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Lectin binding by resting and reactive microglia.

Conjugates of the B4 isolectin from Griffonia simplicifolia seeds and horseradish peroxidase were used as a histochemical reagent for the specific visualization of microglial cells in the rat CNS. Resident microglia bearing galactose-containing glycoconjugates were stained throughout the brainstem and cerebellum. In the first week following axotomy of the facial nerve, a profound and rapid accumulation of reactive microglia, as evidenced by increasing lectin reactivity, was seen to take place in the facial nucleus. Light microscopy of paraffin sections demonstrated binding of lectin-horseradish peroxidase conjugates to microglial cytoplasmic processes. When ultrastructural cytochemistry was performed, reaction product was found localized on microglial plasma membranes, as well as on intracytoplasmic membranes. The glial reaction to axotomy was studied further with double labelling of microglia and astrocytes by lectin histochemistry and immunostaining for glial fibrillary acidic protein, respectively. Our results demonstrated the presence of membrane-associated glycoconjugates containing terminal alpha-D-galactose residues on microglia, but not on other glial cell types. The possible nature and function of these glycoconjugates are discussed.

Animals↗

Reactive microglia express class I and class II major histocompatibility complex antigens in Alzheimer's disease.

Expression of major histocompatibility complex (MHC) class I (HLA-A,B,C) and class II (HLA-DR) antigens was studied in postmortem brain tissue of Alzheimer's disease and age-matched control cases. Monoclonal antibodies to class I antigens stained reactive microglia weakly to moderately and vascular endothelial cells strongly in Alzheimer's gray and white matter. Capillaries were also stained strongly in control brains, but only a few positively stained microglia could be observed in white matter. In Alzheimer cases, HLA-DR positive reactive microglia were more numerous and more strongly staining than MHC class I positive reactive microglia but many cells appeared to express both classes of antigen. Rare large cells of uncertain origin also stained positively for class I. Double immunostaining established that MHC class I positive cells were a separate population from glial fibrillary acidic-protein-positive astrocytes.

Antibodies, Monoclonal↗

Morphological transformation of microglia in vitro.

Isolated microglia underwent in vitro morphological transformation to ameboid, rod-shaped and ramified forms in response to several cytokines, factors from astrocytes, or factors which affect macrophage function. These morphological transformations were reversible. Examination of the enzymatic activity, superoxide anion formation and proliferative activity of these transformed microglia indicated that the ameboid forms were activated, rod-shaped microglia were proliferating, and the ramified forms were resting microglia.

Animals↗

MHC antigen expression on bulk isolated macrophage-microglia from newborn mouse brain: induction of Ia antigen expression by gamma-interferon.

Macrophage-microglia were isolated from primary mixed brain cell cultures of normal newborn mice. They were successfully maintained in vitro for at least 8 weeks. Purity of the cultures was 97-100%, as determined by endocytosis of latex beads, non-specific staining through Fc receptors, EA and EAC rosette formation. These cells were non-specific esterase-positive, but peroxidase-negative. Electron-microscope observations revealed morphological similarities to mature macrophages. Isolated macrophage-microglia seldom incorporated [3H]thymidine in vitro. By means of 51Cr release assay, using monoclonal antibodies against mouse major histocompatibility complex (MHC) antigens and complement, we detected class I MHC (H-2) antigen on unstimulated macrophage-microglia, and both class I and class II (Ia) antigens on gamma-interferon-treated cells. These observations suggest possible immunoregulatory functions of macrophage-microglia in the central nervous system, as is characteristic of other cells of monocyte lineage.

Animals↗

Differential suppression of interferon-gamma-induced Ia antigen expression on cultured rat astroglia and microglia by second messengers.

The roles of intracellular second messengers in interferon-gamma (IFN-gamma)-induced Ia antigen (Ag) expression by astroglia and microglia were examined. Ia Ag on both glia types was induced by IFN-gamma. Reagents known to increase intracellular cAMP or activate intracellular protein kinase C (PKC) reduced IFN-gamma-induced Ia Ag expression by astroglia. In contrast, increasing intracellular cAMP had no suppressive effect on Ia Ag expression by microglia. These results indicate (1) cAMP and PKC negatively regulate IFN-gamma-induced Ia expression on astroglia, and (2) Ia expression is regulated differentially in astroglia vs. microglia. These findings may explain the frequent observation of Ia+ microglia (or macrophages) but not astroglia in various neurodegenerative diseases.

Animals↗

Detection of interleukin-1 and interleukin-6 in adult rat brain, following mechanical injury, by in vivo microdialysis: evidence of a role for microglia in cytokine production.

