Pathological alterations were characterized by mild (FB29) to extensive (NT40) noninflammatory spongiform degeneration, mainly of brain-stem areas. Infectious center assays revealed that viral titers in brain tissues of NT40-infected rats were 100-fold higher than those of FB29-infected animals. Employing immunohistochemistry, in situ hybridization, and flow cytometry, NT40 was found to infect many endothelial cells of brain blood vessels and microglia, whereas FB29 infected only microglia and those to a lower extent. However, when isolated from adult diseased rats, microglial cells turned out in both cases to be nonproductively infected with either FB29 or NT40. Of peripheral organs, we found enhanced levels of NT40 in peritoneal macrophages but not in spleen, thymus, or serum when compared to FB29. Altogether these data suggest that an expanded cellular tropism within the CNS and elevated viral titers in macrophages and microglia correlated with enhancement of neuropathogenicity.

3T3 Cells↗

White matter microglia produce membrane-type matrix metalloprotease, an activator of gelatinase A, in human brain tissues.

Membrane-type matrix metalloprotease (MT-MMP) is an activator of gelatinase A (MMP-2), which has previously been found in carcinoma cells. We examined non-neurological and Alzheimer's disease brain tissues for MT-MMP by immunohistochemistry and in situ hybridization. The anti-MT-MMP antibodies gave positive staining of brain microglial cells in all the brain tissues. Positively stained microglia were found only in the white matter. The cells producing MT-MMP protein were also shown to be white matter microglia. These results provide further evidence that activated gelatinase A, which may be a processing enzyme for degradation of beta-amyloid protein, may be produced in white matter microglia.

Aged↗

Properties of voltage-gated currents of microglia developed using macrophage colony-stimulating factor.

Microglia were isolated from a murine neonatal brain cell culture in which their development had been stimulated by supplementation with the macrophage/microglial growth factor macrophage colony-stimulating factor (M-CSF). Using the whole-cell configuration of the patch-clamp technique, voltage-gated membrane currents were recorded from these microglial cells. Hyperpolarization induced inward rectifying K+ currents, as described for microglia from untreated cultures. These currents activated negative to the K+ equilibrium potential and, with a strong hyperpolarization, displayed time-dependent inactivation. The inactivation was abolished when extracellular NaCl was replaced by N-methyl-D-glucamine (NMG), thereby indicating a partial block of this K+ conductance by Na+. Inward rectifying currents were also blocked by extracellularly applied Cs+ or Ba2+. They were slightly diminished following treatment with extracellular tetraethylammonium chloride (TEA) but were not affected by 4-aminopyridine (4-AP). Upon long lasting depolarizing voltage pulses to potentials positive to 0 mV, the cells exhibited a slowly activating H+ current which could be reduced by application of inorganic polyvalent cations (Ba2+, Cd2+, Co2+, La3+, Ni2+, Zn2+) as well as by 4-AP or TEA. Based on their kinetics and pharmacological characteristics, both currents detected on M-CSF-grown microglia are suggested to correspond to the inward rectifier and the H+ current of macrophages.

Animals↗