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Although del Río-Hortega originally reported that leptomeningeal cells are the source of ramified microglia in the developing brain, recent views do not seem to pay much attention to this notion. In this study, in vitro experiments were conducted to determine whether leptomeninges generate ramified microglia. The leptomeninges of neonatal rats containing Iba1 + macrophages were peeled off the brain surface. Leptomeningeal macrophages strongly expressed CD68 and CD163, but microglia in the brain parenchyma did not. Leptomeningeal macrophages expressed epidermal growth factor receptor (EGFR) as revealed by RT-PCR and immunohistochemical staining. Cells obtained from the peeled-off leptomeninges were cultured in a serum-free medium containing EGF, resulting in the formation of large cell aggregates in which many proliferating macrophages were present. In contrast, colony-stimulating factor 1 (CSF1) did not enhance the generation of Iba1 + cells from the leptomeningeal culture. The cell aggregates generated ramified Iba1 + cells in the presence of serum, which express CD68 and CD163 at much lower levels than primary microglia isolated from a mixed glial culture. Therefore, the leptomeningeal-derived cells resembled parenchymal microglia better than primary microglia. This study suggests that microglial progenitors expressing EGFR reside in the leptomeninges and that there is a population of microglia-like cells that grow independently of CSF1.

Brain edema following brain infarction affects mobility and mortality. The mechanisms underlying this process remain to be elucidated. Animal studies have shown that aquaporin-4 (AQP4) expression in astrocytes increases after stroke, and its deletion significantly reduces brain swelling. Recently, two kinds of cells, resident microglia-derived macrophage-like cells (MG-MΦ) and bone marrow-derived macrophages (BM-MΦ), have been reported to accumulate in the ischemic core and stimulate adjacent astrocytes. Therefore, we hypothesized that these cells play crucial roles in the expression of AQP4 and ultimately lead to exacerbated brain edema. To verify this hypothesis, we investigated the role of MG- or BM-MΦ in brain edema using a rat model of transient middle cerebral artery occlusion and rat astrocyte primary cultures. AQP4 expression significantly increased in the peri-infarct tissue at 3-7 days post-reperfusion (dpr) and in the core tissue at 5 and 7 dpr, which synchronized with the expression of Iba1, Il1a, Tnf, and C1qa mRNA. Interleukin (IL)-1α treatment or coculture with MG- and BM-MΦ increased AQP4 expression in astrocytes, while an IL-1 receptor type I antagonist reduced these effects. Furthermore, aggravated animals exhibited high expression of Aqp4 and Il1a mRNA in the ischemic core at 7 dpr, which led to the exacerbation of brain edema. MG-MΦ signature genes were highly expressed in the ischemic core in aggravated rats, while BM-MΦ signature genes were weakly expressed. These findings suggest that IL-1α produced by MG-MΦ induces astrocytic AQP4 expression in the peri-infarct and ischemic core tissues, thereby exacerbating brain edema. Therefore, the regulation of MG-MΦ may prevent the exacerbation of brain edema., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Microglia, the immunocompetent cells in the central nervous system (CNS), have long been studied as pathologically deteriorating players in various CNS diseases. However, microglia exert ameliorating neuroprotective effects, which prompted us to reconsider their roles in CNS and peripheral nervous system (PNS) pathophysiology. Moreover, recent findings showed that microglia play critical roles even in the healthy CNS. The microglial functions that normally contribute to the maintenance of homeostasis in the CNS are modified by other cells, such as astrocytes and infiltrated myeloid cells; thus, the microglial actions on neurons are extremely complex. For a deeper understanding of the pathophysiology of various diseases, including those of the PNS, it is important to understand microglial functioning. In this review, we discuss both the favorable and unfavorable roles of microglia in neuronal survival in various CNS and PNS disorders. We also discuss the roles of blood-borne macrophages in the pathogenesis of CNS and PNS injuries because they cooperatively modify the pathological processes of resident microglia. Finally, metabolic changes in glycolysis and oxidative phosphorylation, with special reference to the pro-/anti-inflammatory activation of microglia, are intensively addressed, because they are profoundly correlated with the generation of reactive oxygen species and changes in pro-/anti-inflammatory phenotypes.

