Motility and ramification of human fetal microglia in culture: an investigation using time-lapse video microscopy and image analysis

Microglia are mononuclear phagocytes of the central nervous system and are considered to derive from circulating bone marrow progenitors that colonize the developing human nervous system in the second trimester. They first appear as ameboid forms and progressively differentiate to process-bearing "ramified" forms with maturation. Signals driving this transformation are known to be partly derived from astrocytes. In this investigation we have used cocultures of astrocytes and microglia to demonstrate the relationship between motility and morphology of microglia associated with signals derived from astrocytes. Analysis of progressive cultures using time-lapse video microscopy clearly demonstrates the dynamic nature of microglia. We observe that ameboid microglial cells progressively ramify when cocultured with astrocytes, mirroring the "differentiation" of microglia in situ during development. We further demonstrate that individual cells undergo morphological transformations from "ramified" to "bipolar" to "tripolar" and "ameboid" states in accordance with local environmental cues associated with astrocytes in subconfluent cultures. Remarkably, cells are still capable of migration at velocities of 20-35 microm/h in a fully ramified state overlying confluent astrocytes, as determined by image analysis of motility. This is in keeping with the capacity of microglia for a rapid response to inflammatory cues in the CNS. We also demonstrate selective expression of the chemokines MIP-1alpha and MCP-1 by confluent human fetal astrocytes in cocultures and propose a role for these chemotactic cytokines as regulators of microglial motility and differentiation. The interchangeable morphological continuum of microglia supports the view that these cells represent a single heterogeneous population of resident mononuclear phagocytes capable of marked plasticity.     

Role of the Cell Cycle in the Pathobiology of Central Nervous System Trauma

Up-regulation of cell cycle proteins occurs in both mitotic and post-mitotic neural cells after central nervous system (CNS) injury in adult animals. In mitotic cells, such as astroglia and microglia, they induce proliferation, whereas in post-mitotic cells such as neurons they initiate caspase-related apoptosis. We recently reported that early central administration of the cell cycle inhibitor flavopiridol after experimental traumatic brain injury (TBI) significantly reduced lesion volume, scar formation and neuronal cell death, while promoting near complete behavioral recovery. Here we show that in primary neuronal or astrocyte cultures structurally different cell cycle inhibitors (flavopiridol, roscovitine, and olomoucine) significantly reduce up-regulation of cell cycle proteins, attenuate neuronal cell death induced by etoposide, and decrease astrocyte proliferation. Flavopiridol, in a concentration dependent manner, also attenuates proliferation/activation of microglia. In addition, we demonstrate that central administration of flavopiridol improves functional outcome in dose-dependent manner after fluid percussion induced brain injury in rats. Moreover, delayed systemic administration of flavopiridol significantly reduces brain lesion volume and edema development after TBI. These data provide further support for the therapeutic potential of cell cycle inhibitors for the treatment of clinical CNS injury and that protective mechanisms likely include reduction of neuronal cell death, inhibition of glial proliferation and attenuation of microglial activation.     

Complexity of the microglial activation pathways that drive innate host responses during lethal alphavirus encephalitis in mice

Microglia express multiple TLRs (Toll-like receptors) and provide important host defence against viruses that invade the CNS (central nervous system). Although prior studies show these cells become activated during experimental alphavirus encephalitis in mice to generate cytokines and chemokines that influence virus replication, tissue inflammation and neuronal survival, the specific PRRs (pattern recognition receptors) and signalling intermediates controlling microglial activation in this setting remain unknown. To investigate these questions directly in vivo, mice ablated of specific TLR signalling molecules were challenged with NSV (neuroadapted Sindbis virus) and CNS viral titres, inflammatory responses and clinical outcomes followed over time. To approach this problem specifically in microglia, the effects of NSV on primary cells derived from the brains of wild-type and mutant animals were characterized in vitro. From the standpoint of the virus, microglial activation required viral uncoating and an intact viral genome; inactivated virus particles did not elicit measurable microglial responses. At the level of the target cell, NSV triggered multiple PRRs in microglia to produce a broad range of inflammatory mediators via non-overlapping signalling pathways. In vivo, disease survival was surprisingly independent of TLR-driven responses, but still required production of type-I IFN (interferon) to control CNS virus replication. Interestingly, the ER (endoplasmic reticulum) protein UNC93b1 facilitated host survival independent of its known effects on endosomal TLR signalling. Taken together, these data show that alphaviruses activate microglia via multiple PRRs, highlighting the complexity of the signalling networks by which CNS host responses are elicited by these infections.     

