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Microglia are the innate immune cells of the central nervous system (CNS). In the adult uncompromised CNS, they have a highly ramified morphology and continuously extend and retract their processes. A subpopulation of microglial cells forms close soma-to-soma contacts with neurons and have been termed satellite microglia, yet the role of such interaction is largely unknown. Here, we analyzed the distribution of satellite microglia in different areas of the CNS of adult male mice applying transgenic- and immunolabeling of neuronal subtypes and microglia followed by three-dimensional imaging analysis. We quantified satellite microglia associated with GABAergic and glutamatergic neurons in the somatosensory cortex, striatum, and thalamus; with dopaminergic and serotonergic neurons in the basal forebrain and raphe nucleus, respectively; and with cerebellar Purkinje cell neurons. Satellite microglia in the retina were assessed qualitatively. Microglia form satellites with all neuronal subtypes studied, whereas a preference for a specific neuron subtype was not found. The occurrence and frequency of satellite microglia is determined by the histo-architectural organization of the brain area and the densities of neuronal somata therein., (© 2022 The Authors. Journal of Neuroscience Research published by Wiley Periodicals LLC.)

Ground state depletion followed by individual molecule return microscopy (GSDIM) has been used in the past to study the nanoscale distribution of protein co-localization in living cells. We now demonstrate the successful application of GSDIM to archival human brain tissue sections including from Alzheimer's disease cases as well as experimental tissue samples from mouse and zebrafish larvae. Presynaptic terminals and microglia and their cell processes were visualized at a resolution beyond diffraction-limited light microscopy, allowing clearer insights into their interactions in situ. The procedure described here offers time and cost savings compared to electron microscopy and opens the spectrum of molecular imaging using antibodies and super-resolution microscopy to the analysis of routine formalin-fixed paraffin sections of archival human brain. The investigation of microglia-synapse interactions in dementia will be of special interest in this context., (© 2021 The Authors. Journal of Neuroscience Research published by Wiley Periodicals LLC.)

Neonatal hypoxic-ischemic (H-I) injury, which mainly causes neuronal damage and white matter injury (WMI), is among the predominant causes of infant morbidity (cerebral palsy, cognitive and persistent motor disabilities) and mortality. Disruptions to the oxygen and blood supply in the perinatal brain affect the cerebral microenvironment and may affect microglial activation, excitotoxicity, and oxidative stress. Microglia are significantly associated with axonal damage and myelinating oligodendrocytes, which are major pathological components of WMI. However, the effects of H-I injury on microglial functions and underlying transformation mechanisms remain poorly understood. The historical perception that these cells are major risk factors for ischemic stroke has been questioned due to our improved understanding of the diversity of microglial phenotypes and their alterable functions, which exacerbate or attenuate injuries in different regions in response to environmental instability. Unfortunately, although therapeutic hypothermia is an efficient treatment, death and disability remain the prognosis for a large proportion of neonates with H-I injury. Hence, novel neuroprotective therapies to treat WMI following H-I injury are urgently needed. Here, we review microglial mechanisms that might occur in the developing brain due to neonatal H-I injury and discuss whether microglia function as a double-edged sword in WMI. Then, we emphasize microglial heterogeneity, notably at the single-cell level, and sex-specific effects on the etiology of neurological diseases. Finally, we discuss current knowledge of strategies aiming to improve microglia modulation and remyelination following neonatal H-I injury. Overall, microglia-targeted therapy might provide novel and valuable insights into the treatment of neonatal H-I insult., (© 2021 Wiley Periodicals LLC.)

Microglia populate the early developing brain and mediate pruning of the central synapses. Yet, little is known on their functional significance in shaping the developing cortical circuits. We hypothesize that the developing cortical circuits require microglia for proper circuit maturation and connectivity, and as such, ablation of microglia during the cortical critical period may result in a long-lasting circuit abnormality. We administered PLX3397, a colony-stimulating factor 1 receptor inhibitor, to mice starting at postnatal day 14 and through P28, which depletes >75% of microglia in the visual cortex (VC). This treatment largely covers the critical period (P19-32) of VC maturation and plasticity. Patch clamp recording in VC layer 2/3 (L2/3) and L5 neurons revealed increased mEPSC frequency and reduced amplitude, and decreased AMPA/NMDA current ratio, indicative of altered synapse maturation. Increased spine density was observed in these neurons, potentially reflecting impaired synapse pruning. In addition, VC intracortical circuit functional connectivity, assessed by laser scanning photostimulation combined with glutamate uncaging, was dramatically altered. Using two photon longitudinal dendritic spine imaging, we confirmed that spine elimination/pruning was diminished during VC critical period when microglia were depleted. Reduced spine pruning thus may account for increased spine density and disrupted connectivity of VC circuits. Lastly, using single-unit recording combined with monocular deprivation, we found that ocular dominance plasticity in the VC was obliterated during the critical period as a result of microglia depletion. These data establish a critical role of microglia in developmental cortical synapse pruning, maturation, functional connectivity, and critical period plasticity., (© 2020 Wiley Periodicals, Inc.)

Microglial cells (resident macrophages) feature rapid activation in CNS disease and can acquire multiple phenotypes exerting neuroprotection or neurotoxicity. The functional impact of surveying ("resting") microglia on neural excitability and neurotransmission in physiology is widely unknown, however. We addressed this issue in male rat hippocampal slice cultures (in situ) by pharmacological microglial ablation within days and by characterizing neuronal gamma-band oscillations (30-70 Hz) that are highly sensitive to neuromodulators and disturbances in ion and energy regulation. Gamma oscillations support action potential timing and synaptic plasticity, associate with higher brain functions like perception and memory, and require precise communication between excitatory pyramidal cells and inhibitory (GABAergic) interneurons. The slice cultures featured well-preserved hippocampal cytoarchitecture and parvalbumin-positive interneuron networks, microglia with ramified morphology, and low basal levels of IL-6, TNF-α, and nitric oxide (NO). Stimulation of slice cultures with the pro-inflammatory cytokine IFN-γ or bacterial LPS serving as positive controls for microglial reactivity induced MHC-II expression and increased cytokine and NO release. Chronic exposure of slice cultures to liposome-encapsulated clodronate reduced the microglial cell population by about 96%, whereas neuronal structures, astrocyte GFAP expression, and basal levels of cytokines and NO were unchanged. Notably, the properties of gamma oscillations reflecting frequency, number and synchronization of synapse activity were regular after microglial depletion. Also, electrical stimulus-induced transients of the extracellular potassium concentration ([K + ] o ) reflecting cellular K + efflux, clearance and buffering were unchanged. This suggests that nonreactive microglia are dispensable for neuronal homeostasis and neuromodulation underlying network signaling and rhythm generation in cortical tissue., (© 2020 The Authors. Journal of Neuroscience Research published by Wiley Periodicals LLC.)

In contrast to peripheral macrophages, microglia in the central nervous system (CNS) exhibit a specific deactivated phenotype; however, it is not clear how this phenotype is maintained. Two alternative hypotheses were postulated recently: (a) microglia differ from peripheral macrophages being derived from the yolk sac (YS), whereas peripheral macrophages originate from bone marrow (BM); (b) microglia acquire a specific phenotype under the influence of the CNS microenvironment. We have previously shown that microglia express miR-124, which was also induced in BM-derived macrophages co-cultured with a neurons. We here investigated the possibility of horizontal transfer of the neuron-specific microRNAs miR-124 and miR-9 from primary neurons to microglia/macrophages. We found that after incubation with neuronal conditioned media (NCM), macrophages downregulated activation markers MHC class II and CD45. Neither cultured adult microglia nor YS- and BM-derived macrophages demonstrated intrinsic levels of miR-124 expression. However, after incubation with NCM, miR-124 was induced in both YS- and BM-derived macrophages. Biochemical analysis demonstrated that the NCM contained miR-124 and miR-9 in complex with small proteins, large high-density lipoproteins (HDLs), and exosomes. MiR-124 and miR-9 were promptly released from neurons, and this process was inhibited by tetrodotoxin, indicating an important role of neuronal electric activity in secretion of these microRNAs. Incubation of macrophages with exogenous miR-124 resulted in efficient translocation of miR-124 into the cytoplasm. This study demonstrates an important role of neuronal miRNAs in communication of neurons with microglia, which favors the hypothesis that microglia acquire a specific phenotype under the influence of the CNS microenvironment., (© 2018 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.)