Chronic constriction injury of the sciatic nerve is frequently considered as a cause of chronic neuropathic pain. Marked activation of microglia in the posterior horn (PH) has been well established with regard to this pain. However, microglial activation in the anterior horn (AH) is also strongly induced in this process. Therefore, in this study, we compared the differential activation modes of microglia in the AH and PH of the lumbar cord 7 days after chronic constriction injury of the left sciatic nerve in Wistar rats. Microglia in both the ipsilateral AH and PH demonstrated increased immunoreactivity of the microglial markers Iba1 and CD11b. Moreover, abundant CD68 + phagosomes were observed in the cytoplasm. Microglia in the AH displayed elongated somata with tightly surrounding motoneurons, whereas cells in the PH displayed a rather ameboid morphology and were attached to myelin sheaths rather than to neurons. Microglia in the AH strongly expressed NG2 chondroitin sulfate proteoglycan. Despite the tight attachment to neurons in the AH, a reduction in synaptic proteins was not evident, suggesting engagement of the activated microglia in synaptic stripping. Myelin basic protein immunoreactivity was observed in the phagosomes of activated microglia in the PH, suggesting the phagocytic removal of myelin. CCI caused both motor deficit and hyperalgesia that were evaluated by applying BBB locomotor rating scale and von Frey test, respectively. Motor defict was the most evident at postoperative day1, and that became less significant thereafter. By contrast, hyperalgesia was not severe at day 1 but it became worse at least by day 7. Collectively, the activation modes of microglia were different between the AH and PH, which may be associated with the difference in the course of motor and sensory symptoms., Competing Interests: Declaration of competing interest None., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Microglia and blood-borne macrophages in injured or diseased brains are difficult to distinguish because they share many common characteristics. However, the identification of microglia-specific markers and the use of flow cytometry have recently made it easy to discriminate these types of cells. In this study, we analyzed the features of blood-borne macrophages, and activated and resting microglia in a rat traumatic brain injury (TBI) model. Oxidative injury was indicated in macrophages and neurons in TBI lesions by the presence of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Generation of mitochondrial reactive oxygen species (ROS) was markedly observed in granulocytes and macrophages, but not in activated or resting microglia. Dihydroethidium staining supported microglia not being the major source of ROS in TBI lesions. Furthermore, macrophages expressed NADPH oxidase 2, interleukin-1β (IL-1β), and CD68 at higher levels than microglia. In contrast, microglia expressed transforming growth factor β1 (TGFβ1), interleukin-6 (IL-6), and tumor necrosis factor α at higher levels than macrophages. A hypnotic, bromovalerylurea (BU), which has anti-inflammatory effects, reduced both glycolysis and mitochondrial oxygen consumption. BU administration inhibited chemokine CCL2 expression, accumulation of monocytes/macrophages, 8-OHdG generation, mitochondrial ROS generation, and proinflammatory cytokine expression, and markedly ameliorated the outcome of the TBI model. Yet, BU did not inhibit microglial activation or expression of TGFβ1 and insulin-like growth factor 1 (IGF-1). These results indicate that macrophages are the major aggravating cell type in TBI lesions, in particular during the acute phase. Activated microglia may even play favorable roles. Reduction of cellular energy metabolism in macrophages and suppression of CCL2 expression in injured tissue may lead to amelioration of TBI., (© 2018 Wiley Periodicals, Inc.)