Tumor necrosis factor receptor-associated death domain mediated neuronal death contributes to the glial activation and subsequent neuroinflammation in Japanese encephalitis

While a number of studies have documented the importance of microglia in central nervous system (CNS) response to injury, infection and in disease state, little is known regarding how the neuronal death initiates the cascades of secondary neuroinflammation. We have exploited an experimental model of Japanese encephalitis to better understand how neuronal death following viral infection initiates microglial activation following Japanese encephalitis virus infection. We have earlier shown that the altered expression of tumor necrosis factor receptor-1 (TNFR-1) and TNFR associated death domain (TRADD) following Japanese encephalitis virus infection regulates the downstream apoptotic cascades. Here we have reported that silencing TRADD expression with small-interfering RNA reduced neuronal apoptosis and subsequent microglial and astroglial activation and release of various pro-inflammatory mediators. Our findings suggest that the engagement of TNFR-1 and TRADD following Japanese encephalitis virus infection plays a crucial role in glial activation also and influences the outcome of viral pathogenesis.     

Microglia in neuropathology caused by protozoan parasites

Involvement of the central nervous system (CNS) is the most severe consequence of some parasitic infections. Protozoal infections comprise a group of diseases that together affect billions of people worldwide and, according to the World Health Organization, are responsible for more than 500000 deaths annually. They include African and American trypanosomiasis, leishmaniasis, malaria, toxoplasmosis, and amoebiasis. Mechanisms underlying invasion of the brain parenchyma by protozoa are not well understood and may depend on parasite nature: a vascular invasion route is most common. Immunosuppression favors parasite invasion into the CNS and therefore the host immune response plays a pivotal role in the development of a neuropathology in these infectious diseases. In the brain, microglia are the resident immune cells active in defense against pathogens that target the CNS. Beside their direct role in innate immunity, they also play a principal role in coordinating the trafficking and recruitment of other immune cells from the periphery to the CNS. Despite their evident involvement in the neuropathology of protozoan infections, little attention has given to microglia–parasite interactions. This review describes the most prominent features of microglial cells and protozoan parasites and summarizes the most recent information regarding the reaction of microglial cells to parasitic infections. We highlight the involvement of the periphery–brain axis and emphasize possible scenarios for microglia–parasite interactions.     

Microglia promote the death of developing Purkinje cells

The loss of neuronal cells, a prominent event in the development of the nervous system, involves regulated triggering of programmed cell death, followed by efficient removal of cell corpses. Professional phagocytes, such as microglia, contribute to the elimination of dead cells. Here we provide evidence that, in addition to their phagocytic activity, microglia promote the death of developing neurons engaged in synaptogenesis. In the developing mouse cerebellum, Purkinje cells die, and 60% of these neurons that already expressed activated caspase-3 were engulfed or contacted by spreading processes emitted by microglial cells. Apoptosis of Purkinje cells in cerebellar slices was strongly reduced by selective elimination of microglia. Superoxide ions produced by microglial respiratory bursts played a major role in this Purkinje cell death. Our study illustrates a mammalian form of engulfment-promoted cell death that links the execution of neuron death to the scavenging of dead cells.     

Microglial functions and proteases

There is accumulating evidence that intracellular and extracellular proteases of microglia contribute to various events in the central nervous system (CNS) through both nonspecific and limited proteolysis. Cathepsin E and cathepsin S, endosomal/lysosomal proteases, have been shown to play important roles in the major histocompatibility complex (MHC) class II-mediated antigen presentation of microglia by processing of exogenous antigens and degradation of the invariant chain associated with MHC class II molecules, respectively. Some members of cathepsins are also involved in neuronal death after secreted from microglia and clearance of phagocytosed amyloid-β peptides. Tissue-type plasminogen activator, a serine protease, secreted from microglia participates in neuronal death, enhancement of N-methyl-d-aspartate receptor-mediated neuronal responses, and activation of microglia via either proteolytic or nonproteolytic activity. Calpain, a calcium-dependent cysteine protease, has been shown to play a pivotal role in the pathogenesis of multiple sclerosis by degrading myelin proteins extracellulary. Furthermore, matrix metalloproteases secreted from microglia also receive great attention as mediators of inflammation and tissue degradation through processing of pro-inflammatory cytokines and damage to the blood-brain barrier. The growing knowledge about proteolytic events mediated by microglial proteases will not only contribute to better understanding of microglial functions in the CNS but also may aid in the development of protease inhibitors as novel neuroprotective agents.     