In vivo levels of interleukin-1 (IL-1) and IL-6, present in the interstitial spaces of brain, have been repeatedly monitored up to 7 days after insertion of a microdialysis probe, designed to induce mechanical trauma to the brain. IL-1 is barely detectable immediately after implantation but over a 24-48 h period a 15-fold increase is seen. In contrast IL-6 levels at day 0 are high, increasing slightly (10%) by day 1 but decreasing to 40% by day 2. The temporal pattern of IL-6 recovery in the cerebrospinal fluid was similar to that in the dialysate but the levels were significantly lower and may reflect diffusion from the site of the probe lesion. Cellular sources of these cytokines include macrophages and neutrophils, which have infiltrated the lesion and microglia resident in the brain, which can be identified at the lesion site within 24 h of probe implantation. The astrocytic response to injury, evidenced by increased glial fibrillary acidic protein staining occurs much later, by day 7, and is unlikely to be responsible for IL-1 and IL-6 production found at 24-48 h. Since upon isolation and stimulation of microglia in vitro with lipopolysaccharide IL-1 and IL-6 can be measured in the supernatant, it would appear that they have the capacity to produce cytokines in vivo. Localised synthesis of cytokines at sites of brain injury by microglia would further stimulate microglia in an autocrine manner and also propagate the astrocytic reaction.

Animals↗

Functional dichotomy of mouse microglia developed in vitro: differential effects of macrophage and granulocyte/macrophage colony-stimulating factor on cytokine secretion and antitoxoplasmic activity.

After differentiation either with exogenous macrophage (M) or with granulocyte/macrophage (GM) colony-stimulating factor (CSF) microglial cells were isolated from neonatal mouse brain cell cultures and were comparatively tested for secretory immune effector cell functions. Both factors obviously do not promote the development of cells with biased growth requirement; however, the two microglia populations displayed distinct potentials to produce inflammatory cytokines. Upon gradual stimulation by lipopolysaccharide, the cells harvested from M-CSF-driven culture released more interleukin-1 and tumor necrosis factor activity, GM-CSF-grown cells on the contrary proved superior in interleukin-6 secretion. This pattern was paralleled by corresponding different kinetics of cytokine release in both types of microglial cells. When infected with Toxoplasma gondii only GM-CSF-differentiated cells were able to restrict the intracellular multiplication of tachyzoites in the absence of external stimuli. As described for interferon-gamma-treated macrophages, the antiparasitic activity of this microglia population is due to the synthesis of reactive nitrogen intermediates, since it was antagonized by NG-monomethyl-L-arginine, a competitive inhibitor of the arginine-dependent metabolic pathway. Complementary to previous data which attest an intrinsic capability for antigen presentation to GM-CSF-grown microglia, the functional state of the cells elicited by M-CSF and GM-CSF, respectively, may correspond to the resting and an activated form of microglia as distinguished in vivo.

Animals↗

Abnormal production of interleukin-1 by microglia from trisomy 16 mice.

The production of interleukin-1 (IL-1) was examined in cultured CNS microglia obtained from trisomy 16 (Ts16) fetal mouse brain, a model system for studies relevant to Down syndrome (DS). When compared to microglia from their normal littermates, Ts16 microglia produced significantly higher levels of IL-1 activity both before and following stimulation with lipopolysaccharide (LPS). IL-1 release was stimulated by alpha/beta interferon (IFN) in the normal but not Ts16 microglial cultures. The overall level of IL-1 production in normal littermates, however, was still less than that seen in Ts16. Thus, microglia from Ts16 mice may function in an inappropriate manner and, if this abnormality occurs in vivo, may have wide ranging effects on a developing nervous system.

Animals↗

Microglia and the developing olfactory bulb.

The developmental appearance of microglia in the rat olfactory bulb was investigated through the use of selective staining with the B4-isolectin from Griffonia simplicifolia. No changes in the density or distribution of either the spherical, macrophage "ameboid" form or the highly arborized "ramified" variety of microglia were observed in the superficial layers of the bulb between postnatal days 10 and 30. The subependymal zone exhibited the only substantial population of ameboid cells and the only developmental increases in ramified cell density during this time-period. External single naris closure, which enhances cell death in the ipsilateral bulb, did not affect microglia density, presumably due to the unusually high numbers of microglia normally present in the bulb. The olfactory bulb has a dense and relatively uniform population of microglial cells from very early stages of postnatal life, perhaps because of the constant turnover of cells and processes.

Aging↗

Single-nucleus transcriptomics reveals cell type-specific remodeling and epilepsy-associated microglia.