Ischemic brain injuries caused release of damage-associated molecular patterns (DAMPs) that activate microglia/macrophages (MG/MPs) by binding to Toll-like receptors. Using middle cerebral artery transiently occluded rats, we confirmed that MG/MPs expressed inducible nitric oxide synthase (iNOS) on 3days after reperfusion (dpr) in ischemic rat brain. iNOS expression almost disappeared on 7dpr when transforming growth factor-β1 (TGF-β1) expression was robustly increased. After transient incubation with TGF-β1 for 24h, rat primary microglial cells were incubated with lipopolysaccharide (LPS) and released NO level was measured. The NO release was persistently suppressed even 72h after removal of TGF-β1. The sustained TGF-β1 effects were not attributable to microglia-derived endogenous TGF-β1, as revealed by TGF-β1 knockdown and in vitro quantification studies. Then, boiled supernatants prepared from ischemic brain tissues showed the similar sustained inhibitory effects on LPS-treated microglial cells that were prevented by the TGF-β1 receptor-selective blocker SB525334. After incubation with TGF-β1 for 24h and its subsequent removal, LPS-induced phosphorylation of IκB kinases (IKKs), IκB degradation, and NFκB nuclear translocation were inhibited in a sustained manner. SB525334 abolished all these effects of TGF-β1. In consistent with the in vitro results, phosphorylated IKK-immunoreactivity was abundant in MG/MPs in ischemic brain lesion on 3dpr, whereas it was almost disappeared on 7dpr. The findings suggest that abundantly produced TGF-β1 in ischemic brain displays sustained anti-inflammatory effects on microglial cells by persistently inhibiting endogenous Toll-like receptor ligand-induced IκB degradation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

CD200 induces immunosuppression in myeloid cells expressing its receptor CD200R, which may have consequences for tumor immunity. We found that human carcinoma tissues express not only full-length CD200 (CD200L) but also its truncated form, CD200S. Although CD200S is reported to antagonize the immunosuppressive actions of CD200L, the role of CD200S in tumor immunity has never been investigated. We established rat C6 glioma cell lines that expressed either CD200L or CD200S; the original C6 cell line did not express CD200 molecules. The cell lines showed no significant differences in growth. Upon transplantation into the neonatal Wistar rat forebrain parenchyma, rats transplanted with C6-CD200S cells survived for a significantly longer period than those transplanted with the original C6 and C6-CD200L cells. The C6-CD200S tumors were smaller than the C6-CD200L or C6-original tumors, and many apoptotic cells were found in the tumor cell aggregates. Tumor-associated macrophages (TAMs) in C6-CD200S tumors displayed dendritic cell (DC)-like morphology with multiple processes and CD86 expression. Furthermore, CD3(+), CD4(+) or CD8(+) cells were more frequently found in C6-CD200S tumors, and the expression of DC markers, granzyme, and perforin was increased in C6-CD200S tumors. Isolated TAMs from original C6 tumors were co-cultured with C6-CD200S cells and showed increased expression of DC markers. These results suggest that CD200S activates TAMs to become DC-like antigen presenting cells, leading to the activation of CD8(+) cytotoxic T lymphocytes, which induce apoptotic elimination of tumor cells. The findings on CD200S action may provide a novel therapeutic modality for the treatment of carcinomas., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Two types of macrophages in lesion core of rat stroke model were identified according to NG2 chondroitin sulfate proteoglycan (NG2) and CD200 expression. NG2(+) macrophages were CD200(-), and vice versa. NG2(-) macrophages expressed two splice variants of CD200 that are CD200L and CD200S. CD200(+) macrophages expressed CD8, CD68, CD163, CCL2, inducible nitric oxide synthase, interleukin-1β, Toll-like receptor 4 and transforming growth factor β, whilst NG2(+) cells expressed a costimulatory factor CD86. Both cell types expressed insulin-like growth factor 1 and CD200R. These results demonstrate that the two macrophage types cannot be classified as either M1 or M2., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Some macrophages expressing NG2 chondroitin sulfate proteoglycan (NG2) and the macrophage marker Iba1 accumulate in the ischemic core of a rat brain subjected to transient middle cerebral artery occlusion (MCAO) for 90 min. These cells are termed BINCs (for brain Iba1(+) /NG2(+) cells) and may play a neuroprotective role. Because BINCs are bone marrow-derived cells, they are able to invade ischemic tissue after the onset of an ischemic insult. In this study, chemokine-based mechanisms underlying the invasion of BINCs or their progenitor cells were investigated. We found that isolated BINCs expressed mRNA encoding CCR2 and CX3CR1 at high levels. Cultured astrocytes expressed mRNA encoding their ligands, MCP-1 and fractalkine. Recombinant MCP-1 and/or fractalkine, as well as astrocytes, induced the migration of BINCs in vitro. mRNA for MCP-1, fractalkine, CCR2, and CX3CR1 was expressed in the ischemic core during the acute phase of the ischemic event. Immunohistochemical studies revealed that vascular endothelial cells and astrocytic endfeet expressed MCP-1 and fractalkine, respectively, in the ischemic core during the acute phase. CCR2(+) /Iba1(+) monocytes attached to the inside of the vascular wall at 1 day postreperfusion (dpr), and there were CCR2(+) /CX3CR1(+) macrophage-like cells in the parenchyma in the ischemic lesion core at 2 dpr, which may be the progenitors for BINCs. These results suggest that CCR2(+) monocytes are first attracted to the ischemic lesion by MCP-1(+) endothelial cells and migrate toward fractalkine(+) astrocytic endfeet through the disrupted blood-brain barrier. Thus, chemokines may play a critical role in the accumulation of neuroprotective BINCs. © 2013 Wiley Periodicals, Inc., (Copyright © 2013 Wiley Periodicals, Inc.)