The importance of a neuroinflammatory response to the development and maintenance of inflammatory and neuropathic pain have been highlighted in recent years. Inflammatory cells contributing to this response include circulating immune cells such as monocytes, T and B lymphocytes, and neutrophils, as well as microglia in the central nervous system. Pain signals are transmitted via sensory neurons in the peripheral nervous system, which express various receptors and channels that respond to mediators secreted from these inflammatory cells. Chronobiological rhythms, which include the 24-hr circadian cycle, have recently been shown to regulate both nervous and immune cell activity and function. This review examines the current literature on chronobiological control of neuroinflammatory processes, with a focus on inflammatory and neuropathic pain states. While the majority of this work has stemmed from observational studies in humans, recent advances in using animal models have highlighted distinct mechanisms underlying these interactions. Better understanding interactions between the circadian and neuroimmune systems can help guide the development of new treatments and provide improved care for patients suffering from acute and chronic pain., (© 2017 Wiley Periodicals, Inc.)

Traumatic brain injury (TBI) is one of the leading causes of morbidity and mortality for both males and females and is, thus, a major focus of current study. Although the overall death rate of TBI for males is roughly three times higher than that for females, males have been disproportionately represented in clinical and preclinical studies. Gender differences are known to exist in many neurologic disorders, such as multiple sclerosis and stroke, and differences appear to exist in TBI. Furthermore, it is known that microglia have sexually dimorphic roles in CNS development and other neurologic conditions; however, most animal studies of microglia and TBI have focused on male subjects. Microglia are a current target of many preclinical and clinical therapeutic trials for TBI. Understanding the relationship among sex, sex hormones, and microglia is critical to truly understanding the pathophysiology of TBI. However, the evidence for sex differences in TBI centers mainly on sex hormones, and evidenced-based conclusions are often contradictory. In an attempt to review the current literature, it is apparent that sex differences likely exist, but the contradictory nature and magnitude of such differences in the existing literature does not allow definite conclusions to be drawn, except that more investigation of this issue is necessary. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Microglia are versatile immune effector cells of the CNS and are sensitive to various stimuli. The different methods used to isolate microglia may affect some of their characteristics, such as their polarization state. The influence of cell sorting methods on the polarization state of microglia has never been studied. Mixed glial culture system (MGCS) and magnetic activated cell sorting (MACS) are two methods that are commonly used to purify microglia. This study compares the immunological states between microglia isolated by MGCS and microglia isolated by MACS. We show that microglia isolated by MGCS exhibit a stronger immune-activated state than microglia isolated by MACS. They present an elevated phagocytic ability and high levels of markers associated with classical activation (M1) and alternative activation (M2). In addition, high levels of M1-type and M2-type chemokine (C-C motif) ligand 2 and transforming growth factor-β1 were detected in the culture medium of mixed glial cells. Our results show that microglia isolated by MGCS are in an immune-activated state, whereas microglia isolated by MACS appear to be closer to their primary in vivo state. Therefore, the immune status of microglia, depending on the protocol used to purify them, should be carefully considered in neuropathology research., (© 2015 Wiley Periodicals, Inc.)

Amyotrophic lateral sclerosis (ALS) is a progressive disease associated with motor neuron death. Several experimental treatments, including cell therapy using hematopoietic or neuronal stem cells, have been tested in ALS animal models, but therapeutic benefits have been modest. Here we used a new therapeutic strategy, bone marrow transplantation (BMT) with stem cell factor (SCF)- or FMS-like tyrosine kinase 3 (flt3)-activated bone marrow (BM) cells for the treatment of hSOD1(G93A) transgenic mice. Motor function and survival showed greater improvement in the SCF group than in the group receiving BM cells that had not been activated (BMT alone group), although no improvement was shown in the flt3 group. In addition, larger numbers of BM-derived cells that expressed the microglia marker Iba1 migrated to the spinal cords of recipient mice compared with the BMT alone group. Moreover, after SCF activation, but not flt3 activation or no activation, the migrating microglia expressed glutamate transporter-1 (GLT-1). In spinal cords in the SCF group, inflammatory cytokines tumor necrosis factor-α and interleukin-1β were suppressed and the neuroprotective molecule insulin-like growth factor-1 increased relative to nontreatment hSOD1(G93A) transgenic mice. Therefore, SCF activation changed the character of the migrating donor BM cells, which resulted in neuroprotective effects. These studies have identified SCF-activated BM cells as a potential new therapeutic agent for the treatment of ALS.

Cochlear ablation triggers cellular and molecular reactions in the adult mammalian central auditory system, leading to complex rearrangements in the cellular networks of the auditory brainstem. The role of microglial cells in these processes is largely unknown. We analyzed morphological and molecular responses as well as cellular affiliations of microglia in the auditory brainstem 1 and 7 days after unilateral sensory deafferentation of the cochlear nucleus. In the ventral cochlear nucleus (VCN), morphological changes of microglial cells were evident following cochlear ablation. Microglial activation preceded astroglial hypertrophy in VCN and lateral superior olive (LSO). During axonal degeneration in VCN early after cochlear ablation, p-ERK1/2- and p-p38-immunoreactive microglia displayed a hypertrophied phenotype, with processes partially surrounding glutamatergic but not GABAergic synapses. During the peak of VCN reinnervation 1 week after cochlear ablation, the number of microglial cells increased massively. Microglia now displayed dense ramifications juxtaposed to Gap43-immunoreactive axons and their terminals. Moreover, we identified lesion-dependent changes in the populations of microglia and astrocytes in LSO and inferior colliculus. By covisualizing cytological markers such as NeuN, GFAP, CD11b, vGluT-1, GAD-65, and Gap43 with the prominent MAP kinases ERK1/2 and p38, we show that MAPK signaling is affected by sensory deafferentation in microglia but not in astroglia or in neurons. In conclusion, microglia displaying MAPK signaling appear to contribute to an adaptive response in central auditory regions that was directly or indirectly affected by sensory deafferentation. Moreover, microglial cells are temporally and spatially in place to participate in synaptogenesis inside VCN., (Copyright © 2014 Wiley Periodicals, Inc.)

Retinal microglia play an important role as resident immunocompetent and phagocytic cells in the event of injury and disease. Retinal microglia and microglia precursor transplantation show a rescue effect in ischemic retina and retinal degeneration. However, studies of retinal microglia have been hampered by the difficulty of obtaining sufficient numbers of microglia. One way to circumvent this difficulty is to establish permanent retinal microglia cell lines. In the present study, we report the generation of immortalized retinal microglia, T-MG cells, from postnatal day 3 rat retinal tissue using a lentiviral vector encoding SV40 large T antigen. The T-MG cells exhibited cell-type-specific antigens for monocyte/macrophage lineage cells, including CD11b (OX42), ED1 (OX6), and Iba1, and actively phagocytosed latex beads. In addition to primary retinal microglia, T-MG cells also have the ability to recruit into chemokines. Treatment of T-MG cells with lipopolysaccharide (LPS) led to increased levels of tumor necrosis factor-α, interleukin-1β, and inducible nitric oxide synthase. Genome-wide microarray analysis showed a less than 1% difference in the genes between the T-MG cells and the control primary retinal microglia. The T-MG cells exhibited properties similar to those of the primary retinal microglia and should have considerable utility as an in vitro model for the study of retinal microglia in health and as a curative therapy and an in vivo model for the study of retinal microglia in disease., (Copyright © 2014 Wiley Periodicals, Inc.)