Temporal lobe epilepsy (TLE) is the most common acquired epilepsy, causing refractory seizures and cognitive deficits. We performed single-nucleus RNA sequencing on hippocampal tissue from mice 3 and 6 weeks following pilocarpine-induced status epilepticus, a robust model of TLE. Epilepsy samples showed reductions in Cck and Lamp5-Lhx6 interneuron subclusters, alongside increases in Cajal-Retzius cells, dentate granule (DG) cell precursors, and a mature DG cell subcluster. Among glia, an astrocyte subcluster and a markedly expanded microglia sublcuster were increased. We term this microglia population epilepsy-associated microglia (EAM). The transcriptomic profile of EAM overlaps with microglia described in models of Alzheimer's disease and traumatic brain injury, including enrichment of Myo1e and Igf1. EAM display amoeboid morphology, can be found in clumps around pyramidal and granule cell body layers, and exhibit enlarged vesicles and mitochondria. Cell-cell interaction analysis predicts DG cells as their primary interaction partners. This dataset defines transcriptomic programs underlying key cellular alterations in TLE, enabling mechanistic dissection of epileptogenesis.

TLE↗

TNF alpha induces IL-6 production by astrocytes but not by microglia.

Astrocytes and microglia, both produced interleukin-6 (IL-6) in culture by lipopolysaccharide (LPS) stimulation. IL-6 activity was detected 3-5 h after LPS stimulation and reached a maximum at 10 h. Microglia responded faster than astrocytes. Tumor necrosis factor alpha and interleukin 1 also induced IL-6 mRNA and biological activity in astrocytes, but not in microglia. Among these stimuli, LPS was the most potent inducer of IL-6 production by astrocytes. Our results suggest that different regulatory mechanisms for cytokine production exist in glial cells. The possible roles of astrocytes and microglia in CNS immune responses are also discussed.

Animals↗

Are resting and/or reactive microglia macrophages?

According to recent submicroscopic, cytokinetics, and functional (particularly cytoimmunologic) investigations, no relationship exists between "resting" microglia (the small argyrophilic cells appearing in undamaged brain tissue, first described by Rio Hortega) and "reactive" microglia (the argyrophilic cells appearing under pathologic conditions). While "resting" microglia are apparently cells of neuro-ectodermal origin, all observations tend to indicate that "reactive" microglia are derived from extravasated blood monocytes and should be called brain macrophages. In the intact brain parenchyma, no macrophages are demonstrable. Free subarachnoidal cells in the cerebrospinal fluid (CSF), perivascular cells, and epiplexus and/or supraependymal cells in the CSF-containing spaces of the normal central nervous system are cells of the mononuclear phagocyte system and must be considered as CSF macrophages. According to rough estimates, the normal adult central nervous system contains a maximum of 280,000 CSF macrophages.

Animals↗

Regulators of interferon-responsive microglia uncovered by Genome-wide CRISPRi screening.

Microglia dynamically support brain health through the induction of specialized activation states in response to injury or disease. Activation of the interferon-responsive microglia (IRM) state has been identified across neurodevelopmental windows, age-related cognitive decline, and neurodegenerative diseases. Functionally, IRM have been linked to synaptic pruning, dead cell removal, and neuroinflammation, making this state critical to brain homeostasis. While the functional importance of this state is becoming increasingly clear, our understanding of the regulatory networks that govern IRM induction remain incomplete. To systematically identify genetic regulators of the IRM state, we conducted a genome-wide CRISPR interference screen in human iPSC-derived microglia using IFIT1 as a representative IRM marker. We identified 772 genes that modulate IRM, including canonical type I interferon signaling genes (IFNAR2, TYK2, STAT1/2, USP18) and newly described regulators. We uncovered a non-canonical role for the CCR4-NOT transcription complex subunit 10, CNOT10, in IRM activation. This work provides a comprehensive resource that can be applied to dissect the functions of interferon-responsive microglia and highlights both established and novel targets for modulating microglial interferon signaling in health and disease.

Computational biology and bioinformatics↗

Individual microglia move rapidly and directly to nerve lesions in the leech central nervous system.

Small cells called microglia, which collect at nerve lesions, were tracked as they moved within the leech nerve cord to crushes made minutes or hours before. The aim of this study was to determine whether microglia respond as a group and move en masse or instead move individually, at different rates, and whether they move along axons directly to the lesion or take another route, such as along the edges of the nerve cord. Cell nuclei in living nerve cords were stained with Hoechst 33258 dye and observed under dim ultraviolet illumination using fluorescence optics, a low-light video camera, and computer-assisted signal enhancement. Muscular movements of the cord were selectively reduced by bathing in 23 mM MgCl2. Regions of nerve cord within 300 microns of the crush were observed for 2-6 hr. Only a fraction of microglia, typically less than 50%, moved at any time, traveling toward the lesion at speeds up to 7 microns/min. Cells were moving as soon as observation began, within 15 min of crushing, and traveled directly toward the lesion along axons or axon tracts. Movements and roles of leech microglia are compared with their vertebrate counterparts, which are also active and respond to nerve injury.