In this study, we perform a detailed analysis of the microglial and macrophage responses in a model of spinal cord ischemia and reperfusion (SCI/R) injury in Wistar rats. The rats underwent occlusion across the descending aorta for 13min, causing paraplegia or paresis of varying severity. They were divided into four groups based on neurological assessment: sham, mild paresis, moderate paresis, and severe (complete) paraplegia. To examine the origin of microglia and macrophages in the ischemic lesion, bone marrow from rats expressing green fluorescent protein (GFP) was transplanted into test subjects one month before performing SCI/R. Many GFP(+)/CD68(+) microglia and macrophages were present 7d after SCI/R. Resident (GFP(-)/Iba1(+)/CD68(-)) microglia and bone marrow-derived macrophages (BMDMs; GFP(+)/Iba1(+)/CD68(+)) colocalized in the mild group 7d after SCI/R. In the moderate group, BMDMs outnumbered resident microglia. A greater accumulation of BMDMs expressing insulin-like growth factor-1 (IGF-1) was observed in lesions in the severe group, relative to the moderate group. BMDMs in the severe group strongly expressed tumor necrosis factor α, interleukin-1β, and inducible nitric oxide synthase, in addition to IGF-1. A robust accumulation of BMDMs occupying the entire ischemic gray matter was observed only in the severe group. These results demonstrate that the magnitude of the microglial and BMDM responses varies considerably, and that it correlates with the severity of the neurological dysfunction. Remarkably, BMDMs appear to have a beneficial effect on the spinal cord in paresis. In contrast, BMDMs seem to exhibit both beneficial and harmful effects in severe paraplegia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In a transient 90-min middle cerebral artery occlusion (MCAO) model of rats, a large ischemic lesion is formed where macrophage-like cells massively accumulate, many of which express a macrophage marker, Iba1, and an oligodendrocyte progenitor cell marker, NG2 chondroitin sulfate proteoglycan (NG2); therefore, the cells were termed BINCs (Brain Iba1(+)/NG2(+) Cells). A bone marrow transplantation experiment using green-fluorescent protein-transgenic rats showed that BINCs were derived from bone marrow. 5-Fluorouracil (5FU) injection at 2 days post reperfusion (2 dpr) markedly reduced the number of BINCs at 7 dpr, causing enlargement of necrotic volumes and frequent death of the rats. When isolated BINCs were transplanted into 5FU-aggravated ischemic lesion, the volume of the lesion was much reduced. Quantitative real-time RT-PCR showed that BINCs expressed mRNAs encoding bFGF, BMP2, BMP4, BMP7, GDNF, HGF, IGF-1, PDGF-A, and VEGF. In particular, BINCs expressed IGF-1 mRNA at a very high level. Immunohistochemical staining showed that IGF-1-expressing BINCs were found not only in rat but also human ischemic brain lesions. These results suggest that bone marrow-derived BINCs play a beneficial role in ischemic brain lesions, at least in part, through secretion of neuroprotective factors.