The complex process of microglial activation encompasses several functional activation states associated either with neurotoxic/antineurogenic or with neurotrophic/proneurogenic properties, depending mainly on the extent of activation and the nature of the activating stimuli. Several studies have demonstrated that acute exposure to the prototypical activating agent lipopolysaccharide (LPS) confers antineurogenic properties upon microglial cells. Acutely activated microglia ortheir conditioned media (CM) reduce neural stem progenitor cell (NPC) survival and prevent NPC differentiation into neurons. The present study tested the hypothesis that docosahexaenoic acid (DHA), a long-chain polyunsatured fatty acid (L-PUFA) with potent immunomodulatory properties, could dampen microglial proinflammatory functions and modulate their antineurogenic effect. We demonstrate that DHA dose dependently inhibits the synthesis of inflammatory products in activated microglia without inducing an alternative antiinflammatory phenotype. Among the possible DHA mechanisms of action, we propose the inhibition of p38 MAPK phosphorylation and the activation of the nuclear receptor peroxisome proliferator activated receptor (PPAR)-γ. The attenuation of M1 proinflammatory phenotype has relevant consequences for the survival and differentiation of NPC, because DHA reverses the antineurogenic activities of conditioned media from LPS-activated microglia. Our study identifies new relevant potentially protective and proneurogenic functions of DHA, exerted through the modulation of microglial functions, that could be exploited to sustain or promote neuroregenerative processes in damaged/aged brain., (Copyright © 2011 Wiley Periodicals, Inc.)

Microglia play important roles in the damaged or degenerating adult nervous system. However, the role of microglia in embryonic brain development is still largely uncharacterized. Here we show that microglia are present in regions of the developing brain that contain neural precursors from E11 onward. To determine whether these microglia are important for neural precursor maintenance or self-renewal, we cultured embryonic neural precursors from the cortex of PU.1(-/-) mice, which we show lack resident microglia during embryogenesis. Cell survival and neurogenesis were similar in cultures from PU.1(-/-) vs. PU.1(+/+) mice, but precursor proliferation and astrogenesis were both reduced. Cortical precursors depleted of microglia also displayed decreased precursor proliferation and astrogenesis, and these deficits could be rescued when microglia were added back to the cultures. Moreover, when the number of microglia present in cortical precursor cultures was increased above normal levels, astrogenesis but not neurogenesis was increased. Together these results demonstrate that microglia present within the embryonic neural precursor niche can regulate neural precursor development and suggest that alterations in microglial number as a consequence of genetic or pathological events could perturb neural development by directly affecting embryonic neural precursors., (Copyright © 2011 Wiley-Liss, Inc.)

Although several studies have provided evidence for the therapeutic potential of bone marrow-derived mononuclear cells (MNCs) in animal models of stroke, the mechanisms underlying their benefits remain largely unknown. We have determined the neuroprotective potential of MNCs in primary neuronal cultures exposed to various injuries in vitro. Cortical neurons in culture were exposed to oxygen-glucose deprivation, hypoxia, or hydrogen peroxide, and cell death was assayed by MTT, caspase-3 activation or TUNEL labelling at 24 hrs. Cultures were randomized to cotreatment with MNC-derived supernatants or media before injury exposure. In separate experiments, macrophage or microglial cultures were exposed to lipopolypolysacharide (LPS) in the presence and absence of MNC-derived supernatants. Neuronal cultures were then exposed to conditioned media derived from activated macrophages or microglia. Cytokines from the supernantants of MNC cultures exposed to normoxia or hypoxia were also estimated by enzyme-linked immunosorbant assay (ELISA). MNC-derived supernatants attenuated neuronal death induced by OGD, hypoxia, hydrogen peroxide, and conditioned macrophage/microglial media and contain a number of trophic factors, including interleukin-10, insulin-like growth factor-1, vascular endothelial growth factor, and stromal cell-derived factor-1. MNCs provide broad neuroprotection against a variety of injuries relevant to stroke., ((c) 2010 Wiley-Liss, Inc.)

As CNS macrophages, microglia show a high spontaneous motility of their processes, continuously surveying their microenvironment. Upon CNS injury, microglia react by immediate cellular polarization and process extension toward the lesion site as well as by subsequent amoeboid lesion-directed migration and phagocytosis. To determine the ability of microglia to fulfill their role within distinctively lesioned tissue in the absence of life support, we investigated microglial activity and responsiveness to laser-induced axonal injuries in the spinal dorsal columns in situ after cardiac and respiratory arrest, i.e., post-mortem, in the progressively degrading nervous tissue. For this purpose, we used time-lapse two-photon laser scanning microscopy in double transgenic mice expressing enhanced green fluorescent protein in microglia and enhanced yellow fluorescent protein in projection neurons. Depending on the premortal condition of the animal, microglial activity and responsiveness remain for up to5-10 hr post-mortem. Thereby, the continuously decreasing glial reaction is independent of oxygen and glucose supply but requires residual ATP, suggesting a parasitic form of energy, such as a transmembrane uptake of ATP released from injured nervous tissue. Even though initially microglia are able to detect axonal injury after disruption of the blood supply, the later aspects of glial reaction, for example amoeboid conversion and migration, are absent post- mortem, corresponding to the failure of microglia to prevent secondary damage after injury of nervous tissue., ((c) 2010 Wiley-Liss, Inc.)

Intracerebral hemorrhage-associated tissue damage is triggered by blood-derived serine proteases such as thrombin. In addition, our previous studies have suggested that mitogen-activated protein (MAP) kinases contribute to intracerebral hemorrhage- and thrombin-induced striatal tissue damage in vivo. Here we addressed the mechanisms of MAP kinase involvement in thrombin cytotoxicity in rat corticostriatal slice culture, focusing on striatal tissue damage. Thrombin induced apoptotic nuclear condensation and fragmentation in striatal cells, which was suppressed by DEVD-CHO, a caspase-3 inhibitor. DEVD-CHO also prevented shrinkage of the striatal tissue induced by thrombin. Phagocytotic activity may be involved in tissue deterioration, because a phagocytosis inhibitor (cytochalasin D) and an inhibitor of phagocytosis of apoptotic cells (O-phospho-L-serine) suppressed shrinkage of the striatal tissue. OX42 immunostaining revealed that apoptosis-like microglial cell death was induced only when thrombin treatment was combined with application of inhibitors of MAP kinase/extracellular signal-regulated kinase kinase (PD98059), p38 MAP kinase (SB203580), or c-Jun N-terminal kinase (SP600125). Thrombin-induced increase in the number of microglia was also prevented by these inhibitors of MAP kinase pathways. We also found that thrombin-induced production of tumor necrosis factor (TNF)-alpha was inhibited by PD98059, SB203580, and SP600125. Finally, thrombin-induced neuronal apoptosis and shrinkage of the striatal tissue were significantly inhibited by anti-TNF-alpha neutralizing antibody. These results suggest that MAP kinases contribute to thrombin-induced striatal damage by supporting survival of activated microglia, which induce neuron death by producing TNF-alpha and cause tissue shrinkage by phagocytosing apoptotic cells.