Microglia - insights into immune system structure, function, and reactivity in the central nervous system

Microglia are essential cellular components of a well-functioning central nervous system (CNS). The development and establishment of the microglial population differs from the other major cell populations in the CNS i.e. neurons and macroglia (astrocytes and oligodendrocytes). This different ontogeny gives microglia unique properties. In recent years detailed studies of the microglial population have been greatly facilitated by the use of bone marrow (BM) chimeric animals. Experimental BM transplants have provided the opportunity to trace and investigate how BM cells migrate into the CNS and settle to become microglia. Furthermore various functional properties of microglia in the normal and pathological CNS are now being revealed because of combinations of BM transplantations and experimental disease models. Here, we describe some of the latest findings in microglial biology and discuss the potential for using microglia in therapeutic interventions.     

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1^GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.     

Age-related alterations in the dynamic behavior of microglia

Microglia, the primary resident immune cells of the central nervous system (CNS), exhibit dynamic behavior involving rapid process motility and cellular migration that is thought to underlie key functions of immune surveillance and tissue repair. Although age-related changes in microglial activation have been implicated in the pathogenesis of neurodegenerative diseases of aging, how dynamic behavior in microglia is influenced by aging is not fully understood. In this study, we employed live imaging of retinal microglia in situ to compare microglial morphology and behavioral dynamics in young and aged animals. We found that aged microglia in the resting state have significantly smaller and less branched dendritic arbors, and also slower process motilities, which probably compromise their ability to survey and interact with their environment continuously. We also found that dynamic microglial responses to injury were age-dependent. While young microglia responded to extracellular ATP, an injury-associated signal, by increasing their motility and becoming more ramified, aged microglia exhibited a contrary response, becoming less dynamic and ramified. In response to laser-induced focal tissue injury, aged microglia demonstrated slower acute responses with lower rates of process motility and cellular migration compared with young microglia. Interestingly, the longer term response of disaggregation from the injury site was retarded in aged microglia, indicating that senescent microglial responses, while slower to initiate, are more sustained. Together, these altered features of microglial behavior at rest and following injury reveal an age-dependent dysregulation of immune response in the CNS that may illuminate microglial contributions to age-related neuroinflammatory degeneration.     

Microglial Depletion Causes Region‐Specific Changes to Developmental Neuronal Cell Death in the Mouse Brain

Developmental neuronal cell death has been characterized as a cell autonomous “suicide” program, but recent findings suggest that microglia play an active role in determining the survival of developing neurons. Results have been contradictory, however, with some studies concluding that microglia promote cell death, while others report that microglia are neuroprotective. Here, we depleted microglia throughout the newborn mouse brain using intracerebroventricular injections of clodronate liposomes, and examined effects on naturally occurring cell death across multiple brain areas. Microglial density varied significantly by brain region, and clodronate liposome treatment at birth reduced the number of microglia in all regions examined. The effect of microglia reduction on cell death, however, varied by region: the number of dying cells was reduced in the medial septum and medial amygdala in clodronate treated animals, but was increased in the oriens layer of the hippocampus, and unchanged in several other brain regions. In most brain regions, the average size of microglia was greater in microglia‐depleted than in control animals, suggesting that the remaining microglia compensate to some extent for a reduction in microglial number. The hippocampal oriens was exceptional in this regard, in that microglial size was reduced following treatment with clodronate. Microglia produce cytokines which mediate many of their effects, and we found higher expression of inflammatory cytokines in the hippocampus than in the septum, independent of clodronate treatment. Thus, microglial depletion has opposite effects on cell death in different brain regions of the newborn brain, which may be related to regional heterogeneity in microglia.     