Although neurons and glia inevitably undergo degeneration in the core of ischemic lesions, many cells, particularly immune cells, infiltrate the core and survive in it. Such infiltrating cells may play certain roles in the regeneration and repair of damaged brain tissues. In this study, we characterized macrophage-like cells that accumulated in the ischemic core of a rat brain whose right middle cerebral artery was transiently occluded for 90 mins. Many of the accumulated macrophage-like cells expressed Iba1, a marker of macrophages/microglia, as well as NG2 chondroitin sulfate proteoglycan (NG2), which has been recognized as a marker of oligodendrocyte progenitor cells. Such macrophage-like cells were termed BINCs (brain Iba1(+)/NG2(+) cells) to distinguish them from NG2(-)/Iba1(+) or NG2(+)/Iba1(-) cells that were also present in the perilesion and the contralateral hemisphere. Electron microscopy showed the localization of NG2 along the plasma membrane of cells that had many phagosomes and irregular-shaped or reniform heterochromatin-rich nuclei, which are characteristics of monocytes/macrophages. Brain Iba1(+)/NG2(+) cells were highly proliferative and their number peaked at 7 days post-reperfusion. An immunoblot analysis of NG2 revealed the presence of two NG2s: one expressed by BINCs with a molecular weight of 300 kDa, and the other found in the contralateral hemisphere with a molecular weight of 290 kDa. Taken the various functions of NG2, BINCs may be involved in not only phagocytosis of degenerated cells but also the healing and regeneration of lesion cores.

Brain ischemia causes the death of neurons and glial cells. Such brain cells are believed to inevitably undergo degeneration in the core of ischemic lesions, whereas neurons and glial cells may survive in the region surrounding the core that is often referred to as the ischemic penumbra. However, many cells, particularly immune cells infiltrate and survive in the core. In this study, we characterized macrophage-like cells that accumulated in the ischemic core of a rat brain whose right middle cerebral artery was transiently occluded for 90 min. At 7 days post-reperfusion, we observed macrophage-like cells expressing CD200, a cell surface glycoprotein belonging to an immunoglobulin superfamily and that elicits suppressive effects on myeloid cells including microglia by interacting with the CD200 receptor (CD200R). RT-PCR and immunoblot analyses revealed the presence of CD200-mRNA and protein in the ischemic core as well as in the contralateral region. As revealed by immunohistochemistry, CD200 is located on the cell membrane of spherical Iba1(+) cells with many cytoplasmic granules. CD200(-)/Iba1(+) macrophage-like cells were also present, which have a more irregular shape than CD200(+)/Iba1(+) cells. CD200 was detected in isolated spherical Iba1(+) macrophage-like cells. Thus, CD200 is expressed in some populations of macrophage-like cells that may be responsible for the suppression of CD200R(+) myeloid cell functions in the ischemic core.

In a transient 90-min middle cerebral artery occlusion (MCAO) model of rats, a large ischemic lesion is formed where macrophage-like cells massively accumulate, many of which express a macrophage marker, Iba1, and an oligodendrocyte progenitor cell marker, NG2 chondroitin sulfate proteoglycan (NG2); therefore, the cells were termed BINCs (Brain Iba1+/NG2+ Cells). A bone marrow transplantation experiment using green-fluorescent protein-transgenic rats showed that BINCs were derived from bone marrow. 5-Fluorouracil (5FU) injection at 2 days post reperfusion (2 dpr) markedly reduced the number of BINCs at 7 dpr, causing enlargement of necrotic volumes and frequent death of the rats. When isolated BINCs were transplanted into 5FU-aggravated ischemic lesion, the volume of the lesion was much reduced. Quantitative real-time RT-PCR showed that BINCs expressed mRNAs encoding bFGF, BMP2, BMP4, BMP7, GDNF, HGF, IGF-1, PDGF-A, and VEGF. In particular, BINCs expressed IGF-1 mRNA at a very high level. Immunohistochemical staining showed that IGF-1-expressing BINCs were found not only in rat but also human ischemic brain lesions. These results suggest that bone marrow-derived BINCs play a beneficial role in ischemic brain lesions, at least in part, through secretion of neuroprotective factors. [ABSTRACT FROM AUTHOR]