Glucocorticoid-induced tumor necrosis factor receptor family-related protein ligand (GITRL) is a member of the tumor necrosis factor superfamily (TNFSF) and is known to act as a costimulator in the immune system by binding to GITR. GITRL is expressed in endothelial cells, dendritic cells, macrophages, and B cells, but it is not known whether GITRL is expressed in brain microglia cells. Here, we investigated the expression of GITR and GITRL and their potential role in microglia cells. Using BV-2 mouse microglia cells and mouse primary microglia cultures, we have demonstrated that 1) both GITR and GITRL are expressed in microglia cells; 2) stimulation of GITRL induces inflammatory activation of microglia on the basis of production of nitric oxide (NO) and expression of inducible nitric oxide synthase, cyclooxygenase-2, CD40, and matrix metalloproteinase-9; 3) GITRL-mediated microglial NO production partially depends on p38 MAPK, JNK, and nuclear factor-kappaB pathways; and 4) GITRL stimulation also induces microglia cell death. These results indicate that GITR and GITRL are functionally expressed on brain microglia and that the stimulation of GITRL can induce inflammatory activation of microglia. The GITR/GITRL system may play an important role in neuroinflammation.

For several years, a new population of microglia derived from bone marrow has been described in multiple settings such as infection, trauma, and neurodegenerative disease. The aim of this study was to investigate the migration of bone marrow-derived cells to the brain parenchyma after stress exposure. Stress exposure was performed in mice that had received bone marrow transplantation from GFP mice, allowing identification of blood-derived elements within the brain. Electric foot-shock exposure was chosen because of its ability to serve as fundamental and physical stress in mice. Bone marrow-derived GFP(+) cells migrated to the ventral part of the hippocampus and acquired a ramified microglia-like morphology. Microglia marker Iba1 was expressed by 100% of the ramified cells, whereas ramified cells were negative for the astrocyte marker GFAP. Compared with the case in the control group, ramified cells significantly increased after chronic exposure to stress (5 days). One month after 5 days of stress exposure, ramified cells significantly decreased in ventral hippocampus compared with the group examined immediately after the last stress exposure. We report for the first time the migration of bone marrow-derived cells to the ventral hippocampus after stress exposure. These cells have the characteristics of microglia. Mechanisms responsible for this migration and their roles in the brain remain to be determined.

Activated microglia can influence the survival of neural cells through the release of cytotoxic factors. Here, we investigated the interaction between Toll-like receptor 4 (TLR4)-activated microglia and oligodendrocytes or their precursor cells (OPC). Primary rat or N9 microglial cells were activated by exposure to TLR4-specifc lipopolysaccharide (LPS), resulting in mitogen-activated protein kinase activation, increased CD68 and inducible nitric oxide synthase expression, and release of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6). Microglial conditioned medium (MGCM) from LPS-activated microglia attenuated primary OPC proliferation without inducing cell death. The microglial-induced inhibition of OPC proliferation was reversed by stimulating group III metabotropic glutamate receptors in microglia with the agonist L-AP4. In contrast to OPC, LPS-activated MGCM enhanced the survival of mature oligodendrocytes. Further investigation suggested that TNF and IL-6 released from TLR4-activated microglia might contribute to the effect of MGCM on OPC proliferation, insofar as TNF depletion of LPS-activated MGCM reduced the inhibition of OPC proliferation, and direct addition of TNF or IL-6 attenuated or increased proliferation, respectively. OPC themselves were also found to express proteins involved in TLR4 signalling, including TLR4, MyD88, and MAL. Although LPS stimulation of OPC did not induce proinflammatory cytokine release or affect their survival, it did trigger JNK phosphorylation, suggesting that TLR4 signalling in these cells is active. These findings suggest that OPC survival may be influenced not only by factors released from endotoxin-activated microglia but also through a direct response to endotoxins. This may have consequences for myelination under conditions in which microglial activation and cerebral infection are both implicated. , Inc.

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 x 10(6) 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., ((c) 2009 Wiley-Liss, Inc.)

The brain-derived neurotrophic factor (BDNF) plays a critical role in pain hypersensitivity. BDNF is the ligand of P2X4 receptors (P2X4R) in the microglia. The causative factors involving the P2X4R over expression in the microglia remains unclear. Mast cell activation has a close relation with pain hypersensitivity. However, the underlying mechanism between mast cell activation and pain hypersensitivity is unknown. The present study aimed to elucidate the mechanism by which mast cell activation promoted the expression of P2X4R in the microglia. The results of present study showed that mast cell activation markedly promoted the expression of P2X4R and BDNF in microglial cells, which significantly enhanced the release of BDNF from microglial cells upon exposure to adenosine triphosphate. Mast cell-derived tryptase activated PAR2 that resulted in promoting the expression of P2X4R in microglial cells. Pretreatment with antibodies against tryptase or PAR2, or using tryptase-deficient HMC-1 cells or PAR2-deficient microglial cells abolished the increase in P2X4R expression and BDNF release. Increase in mitogen activated protein kinase phosphorylation was observed in the processes of mast cell-induced BDNF release and P2X4R expression. We conclude that mast cell activation has the capacity to promote the expression of P2X4R and BDNF in microglial cells., ((c) 2009 Wiley-Liss, Inc.)

This is the first description of a population of Iba1- and annexin A3-immunopositive cells residing in the peripheral olfactory nerves of adult rats and adult cats. Based on their ramified appearance, positive immunostaining for the monocytic markers Iba1 and annexin A3, and reactivity to bulbectomy (in adult rats), these cells found within the olfactory nerve fascicles of both mammalian species meet several important criteria for their designation as microglia/macrophages. These Iba1-/annexin A3-immunopositive cells may be uniquely positioned to protect against the potential spread of dangerous environmental xenobiotics (such as viruses and toxins) into the brain, where such pathogens may contribute to the development of neurological diseases, such Alzheimer's and Parkinson's diseases.

Cortical or total brain cultures of microglia are commonly used as a model to study the inflammatory processes in Parkinson's disease. Here we characterize microglia cultures from rat ventral midbrain and evaluate their response to zymosan A. We used specific markers of microglia and evaluated the morphology, the phagocytic activity and reactive oxygen species (ROS) levels of the cells. During the first 10 days in vitro (DIV), cultures presented predominantly cells with a round morphology, expressing CD68 and with high phagocytic activity and ROS production. After 13 DIV, this tendency was reversed, with cultures showing higher number of ramified cells and fewer CD68(+) cells along with lower phagocytic and ROS production capability, suggesting that microglia must be kept in vitro for at least 13 days to recover its resting state. The exposure of cultures with less than 10 DIV to zymosan A significantly decreased cell viability. Exposure of cultures with 13 DIV to zymosan A (0.05, 0.5, or 5 microg/ml) increased the total cell number, the percentage of CD68(+) cells, and the phagocytic activity. Concentrations of zymosan A higher than 5 microg/ml were also effective in activating microglia but significantly decreased the number of viable cells. In summary, microglial cells remain in the activated state for several days after the isolation process and, thus, stimulation of microglia recently isolated can compromise interpretation of the results. However, upon 13 DIV, cells achieve properties of nonactivated microglia and present a characteristic response to a proinflammatory agent., (Copyright 2009 Wiley-Liss, Inc.)

Recent evidence showed that epileptic seizures increase hippocampal neurogenesis in the adult rat, but prolonged seizures result in the aberrant hippocampal neurogenesis that often leads to a recurrent excitatory circuitry and thus contributes to epileptogenesis. However, the mechanism underlying the aberrant neurogenesis after prolonged seizures remains largely unclear. In this study, we examined the role of activated astrocytes and microglia in the aberrant hippocampal neurogenesis induced by status epilepticus. Using a lithium-pilocarpine model to mimic human temporal lobe epilepsy, we found that status epilepticus induced a prominent activation of astrocytes and microglia in the dentate gyrus 3, 7, 14, and 20 days after the initial seizures. Then, we injected fluorocitrate stereotaxicly into the dentate hilus to inhibit astrocytic metabolism and found that fluorocitrate failed to prevent the seizure-induced formation of ectopic hilar basal dendrites but instead promoted the degeneration of dentate granule cells after seizures. In contrast, a selective inhibitor of microglia activation, minocycline, inhibited the aberrant migration of newborn neurons at 14 days after status epilepticus. Furthermore, with stereotaxic injection of lipopolysaccharide into the intact dentate hilus to activate local microglia, we found that lipopolysaccharide promoted the development of ectopic hilar basal dendrites in the hippocampus. These results indicate that the activated microglia in the epileptic hilus may guide the aberrant migration of newborn neurons and that minocycline could be a potential drug to impede seizure-induced aberrant migration of newborn neurons., (Copyright 2009 Wiley-Liss, Inc.)