Microglia-astrocyte interaction in Alzheimer's disease: friends or foes for the nervous system?

Brain glial cells secrete several molecules that can modulate the survival of neurons after various types of damage to the CNS. Activated microglia and astrocytes closely associate to amyloid plaques in Alzheimer Disease (AD). They could have a role in the neurotoxicity observed in AD because of the inflammatory reaction they generate. There is controversy regarding the individual part played by the different glial cells, and the interrelationships between them. Both astrocytes and microglia produce several cytokines involved in the inflammatory reaction. Moreover, the same cytokines may have different effects, depending on their concentration and the type of cells in the vicinity. In turn, the events occurring in response to injury may lead to changes in the nature and relative concentration of the various factors involved. To learn about these putative glial interrelationships, we examined some effects of astrocytes on microglial activation.     

Specific immunolabeling of brain macrophages and microglial cells in the developing and mature chick central nervous system.

The present study showed that the HIS-C7 monoclonal antibody, which recognizes the chick form of CD45, is a specific marker for macrophages/microglial cells in the developing and mature chick central nervous system (CNS). HIS-C7-positive cells were characterized according to their morphological features and chronotopographical distribution patterns within developing and adult CNS, similar to those of macrophages/microglial cells in the quail CNS and confirmed by their histochemical labeling with Ricinus communis agglutinin I, a lectin that recognizes chick microglial cells. Therefore, the HIS-C7 antibody is a valuable tool to identify brain macrophage and microglial cells in studies of the function, development, and pathology of the chick brain. CD45 expression differed between chick microglia (as revealed with HIS-C7 antibody) and mouse microglial cells (as revealed with an antibody against mouse form of CD45). Thus, a discontinuous label was seen on mouse microglial cells with the anti-mouse CD45 immunostaining, whereas the entire surface of chick microglial cells was labeled with the anti-chick CD45 staining. The functional relevance of these differences between species has yet to be determined.     

Alzheimer's disease cerebrospinal fluid antibodies display selectivity for microglia

Previous investigations demonstrated that the cerebrospinal fluid (CSF) from Alzheimer's disease (AD) patients contains antibodies that recognize specific neuronal populations in the adult rat central nervous system (CNS). These findings suggest a pathogenic role for immunological aberrations in this disorder. To determine if antibodies may provide a means to differentially diagnose the dementias, CSF from a diversified dementia population was screened against the developing rat CNS and a cell culture system. Markings produced by AD CSF were distinctly different from those of vascular dementias (VAD) against the developing rat CNS. More importantly, some AD CSF recognized amoeboid microglia. The recognition of amoeboid microglia by antibodies in AD CSF is particularly interesting since these cells proliferate in response to nervous system disease and also engulf debris. A cell culture technique was developed to allow the rapid screening of CSF antibodies. Patient CSF produced five different types of markings in the cell culture: microglia, glioblasts, fibers, nonspecific, or negative. Correlations with these structures and the diagnosis of four different dementia populations revealed that, in comparison to the other groups, AD CSF displayed remarkable selectivity toward microglial cells. Cortical biopsies from patients suspected to have AD were incubated with the patient's own CSF and that of confirmed AD patients. Both CSF samples recognized microglial cells in the patient's cortical biopsy. The same CSF samples incubated against normal human cortical autopsy or a biopsy from a 3-mo-old child displayed negative immunoreactivity. These three approaches suggest that the presence of CSF microglial antibodies may be a means to distinguish AD patients from other dementias. The results add further support to the widely growing concept that inflammation and similar immune mechanisms may contribute to AD pathogenesis.     

Regulation of microglia by ionotropic glutamatergic and GABAergic neurotransmission

Recent studies have indicated that constitutive functions of microglia in the healthy adult central nervous system (CNS) involve immune surveillance, synapse maintenance and trophic support. These functions have been related to the ramified structure of 'resting' microglia and the prominent motility in their processes that provide extensive coverage of the entire extracellular milleu. In this review, we examine how external signals, and in particular, ionotropic neurotransmission, regulate features of microglial morphology and process motility. Current findings indicate that microglial physiology in the healthy CNS is constitutively and reciprocally regulated by endogenous ionotropic glutamatergic and GABAergic neurotransmission. These influences do not act directly on microglial cells but indirectly via the activity-dependent release of ATP, likely through a mechanism involving pannexin channels. Microglia in the 'resting' state are not only dynamically active, but also constantly engaged in ongoing communication with neuronal and macroglial components of the CNS in a functionally relevant way.     