Although del Río-Hortega originally reported that leptomeningeal cells are the source of ramified microglia in the developing brain, recent views do not seem to pay much attention to this notion. In this study, in vitro experiments were conducted to determine whether leptomeninges generate ramified microglia. The leptomeninges of neonatal rats containing Iba1+ macrophages were peeled off the brain surface. Leptomeningeal macrophages strongly expressed CD68 and CD163, but microglia in the brain parenchyma did not. Leptomeningeal macrophages expressed epidermal growth factor receptor (EGFR) as revealed by RT-PCR and immunohistochemical staining. Cells obtained from the peeled-off leptomeninges were cultured in a serum-free medium containing EGF, resulting in the formation of large cell aggregates in which many proliferating macrophages were present. In contrast, colony-stimulating factor 1 (CSF1) did not enhance the generation of Iba1+ cells from the leptomeningeal culture. The cell aggregates generated ramified Iba1+ cells in the presence of serum, which express CD68 and CD163 at much lower levels than primary microglia isolated from a mixed glial culture. Therefore, the leptomeningeal-derived cells resembled parenchymal microglia better than primary microglia. This study suggests that microglial progenitors expressing EGFR reside in the leptomeninges and that there is a population of microglia-like cells that grow independently of CSF1. [ABSTRACT FROM AUTHOR]

Abstract: Macrophage-like cells densely accumulate in stab wound-induced brain lesions in rats. Many of these cells express the macrophage marker Iba1 and the oligodendrocyte progenitor cell marker NG2 chondroitin sulfate proteoglycan (NG2), and have been termed BINCs (brain Iba1+/NG2+ cells). Results from our previous study showed that BINCs elicit neuroprotective action, and agents inducing BINC activation or proliferation are expected to ameliorate traumatic brain injuries (TBIs). In the present study, TBI was established by inserting a needle into the cerebrum and moving the needle in a longitudinal, fan-like movement. Isolated BINCs from these stab lesions expressed mRNAs encoding receptors for interleukin-3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF). When this mixture of cytokines was added to the cultured BINCs, expression of mRNAs encoding insulin-like growth factor-1, hepatocyte growth factor, and proliferating cell nuclear antigen increased. The cytokine mixture induced enhanced wound healing in BINCs-brain cell co-cultures in vitro. Stab wounds in the rats resulted in significant brain tissue loss at 2months post-lesion. However, tissue loss was reduced by 40% when the combination of IL-3 and GM-CSF was subcutaneously injected 7 times (once per day) beginning at 2 or 3days post-lesion (dpl). BINCs are highly proliferative and an intraperitoneal injection of 5-fluorouracil (5FU) at 2 dpl eliminated the BINCs, resulting in death of the rats. The cytokine mixture injection significantly reduced mortality of the 5FU-treated rats. These results suggest that the combination of IL-3 and GM-CSF serves as a promising agent to ameliorate TBI via action on BINCs. [Copyright &y& Elsevier]

Abstract: Heparanase, a matrix-degrading enzyme that cleaves heparan sulfate side chains from heparan sulfate proteoglycans (HSPGs), has been shown to facilitate cell invasion, migration, and extravasation of metastatic tumor cells or immune cells. In this study, the expression and functions of heparanase were investigated using rat primary cultured microglia, the resident macrophages in the brain. The microglia were found to express heparanase mRNA and protein. Microglia treated with lipopolysaccharide (LPS) were activated, expressed induced nitric oxide synthase and elevated the expression of heparanase. Heparanase has two molecular weights: a 65 kDa latent form and an active 50 kDa. Both forms were expressed by LPS-treated activated microglia; however, untreated microglia primarily expressed the latent form. Cell lysates from microglia actually degraded Matrigel containing HSPG. Heparanase was colocalized with the actin cytoskeleton in microglial leading edges or ruffled membranes. Microglia transmigrated through a Matrigel-coated pored membrane. This process was inhibited by SF-4, a specific heparanase inhibitor, in a concentration-dependent manner. Degraded HSPG was generated when microglia transmigrated through the coated membrane, and this was also inhibited by SF-4. The results suggest the involvement of heparanase in the migration or invasion of microglia or brain macrophages across basement membrane around brain vasculature. [Copyright &y& Elsevier]