Multiple sclerosis is a demyelinating neurological disease that is influenced by gender, primarily reflected in greater susceptibility to disease development in women than in men. Cuprizone intoxication, an animal model that is used to study demyelination and remyelination, has been extensively characterized in male C57BL/6 mice. Here, we have undertaken a comprehensive characterization of the morphological and cellular processes that occur in female C57BL/6J mice during cuprizone-induced demyelination and subsequent remyelination and compared them with age-matched male mice. We find that the pattern of demyelination and remyelination is similar between genders and that there is little or no difference in the loss or repopulation of mature oligodendrocytes or accumulation of reactive glia. Furthermore, examination of alphaERKO and betaERKO mice suggests that estrogen receptors do not affect the outcome for demyelination or remyelination. Interestingly, we found that cuprizone treatment disrupts estrous cyclicity in female mice, possibly interfering with potential hormone influences on demyelination and remyelination. Therefore, cuprizone-induced demyelination in C57BL/6J mice may have limitations as a model for the study of sex differences., (2009 Wiley-Liss, Inc.)

Although spinal glia acquire a reactive profile in radiculopathy, glial cell proliferation remains largely unstudied. This study investigated spinal glial proliferation in a model simulating painful disc herniation; the C7 nerve root underwent compression and chromic gut suture exposure or sham procedures. A subset of injured rats received minocycline injections prior to injury. Allodynia was assessed and bromodeoxyuridine (BrdU) was injected 2 hr before tissue harvest on day 1 or 3. Spinal cell proliferation and phenotype identification were assayed by fluorescent colabeling with antibodies to BrdU and either glial fibrillary acidic protein (astrocytes) or Iba1 (microglia). At day 1, ipsilateral allodynia was significantly increased (P < 0.001) for injury over sham. Minocycline treatment significantly decreased ipsilateral allodynia to sham levels at day 1 (P < 0.001). At day 3, ipsilateral allodynia remained and contralateral allodynia was also present for injury (P< 0.003) over sham. The number of BrdU-positive cells in the ipsilateral spinal dorsal horn at day 1 after injury was significantly elevated (P < 0.001) over sham. Approximately 70% of BrdU-positive cells labeled positively for Iba1; dividing microglia were significantly increased (P < 0.004) in the ipsilateral dorsal horn at day 1 following injury compared with sham. Spinal cellular proliferation after injury was not changed by minocycline injection. By day 3, the number of BrdU-positive cells had returned to sham levels bilaterally. Data indicate that spinal microglia proliferate after injury but that proliferation is not abolished by minocycline treatment that attenuates allodynia, indicating that spinal microglial proliferation may be related to injury and may not be linked to changes in sensory perception.

The role of microglia during normal development of the nervous system is still not well understood. In the present study, a chick embryo model was used to examine the development of microglia in the spinal cord and characterize their changes in response to naturally occurring and pathological death of motoneurons (MNs). The microglial response to MN axotomy and the effects of microglial activation on MN survival were also studied. We found that: 1) macrophages/microglial cells were present in the spinal cord at early developmental stages (E3) and that they were recruited after normal and induced MN apoptosis; 2) although many microglial cells were seen phagocytosing apoptotic bodies, a proportion of dying cells were devoid of engulfing microglia; 3) axotomy of mature MNs was accompanied by microglial activation in the absence of MN death; 4) excitotoxic (necrotic) MN death provoked a rapid and massive microglial recruitment with phagocytic activity; 5) lipopolysaccharide-induced microglial activation in vivo resulted in the death of immature, but not mature, microglia; and 6) overactivation of microglia modulated the survival of mature MNs, either by killing them or by enhancing their vulnerability to die in response to a mild injury. Taken together, these observations indicate that normal microglia do not play an active role in triggering apoptosis of developing MNs. Rather, they act as phagocytes for the removal of dying cells during the process of programmed cell death.

Proteomic analysis of cerebrospinal fluid (CSF) samples derived from patients with Alzheimer's disease (AD) or Parkinson's disease (PD) was performed. On the basis of liquid chromatography-tandem mass spectrometry, two-dimensional gel electrophoresis analysis, and Western blot validation, it was found that the level of soluble form of monocyte differentiation antigen CD14 precursor was elevated in CSF from AD or PD patients compared with normal subjects. The soluble CD14 protein and mRNA expression was detected in microglia cells, indicating that microglia may be a cellular source of soluble CD14 in CSF. Next, the role of soluble CD14 in the regulation of glial functions was investigated. Soluble CD14 inhibited lipopolysaccharide (LPS)- or LPS/interferon-gamma-induced nitric oxide production and cell death of microglia and astrocytes. Soluble CD14 suppressed glial neurotoxicity in a coculture of glia/neuroblastoma. In addition, soluble CD14 moderately enhanced phagocytic activity of microglia. These results suggest that microglia-derived soluble CD14 is a candidate CSF biomarker for AD and PD, and the soluble CD14 may inhibit glial activation by interfering with LPS effects.

Adult mesenchymal stem cells (MSCs) exhibit neuroprotective properties when introduced into the degenerating central nervous system through different putative mechanisms including secretion of growth factors and transdifferentiation. In the present study, we injected MSCs into the cerebrospinal fluid of symptomatic hSOD1(G93A) rats, a transgenic animal model of familial amyotrophic lateral sclerosis (ALS) expressing a mutated form of the human superoxide dismutase. MSCs were found to infiltrate the nervous parenchyma and migrate substantially into the ventral gray matter, where motor neurons degenerate. Even though overall astrogliosis was not modified, MSCs differentiated massively into astrocytes at the site of degeneration. The intrathecal delivery of MSCs and the subsequent generation of healthy astrocytes at symptomatic stage decreased motor neuron loss in the lumbar spinal cord, preserving motor functions and extending the survival of hSOD1(G93A) rats. This neuroprotection was correlated with decreased inflammation, as shown by the lower proliferation of microglial cells and the reduced expressiontion of COX-2 and NOX-2. Together, these data highlight the protective capacity of adult MSC-derived astrocytes when grafted into the central nervous system and illustrate an attractive strategy to target excessive inflammation in ALS., ((c) 2009 Wiley-Liss, Inc.)

Fluoxetine is a selective serotonin reuptake inhibitor that is widely used in the treatment of major depression including after stroke. In this study, we tested whether fluoxetine protects neuronal death in a rat cerebral ischemia model of middle cerebral artery occlusion (MCAO). The administration of fluoxetine intravenously (10 mg/kg) at 30 min, 3 hr, or 6 hr after MCAO reduced infarct volumes to 21.2+/-6.7%, 14.5+/-3.0%, and 22.8+/-2.9%, respectively, of that of the untreated control. Moreover, the neuroprotective effect of fluoxetine was evident when it was administered as late as 9 hr after MCAO/reperfusion. These neuroprotective effects were accompanied by improvement of motor impairment and neurological deficits. The fluoxetine-treated brain was found to show marked repressions of microglia activation, neutrophil infiltration, and proinflammatory marker expressions. Moreover, fluoxetine suppressed NF-kappaB activity dose-dependently in the postischemic brain and also in lipopolysaccharide-treated primary microglia and neutrophil cultures, suggesting that NF-kappaB activity inhibition explains in part its anti-inflammatory effect. These results demonstrate that curative treatment of fluoxetine affords strong protection against delayed cerebral ischemic injury, and that these neuroprotective effects might be associated with its anti-inflammatory effects.

Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease with severe neurological manifestations due to alpha-N-acetylglucosaminidase (NaGlu) deficiency. The mechanism of neuropathology in MPS IIIB is unclear. This study investigates the role of immune responses in neurological disease of MPS IIIB in mice. By means of gene expression microarrays and real-time quantitative reverse transcriptase-polymerase chain reaction, we demonstrated significant up-regulation of numerous immune-related genes in MPS IIIB mouse brain involving a broad range of immune cells and molecules, including T cells, B cells, microglia/macrophages, complement, major histocompatibility complex class I, immunoglobulin, Toll-like receptors, and molecules essential for antigen presentation. The significantly enlarged spleen and lymph nodes in MPS IIIB mice were due to an increase in splenocytes/lymphocytes, and functional assays indicated that the T cells were activated. An autoimmune component to the disease was further suggested by the presence of putative autoantigen or autoantigens in brain extracts that reacted specifically with serum IgG from MPS IIIB mice. We also demonstrated for the first time that immunosuppression with prednisolone alone can significantly slow the central nervous system disease progression. Our data indicate that immune responses contribute greatly to the neuropathology of MPS IIIB and should be considered as an adjunct treatment in future therapeutic developments for optimal therapeutic effect.

The ability of microglia to produce/secrete glial cell line-derived neurotrophic factor (GDNF) in vitro was examined. Immunoblotting analysis revealed that nonstimulated microglia release limited amounts of GDNF with molecular sizes of 14 and 17 kDa. However, the secreted amounts significantly decreased when the microglia were activated with the endotoxin lipopolysaccharide (LPS). Comparison of the amounts of GDNF in the cells and the conditioned medium between the nonstimulated microglia and LPS-stimulated microglia clarified that the secretion of GDNF, but not its production, is strongly suppressed when the microglia are activated with LPS. The inhibitor experiments suggested that the GDNF secretion is depressed by a signaling cascade associated with protein kinase C alpha (PKCalpha) and/or mitogen-activated protein kinases (MAPKs). As expected from the above results, a PKC activator suppressed the secretion of GDNF in nonstimulated microglia. Taken together, these results demonstrated that microglia have the ability to produce and secrete GDNF in vitro, and that the secretion is suppressed by stimulation with endotoxin, probably due to a signaling mechanism involving PKCalpha and/or MAPKs., (2008 Wiley-Liss, Inc.)

We have previously reported that the transplantation of dendritic cells (DCs) brings about functional recovery after spinal cord injury in mice through the activation of endogenous microglia/macrophages and neural stem/progenitor cells. In this study, the effect of interleukin-12 (IL-12), which is secreted from DCs, was evaluated for the treatment of spinal cord injury in mice. Administration of IL-12 into the injured site significantly increased the number of activated microglia/macrophages and DCs as well as the expression of brain-derived neurotrophic factor surrounding the lesion site. Immunohistochemical analyses showed that de novo neurogenesis and remyelination were induced by IL-12 treatment. Furthermore, an open field test using Basso-Beattie-Brenham scoring revealed a significant improvement of locomotor function in mice treated with IL-12. These results suggest that IL-12 administration into the injured spinal cord results in a functional recovery through the activation of microglia/macrophages and DCs., (2008 Wiley-Liss, Inc.)

HIV-1 patients who abuse opiate-based drugs, including heroin and morphine, are at a higher risk of developing HIV dementia. The effects of opiates are mediated predominantly through opioid receptors, which are expressed on glial cells. As HIV-1 infection in the CNS is restricted to glial cells, experiments were designed to measure the cell-specific effects of HIV Tat and morphine exposure on opioid receptor expression in both astrocytes and microglia. Specifically, the cell-type-specific pattern of mu opioid receptor (MOR), delta opioid receptor (DOR), and kappa opioid receptor (KOR) localization (surface vs. intracellular) and expression of opioid receptor mRNA were determined after exposure to morphine in the presence and the absence of Tat in primary cultured microglia and astrocytes. Data show that morphine treatment caused significantly decreased cell surface expression of opioid receptors in microglia but not in astrocytes. However, morphine treatment in the presence of Tat significantly increased intracellular expression of opioid receptors and prevented morphine-induced cell surface opioid receptor down-regulation in microglia. These findings document that cell surface opioid receptor expression is divergently regulated by morphine in microglia compared with in astrocytes, and further suggest that HIV-Tat could exacerbate opioid receptor signaling in microglia by increasing receptor expression and/or altering ligand-induced trafficking of opioid receptors., (2008 Wiley-Liss, Inc.)

Spinal cord injury (SCI) triggers a robust inflammatory response that contributes in part to the secondary degeneration of spared tissue. Here, we use flow cytometry to quantify the inflammatory response after SCI. Besides its objective evaluation, flow cytometry allows for levels of particular markers to be documented that further aid in the identification of cellular subsets. Analyses of blood from SCI mice for CD45 (common leukocyte antigen), CD11b (complement receptor-3), Gr-1 (neutrophil/monocyte marker), and CD3 (T-cell marker) revealed a marked increase in circulating neutrophils (CD45(high):Gr-1(high)) at 12 hr compared with controls. Monocyte density in blood increased at 24 hr, and in contrast, lymphocyte numbers were significantly decreased. Mirroring the early increase in neutrophils within the blood, flow analysis of the spinal cord lesion site revealed a significant (P < 0.01) and maintained increase in blood-derived leukocytes (CD45(high):CD11b(high)) from 12 to 96 hr compared with sham-injured and naive controls. Importantly, this technique clearly distinguishes blood-derived neutrophils (CD45:Gr-1(high):F4/80(negative)) and monocyte/macrophages (CD45(high)) from resident microglia (CD45(low)) and revealed that the majority of the blood-derived infiltrate were neutrophils. Our results highlight an assumed, but previously uncharacterized, marked and transient increase in leukocyte populations in blood early after SCI followed by the orchestrated invasion of neutrophils and monocytes into the injured cord. In contrast to mobilization of neutrophils, SCI induces lymphopenia that may contribute negatively to the overall outcome after spinal cord trauma., (2008 Wiley-Liss, Inc.)

Microglia play a variety of significant roles in the central nervous system (CNS), and in one of those roles they undergo morphological change in response to neural injury and migrate to the injured region. We previously reported that ATP/ADP promotes microglial chemotaxis via the Gi/o-coupled P2Y12 receptor; however, the intracellular signaling underlying P2Y12-receptor-mediated microglial chemotaxis is not fully understood. In this study, we examined the role of phospholipase C (PLC) and calcium signaling in ADP-induced microglial chemotaxis. A PLC inhibitor, U73122, significantly suppressed the chemotaxis and completely blocked the ADP-evoked intracellular calcium response, and a calcium chelator, BAPTA-AM, inhibited the chemotaxis. These results indicate that ADP-induced microglial chemotaxis is regulated by a PLC-mediated calcium pathway. ADP stimulation induced Akt phosphorylation in microglia, and the phosphorylation was inhibited by a P2Y12 receptor antagonist, AR-C69931MX. The Akt phosphorylation was blocked by U73122 and BAPTA-AM as well as by a phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin, and inhibition of the Akt activation resulted in failure of chemotaxis. These results indicate that Akt activation is dependent on the PI3K pathway and a PLC-mediated increase in intracellular calcium and suggest that Akt activation is involved in ADP-induced microglial chemotaxis.