Prevention of inflammation‐mediated neurotoxicity by butylidenephthalide and its role in microglial activation

Microglial cells are the prime effectors in immune and inflammatory responses of the central nervous system (CNS). During pathological conditions, the activation of these cells helps restore CNS homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory molecules and neurotoxins. Thus, negative regulators of microglial activation have been considered as potential therapeutic candidates to target neurodegeneration, such as that in Alzheimer's and Parkinson's diseases. The rhizome of Ligusticum chuanxiong Hort. (Ligusticum wallichii Franch) has been widely used for the treatment of vascular diseases in traditional oriental medicine. Butylidenephthalide (BP), a major bioactive component from L. chuanxiong, has been reported to have a variety of pharmacological activities, including vasorelaxant, anti‐anginal, anti‐platelet and anti‐cancer effects. The aim of this study was to examine whether BP represses microglial activation. In rat brain microglia, BP significantly inhibited the lipopolysaccharide (LPS)‐induced production of nitric oxide (NO), tumour necrosis factor‐α and interleukin‐1β. In organotypic hippocampal slice cultures, BP clearly blocked the effect of LPS on hippocampal cell death and inhibited LPS‐induced NO production in culture medium. These results newly suggest that BP provide neuroprotection by reducing the release of various proinflammatory molecules from activated microglia. Copyright © 2013 John Wiley & Sons, Ltd.     

Regulation of Microglial Phagocytosis by RhoA/ROCK-Inhibiting Drugs

Inflammation within the central nervous system (CNS) is a major component of many neurodegenerative diseases. The underlying mechanisms of neuronal loss are not fully understood, but the activation of CNS resident phagocytic microglia seems to be a significant element contributing to neurodegeneration. At the onset of inflammation, high levels of microglial phagocytosis may serve as an essential prerequisite for creating a favorable environment for neuronal regeneration. However, the excessive and long-lasting activation of microglia and the augmented engulfment of neurons have been suggested to eventually govern widespread neurodegeneration. Here, we investigated in a functional assay of acute inflammation how the small GTPase RhoA and its main target the Rho kinase (ROCK) influence microglial phagocytosis of neuronal debris. Using BV-2 microglia and human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA activation and microglial phagocytosis of neuronal cell fragments. Inhibition of the downstream effector ROCK with the small-molecule agents Y-27632 and Fasudil reduces the engulfment of neuronal debris and attenuates the production of the inflammatory mediator nitric oxide during stimulation with lipopolysaccharide. Our results support a therapeutic potential for RhoA/ROCK-inhibiting agents as an effective treatment of excessive inflammation and the resulting progression of microglia-mediated neurodegeneration in the CNS.     

Tubulin cofactor B regulates microtubule densities during microglia transition to the reactive states

Microglia are highly dynamic cells of the CNS that continuously survey the welfare of the neural parenchyma and play key roles modulating neurogenesis and neuronal cell death. In response to injury or pathogen invasion parenchymal microglia transforms into a more active cell that proliferates, migrates and behaves as a macrophage. The acquisition of these extra skills implicates enormous modifications of the microtubule and actin cytoskeletons. Here we show that tubulin cofactor B (TBCB), which has been found to contribute to various aspects of microtubule dynamics in vivo, is also implicated in microglial cytoskeletal changes. We find that TBCB is upregulated in post-lesion reactive parenchymal microglia/macrophages, in interferon treated BV-2 microglial cells, and in neonate amoeboid microglia where the microtubule densities are remarkably low. Our data demonstrate that upon TBCB downregulation both, after microglia differentiation to the ramified phenotype in vivo and in vitro, or after TBCB gene silencing, microtubule densities are restored in these cells. Taken together these observations support the view that TBCB functions as a microtubule density regulator in microglia during activation, and provide an insight into the understanding of the complex mechanisms controlling microtubule reorganization during microglial transition between the amoeboid, ramified, and reactive phenotypes.