Animals↗

Immunological analysis of human microglia: lack of monocytic and lymphoid membrane differentiation antigens.

While it is certain that reactive microglia (phagocytes in damaged brain tissue) originate from monocytes, the derivation of resting microglia (argentophilic cells in undamaged brain tissue) is not clear. In order to examine a possible lymphocytic and/or monocytic origin for resting microglia, cryostat sections of presumably normal human brain tissue were treated with anti-lymphocyte, anti-monocyte and anti-beta 2-microglobulin sera and then stained with the immunoperoxidase technique. No positive reaction was obtained. It must be concluded that resting microglia are not derived from mononuclear blood cells.

Antigens, Surface↗

Pinocytosis as a select marker of ramified microglia in vivo and in vitro.

Based on previous observations in tissue culture, we investigated pinocytotic activity as a potential cell marker for brain microglia. This functional activity was assessed in three different preparations derived from rat: primary cultures of mixed cerebral cortical cells, tissue slabs of whole cerebrum, and cultures of isolated or enriched microglial cells. Each preparation was incubated with the fluorescent dye lucifer yellow as a soluble tracer and then processed for light microscopy. Under the conditions utilized, ramified microglia specifically exhibited differentially high pinocytotic labeling in all cases; the dye was mainly localized within the cell somata, where it was sequestered in pinocytotic vesicles. In each preparation, the identity of the labeled cell population was confirmed as microglia through immunohistochemical staining with the monoclonal antibody (MAb) OX-42, a specific microglial marker. Therefore, pinocytotic labeling is proposed as a select cell marker for microglia, which may be extremely useful in the identification and study of ramified microglial cells.

Animals↗

Infiltrative microgliosis: activation and long-distance migration of subependymal microglia following periventricular insults.

BACKGROUND: Subventricular microglia (SVMs) are positioned at the interface of the cerebrospinal fluid and brain parenchyma and may play a role in periventricular inflammatory reactions. However, SVMs have not been previously investigated in detail due to the lack of a specific methodology for their study exclusive of deeper parenchymal microglia. METHODS: We have developed and characterized a novel model for the investigation of subventricular microglial reactions in mice using intracerebroventricular (ICV) injection of high-dose rhodamine dyes. Dynamic studies using timelapse confocal microscopy in situ complemented the histopathological analysis. RESULTS: We demonstrate that high-dose ICV rhodamine dye injection resulted in selective uptake by the ependyma and ependymal death within hours. Phagocytosis of ependymal debris by activated SVMs was evident by 1d as demonstrated by the appearance of rhodamine-positive SVMs. In the absence of further manipulation, labelled SVMs remained in the subventricular space. However, these cells exhibited the ability to migrate several hundred microns into the parenchyma towards a deafferentation injury of the hippocampus. This "infiltrative microgliosis" was verified in situ using timelapse confocal microscopy. Finally, supporting the disease relevance of this event, the triad of ependymal cell death, SVM activation, and infiltrative microgliosis was recapitulated by a single ICV injection of HIV-1 tat protein. CONCLUSIONS: Subependymal microglia exhibit robust activation and migration in periventricular inflammatory responses. Further study of this population of microglia may provide insight into neurological diseases with tendencies to involve the ventricular system and periventricular tissues.

Journal Article↗

The blood-brain barrier regulates the expression of a macrophage sialic acid-binding receptor on microglia.

In vitro the expression of a sialic acid-binding receptor on murine macrophages, sialoadhesin, is regulated by exposure to an inducing agent present in serum. We have used immunocytochemistry to examine the macrophage populations of the nervous system in order to test whether this serum inducing agent (SIA) also regulates sialoadhesin expression in vivo and whether plasma proteins may influence the phenotype of macrophages of the nervous system. Microglia, the resident macrophages of the central nervous system, reside behind the blood-brain barrier and do not express sialoadhesin. Microglia and macrophage populations in the cicumventricular organs, choroid plexus and leptomeninges are exposed to plasma proteins and some macrophages express sialoadhesin at these sites. Injury to the CNS, which damages the blood-brain barrier, induces sialoadhesin expression on a proportion of macrophages and microglia within the parenchyma. The expression of sialoadhesin matches the temporal and spatial distribution of the plasma extravasation into the brain parenchyma. These experiments show that exposure to SIA is necessary for sialoadhesin expression and lend further support to the idea that the phenotype of microglia is in part regulated by the presence of the blood-brain barrier.

Animals↗