Any pathological perturbation to the brain provokes a cascade of molecular and cellular events, which manifests in the form of microglial activation and release of various proinflammatory molecules. This eventually culminates in a profound neuroinflammatory reaction that characterizes the brain's response to stress, injury, or infection. The inflammatory cascade is an attempt by the system to eliminate the challenge imposed on the brain, clear the system of the dead and damaged neurons, and rescue the normal functioning of this vital organ. However, during the process of microglial activation, the proinflammatory mediators released exert certain detrimental effects, and neural stem cells and progenitor cells are likely to be affected. Here we review how the proliferation, maturation, and migration of the neural stem cells are modulated under such an inflammatory condition. The fate of the noncommitted neural stem cells and its differentiation potency are often under strict regulation, and these proinflammatory mediators seem to disrupt this critical balance and finely tune the neurogenesis pattern in the two niches of neurogenesis, the subventricular zone and the subgranular zone of the hippocampus. Moreover, the migration ability of these stem cells, which is important for localization to the proper site, is also affected in a major way by the chemokines released following inflammation., (2007 Wiley-Liss, Inc.)

Mounting evidence has demonstrated that the microenvironment of stem/progenitor cells plays an important role in their proliferation and commitment to their fate. However, it remains unclear how all elements, such as astrocytes, microglia, extracellular matrix molecules, soluble factors, and their cross-talk interactions in the microenvironments, affect neural stem/progenitor cell fate. This work explored the influences of cross-talk between Tenascin-R (TN-R) and microglia on neural stem/progenitor cell proliferation and differentiation. Our results show that microglia triggered by TN-R distinct domains EGF-like repeats (EGFL) and fibronectin 6-8 repeats (FN6-8) significantly enhanced the proliferation of neural stem/progenitor cells and also obviously induced the differentiation into neurons but not oligodendrocytes. Neurite processes of neurons generated from neural progenitor cells were promoted by both EGFL and FN6-8 domains-activated microglia. Microglia triggered by EGFL and FN6-8 secreted brain-derived neurotrophic factor (BDNF) and transforming growth factor-beta (TGF-beta); interestingly, FN6-8 could activate microglia to secrete nerve growth factor in addition to BDNF and TGF-beta, but EGFL domain could not. All these data implied that the cross-talk between TN-R distinct domains EGFL/FN6-8 and microglia promoted neural stem/progenitor cell proliferation and induced their differentiation into neurons.

Cathepsin S (CS) is a lysosomal/endosomal cysteine protease especially expressed in cells of a mononuclear lineage including microglia. To better understand the role of CS in microglia, we investigated microglial responses after a facial nerve axotomy in CS-deficient (CS-/-) and wild-type mice. Microglia in both groups accumulated in the facial motor nucleus following axotomy. However, the mean number of microglia in CS-/- mice on the axotomized side was significantly smaller than that in wild-type mice. Microglia were found to adhere to injured motoneurons in wild-type mice, whereas microglia abutted on injured motoneurons without spreading on their surface in CS-/- mice. At the same time, the axotomy-induced down-regulation of tenasin-R, an antiadhesive perineuronal net for microglia, was partially abrogated in CS-/- mice. Primary cultured microglia prepared from CS-/- mice showed that CS deficiency caused significant suppression of migration and transmigration of microglia. In CS-/- mice, impaired recruitments of circulating monocytes and T lymphocytes and reduced expression of the class II major compatibility complex on the axotomized side were observed. Interestingly, cathepsin B, a typical lysosomal cysteine protease, was markedly expressed on the axotomized side in CS-/- but not in wild-type microglia. Finally, we compared axotomy-induced neuronal death in the two groups and found that the percentage of motoneurons that survived in CS-/- mice was significantly smaller than that in wild-type mice. The present study strongly suggests that CS plays a role in the migration and activation of microglia to protect facial motoneurons against axotomy-induced injury., ((c) 2007 Wiley-Liss, Inc.)

Spinal cord injury (SCI)-induced functional impairment results from secondary apoptosis regulated in part by SCI-induced decreases in the antiapoptotic protein Bcl-x(L). We assessed the role that Bcl-x(L) subcellular rerouting and posttranslational phosphorylation play in Bcl-x(L) decreases in a contusion model of rat SCI. Immunohistochemical analysis showed the presence of Bcl-x(L) in neurons and oligodendrocytes, but not in astrocytes and microglia, whereas phosphorylated Bcl-x(L) (P-ser(62)-Bcl-x(L)) was present only in neurons. Western blot analyses showed Bcl-x(L) present in mitochondria, endoplasmic reticulum, nuclei, and cytosolic extracts, whereas P-ser(62)-Bcl-x(L) was restricted to organelles. During the first 24 hr after SCI, Bcl-x(L) levels decreased in all fractions but with a different time course, suggesting an independent regulation of Bcl-x(L) shuttling from the cytosol to each compartment after SCI. SCI did not affect P-ser(62)-Bcl-x(L) levels in organelles. However, P-ser(62)-Bcl-x(L), which was not detected in the cytosolic fraction of uninjured spinal cord, appeared in the cytosol as early as 15 min postcontusion, suggesting a role for phosphorylation in SCI-induced Bcl-x(L)-decreases. Using an in vitro model, we observed a correlation between levels of cytosolic phosphorylated Bcl-x(L) and neuronal apoptosis, supporting the hypothesis that Bcl-x(L) phosphorylation is proapoptotic. Activated microglia/macrophages robustly expressed Bcl-x(L) 7 days after SCI, and a subpopulation showing nuclear condensation also expressed P-ser(62)-Bcl-x(L). Therefore, phosphorylation of Bcl-x(L) may have opposite effects in injured spinal cords: 1) it may decrease levels of the antiapoptotic Bcl-x(L) in neurons contributing to neuronal death, and 2) it may promote apoptosis in activated microglia/macrophages, thus curtailing the inflammatory cascades associated with SCI.

Apoptosis of autoaggressive T cells in the central nervous system (CNS) and subsequent phagocytosis by microglia is probably crucial in the rapid resolution of the inflammatory infiltrate in T cell mediated neuroinflammatory diseases. In addition to mere clearance, phagocytosis of apoptotic leukocytes results in the downregulation of different microglial immune functions. Chemoattractive functions of Lewis rat microglia and secretion of chemokines and matrix-metalloproteinases (MMPs) were investigated after phagocytosis of apoptotic T cells in vitro. In a modified Boyden chamber assay migration of encephalitogenic T cells toward LPS-stimulated microglial supernatants after phagocytosis of apoptotic thymocytes was reduced by 24.9% in comparison to interaction with viable target cells (P < 0.001). Phagocytosis of apoptotic cells downregulated CC-chemokine ligand (CCL)-5-secretion by LPS-stimulated microglia by 66.2% (P < 0.001), whereas there was only a trend toward decreased CCL2-secretion. As determined by gelatinase-zymography, secretion of MMP-9 by microglia was decreased after phagocytosis of apoptotic cells, whereas MMP-2 secretion was not altered. These mechanisms may reduce further recruitment of pathogenic inflammatory cells into the CNS-lesion and thus contribute to the active resolution of the inflammatory infiltrate and termination of the autoimmune attack., (Copyright 2006 Wiley-Liss, Inc.)

Microglia are bone marrow-derived cells that constitute a facultative macrophage population when activated by trauma or pathology in the CNS. Endogenous CNS-resident microglia as well as exogenous (immigrant) bone marrow-derived cells contribute to reactive microgliosis, raising fundamental questions about the cellular composition, kinetics, and functional characteristics of the reactive microglial cell population. Bone marrow chimeric mice reconstituted with green fluorescent protein-expressing (GFP(+)) donor bone marrow cells were subjected to entorhinal cortex lesion, resulting in selective axonal degeneration and a localized microglial reaction in the hippocampus. Flow cytometric evaluation of individually dissected hippocampi differentiated immigrant GFP(+) microglia from resident GFP(-) microglia (CD11b(+)CD45(dim)) and identified a subset of mainly resident CD11b(+) microglia that was induced to express CD34. The proportion of immigrant GFP(+) microglia (CD11b(+)CD45(dim)) increased signficantly by 3 and 5 days postlesion and reached a maximum of 13% by 7 days. These cells expressed lower CD11b levels than resident microglia, forming a distinct subpopulation on CD11b/CD45 profiles. The proportion of CD34(+)CD11b(+) microglia was significantly increased at 3 days postlesion but had normalized by 5 and 7 days, when the microglial reaction is known to be at its maximum. Our results show that distinct subpopulations of microglia respond to minor CNS injury. The heterogeneity in microglial response may have functional consequences for repair and possibly therapy., (Copyright 2005 Wiley-Liss, Inc.)

Microglia are a major neuroglial component of the CNS, playing an important role as resident immunocompetent and phagocytic cells in the CNS in the event of injury and disease. To understand the role of microglia in the CNS in health and diseases, we have recently established an immortalized clonal cell line of human microglia, HMO6, from human embryonic telencephalon tissue by using a retroviral vector encoding v-myc. This immortalized microglia HMO6 cell line exhibits cell-type-specific antigens for microglia, including CD11b (Mac-1), CD68, CD86 (B7-2), HLA-ABC, HLA-DR, and RCA-1 lectin, and actively phagocytoses latex beads., ((c) 2005 Wiley-Liss, Inc.)

Microglia, one of three glial cell types in the central nervous system (CNS), play an important role as resident immunocompetent and phagocytic cells in the CNS in the event of injury and disease. It was del Rio Hortega in 1927 who determined that microglia belong a distinct glial cell type apart from astrocytes and oligodendrocytes, and since 1970s there has been wide recognition that microglia are immune effectors in the CNS that respond to pathological conditions and participate in initiation and progression of neurological disorders including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and acquired immune deficiency syndrome dementia complex by releasing potentially cytotoxic molecules such as proinflammatory cytokines, reactive oxygen intermediates, proteinases and complement proteins. There is also evidence to suggest that microglia are capable of secreting neurotrophic or neuron survival factors upon activation via inflammation or injury. It is thus timely to review current status of knowledge on biology and immunology of microglia, and consider new directions of investigation on microglia in health and disease., ((c) 2005 Wiley-Liss, Inc.)

Microglia play an important role in the central nervous system, where these cells, it is believed, have both neuroprotective and neurotoxic effects. In response to acute brain injury or during neurodegenerative and neuroinflammatory diseases, activated microglial cells undergo shape changes, migrate to the affected sites of neuronal damage, proliferate, and release a variety of substances, such as cytokines and reactive oxygen species (ROS). This review summarizes the physiological mechanisms underlying microglial activation and deactivation processes, with particular focus on the involvement of microglial ion channels. Microglial ion channels have been shown to be capable, by regulating membrane potential, cell volume, and intracellular ion concentrations, of modulating or facilitating proliferation, migration, cytokine secretion, shape changes, and the respiratory burst of microglial cells., ((c) 2005 Wiley-Liss, Inc.)

Interleukin-1 beta (IL-1beta) is one of the main cytokines involved in the inflammatory response; it has multiple effects that can contribute to cell damage, one of which is the upregulation of the inducible form of nitric oxide (NO) synthase (NOS2) in certain cell types. We demonstrated previously that in vivo, cortical microglial inflammatory responses were increased when noradrenaline (NE) levels were depleted, suggesting that NE can reduce microglial activation. In the present report, we examined the role of IL-1beta in neurotoxicity induced by microglial-conditioned media, and possible neuroprotective effects of NE. Incubation of cortical neurons with conditioned media (CM) obtained from lipopolysaccharide (LPS)-treated microglia induced neuronal NOS2 expression and increased neuronal cell death, and these responses were reduced if the neurons were coincubated with interleukin-1 receptor antagonist. Cotreatment of microglial cells with LPS plus NE potently blocked IL-1beta production and reduced the ability of the CM to induce neuronal NOS2 and cell death. These results suggest that microglial release of IL-1beta is an important activator of neuronal inflammatory responses, and that protective effects of NE upon neurons involve a reduction of microglial-derived IL-1beta., ((c) 2005 Wiley-Liss, Inc.)

Microglia are the resident phagocytes of the brain and are an important source of proinflammatory mediators. Human immunodeficiency virus (HIV)-1 infects the central nervous system early in the course of disease, and it is believed that this occurs, in part, through the transmigration of HIV-1-infected cells across the blood-brain barrier. Infected cells release viral proteins, such as Tat and gp120. After microglia interact with these proteins, they become activated and secrete chemokines; up-regulate key surface receptors, such as CD40, and also activate resident cells. This review focuses on the consequences of microglial activation in NeuroAIDS, with an emphasis on chemokine production and CD40 up-regulation after interaction with tat or gp120. The importance of microglial CD40 in two other neurological diseases, Alzheimer's disease and multiple sclerosis, is also discussed., ((c) 2005 Wiley-Liss, Inc.)

Microglial cells are central nervous system (CNS) resident cells that are thought to become activated and contribute to the inflammation that occurs in the human autoimmune disease multiple sclerosis (MS). This has never been proven, however, because microglial cells cannot be phenotypically distinguished from peripheral macrophages that accumulate in MS inflammatory lesions. To study the kinetics and nature of microglial cell activation in the CNS, we used the animal model of MS, experimental autoimmune encephalomyelitis (EAE), and induced EAE in bone marrow (BM) chimera mice generated using major histocompatibility complex (MHC)-mismatched donor BM, allowing the separation of microglial cells and peripheral monocytes/macrophages. We found that microglial cell activation was evident before onset of disease symptoms and infiltration of peripheral myeloid cells into the CNS. Activated microglial cells underwent proliferation and upregulated the expression of CD45, MHC class II, CD40, CD86, and the dendritic cell marker CD11c. At the peak of EAE disease, activated microglial cells comprised 37% of the total macrophage and dendritic cell populations and colocalized with infiltrating leukocytes in inflammatory lesions. Our findings thus definitively demonstrate that during EAE, microglial cells become activated early in EAE disease and then differentiate into both macrophages and dendritic-like cells, suggesting they play an active role in the pathogenesis of EAE and MS., ((c) 2005 Wiley-Liss, Inc.)

Recent studies have shown that the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) possess antiinflammatory and immunomodulatory properties, distinct from their action of lowering serum lipid levels. Moreover, results of epidemiological studies suggest that long-term use of statins is associated with a decreased risk for Alzheimer's disease (AD). Interestingly, lovastatin (one of the most commonly used anticholesterol drugs) treatment of vascular-derived cells has been reported to antagonize activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway, and it is well known that the JAK/STAT pathway plays a central role in interferon-gamma (IFN-gamma)-induced microglial CD40 expression. We and others have previously reported that microglial CD40 expression is significantly induced by IFN-gamma and amyloid-beta (Abeta) peptide. Moreover, it has been shown that CD40 signaling is critically involved in microglia-related immune responses in the CNS. In this study, we examined the putative role of lovastatin in modulation of CD40 expression and its signaling in cultured microglia. RT-PCR, Western immunoblotting, and flow cytometry data show that lovastatin suppresses IFN-gamma-induced CD40 expression. Additionally, lovastatin markedly inhibits IFN-gamma-induced phosphorylation of JAK/STAT1. Furthermore, lovastatin is able to suppress microglial tumor necrosis factor-alpha, interleukin (IL)-beta1 and IL-6 production promoted either by IFN-gamma or by Abeta peptide challenge in the presence of CD40 cross-linking. To characterize further lovastatin's effect on microglial function, we examined microglial phagocytic capability following CD40 cross-linking. Data reveal that lovastatin markedly attenuates CD40-mediated inhibition of microglial phagocytosis of Abeta. These results provide an insight into the mechanism of the beneficial effects of lovastatin in neurodegenerative disorders, particularly Alzheimer's disease., (Copyright 2004 Wiley-Liss, Inc.)