Hisaaki Takahashi

Activated microglia in a rat stroke model express NG2 proteoglycan in peri-infarct tissue through the involvement of TGF-β1.

We investigated activated microglia in ischemic brain lesions from rats that had been subjected to transient middle cerebral artery occlusion. Activated microglia expressing NG2 chondroitin sulfate proteoglycan (NG2) were found only in the narrow zone (demarcation zone) that demarcated the peri‐infarct tissue and ischemic core. NG2− activated microglia were abundantly distributed in the peri‐infarct tissue outside the demarcation zone. NG2+ microglia but not NG2− microglia expressed both CD68 and a triggering receptor expressed on myeloid cells 2 (TREM‐2), suggesting that NG2+ microglia eliminated apoptotic neurons. In fact, NG2+ microglia often attached to degenerating neurons and sometimes internalized NeuN+ or neurofilament protein+ material. Kinetic studies using quantitative real‐time RT‐PCR revealed that expression of transforming growth factor‐β1 (TGF‐β1) was most evident in the ischemic core; with this marker produced mainly by macrophages located in this region. TGF‐β receptor mRNA expression peaked at 3 days post reperfusion (dpr) in the peri‐infarct tissue, including the demarcation zone. Primary cultured rat microglia also expressed the receptor mRNA. In response to TGF‐β1, primary microglia enhanced the expression of NG2 protein and TREM‐2 mRNA as well as migratory activity. A TGF‐β1 inhibitor, SB525334, abolished these effects. The present results suggest that TGF‐β1 produced in the ischemic core diffused toward the peri‐infarct tissue, driving activated microglial cells to eliminate degenerating neurons. Appropriate control of NG2+ microglia in the demarcation zone might be a novel target for the suppression of secondary neurodegeneration in the peri‐infarct tissue. GLIA 2014;62:185–198

Expression of MCP-1 and fractalkine on endothelial cells and astrocytes may contribute to the invasion and migration of brain macrophages in ischemic rat brain lesions.

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.

Oct-3/4 promotes migration and invasion of glioblastoma cells

As a result of increased glioblastoma migration and invasion into normal brain parenchyma, treatment of local tumor recurrence following initial treatment in glioblastoma patients remains challenging. Recent studies have demonstrated increased Oct‐3/4 expression, a self‐renewal regulator in stem cells, in glioblastomas. However, little is known regarding the influence of Oct‐3/4 in glioblastoma cell invasiveness. The present study established Oct‐3/4‐overexpressing glioblastoma cells, which were prepared from human glioblastoma patients, to assess migration, invasion, and mRNA expression profiles of integrins and matrix metalloproteinases (MMPs). Compared with control cells, Oct‐3/4 expressing‐glioblastoma cells exhibited increased migration and invasion in wound healing and Matrigel invasion assays. Oct‐3/4 overexpression resulted in upregulated FAK and c‐Src expression, which mediate integrin signals. Vinculin accumulated along the leading edges of Oct‐3/4 expressing‐glioblastoma cells and associated with membrane ruffles during cell migration. Oct‐3/4 expressing‐cells exhibited increased MMP‐13 mRNA expression and MMP‐13 knockdown by shRNA suppressed cell invasion into Matrigel and organotypic brain slices. These results suggested that Oct‐3/4 enhanced degradation of surrounding extracellular matrix by increasing MMP‐13 expression and altering integrin signaling. Therefore, Oct‐3/4 might contribute to tumor promoting activity in glioblastomas. J. Cell. Biochem. 113: 508–517, 2012.

A cytokine mixture of GM‐CSF and IL‐3 that induces a neuroprotective phenotype of microglia leading to amelioration of (6‐OHDA)‐induced Parkinsonism of rats

Dopamine (DA) agonists are widely used as primary treatments for Parkinsons disease. However, they do not prevent progressive degeneration of dopaminergic neurons, the central pathology of the disease. In this study, we found that subcutaneous injection of a cytokine mixture containing granulocyte macrophage colony‐stimulating factor and interleukin‐3 (IL‐3) markedly suppressed dopaminergic neurodegeneration in 6‐hydroxydopamine‐lesioned rats, an animal model of Parkinsons disease. The cytokine mixture suppressed the decrease of DA content in the striatum, and ameliorated motor function in the lesioned rats. In response to the cytokine injection, dopaminergic neurons in the substantia nigra pars compacta increased expression of the antiapoptotic protein Bcl‐xL. Microglial activation in the pars compacta was evident in both the saline‐ and cytokine‐injected rats. However, the cytokine mixture suppressed expression of the proinflammatory cytokines IL‐1β and tumor necrosis factors α, and upregulated the neuroprotective factors insulin‐like growth factor‐1 and hepatocyte growth factor. Similar responses were observed in cultured microglia. Detailed morphometric analyses revealed that NG2 proteoglycan‐expressing glial cells increased in the cytokine‐injected rats, while astrocytic activation with increased expression of antioxidative factors was evident only in the saline‐injected rats. Thus, the present findings show that the cytokine mixture was markedly effective in suppressing neurodegeneration. Its neuroprotective effects may be mediated by increased expression of Bcl‐xL in dopaminergic neurons, and the activation of beneficial actions of microglia that promote neuronal survival. Furthermore, this cytokine mixture may have indirect actions on NG2 proteoglycan‐expressing glia, whose role may be implicated in neuronal survival.

Subcutaneous injection containing IL-3 and GM-CSF ameliorates stab wound-induced brain injury in rats.

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 2 months 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 3 days 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.

Transient ischemia-induced paresis and complete paraplegia displayed distinct reactions of microglia and macrophages

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.

CD200+ and CD200− macrophages accumulated in ischemic lesions of rat brain: The two populations cannot be classified as either M1 or M2 macrophages

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.

Anticonvulsive effect of paeoniflorin on experimental febrile seizures in immature rats: possible application for febrile seizures in children.

Febrile seizures (FS) is the most common convulsive disorder in children, but there have been no clinical and experimental studies of the possible treatment of FS with herbal medicines, which are widely used in Asian countries. Paeoniflorin (PF) is a major bioactive component of Radix Paeoniae alba, and PF-containing herbal medicines have been used for neuromuscular, neuropsychiatric, and neurodegenerative disorders. In this study, we analyzed the anticonvulsive effect of PF and Keishikashakuyaku-to (KS; a PF-containing herbal medicine) for hyperthermia-induced seizures in immature rats as a model of human FS. When immature (P5) male rats were administered PF or KS for 10 days, hyperthermia-induced seizures were significantly suppressed compared to control rats. In cultured hippocampal neurons, PF suppressed glutamate-induced elevation of intracellular Ca2+ ([Ca2+]i), glutamate receptor-mediated membrane depolarization, and glutamate-induced neuronal death. In addition, PF partially suppressed the elevation in [Ca2+]i induced by activation of the metabotropic glutamate receptor 5 (mGluR5), but not that mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors. However, PF did not affect production or release of γ-aminobutyric acid (GABA) in hippocampal neurons. These results suggest that PF or PF-containing herbal medicines exert anticonvulsive effects at least in part by preventing mGluR5-dependent [Ca2+]i elevations. Thus, it could be a possible candidate for the treatment of FS in children.

HGF–Met Pathway in Regeneration and Drug Discovery

Hepatocyte growth factor (HGF) is composed of an α-chain and a β-chain, and these chains contain four kringle domains and a serine protease-like structure, respectively. Activation of the HGF–Met pathway evokes dynamic biological responses that support morphogenesis (e.g., epithelial tubulogenesis), regeneration, and the survival of cells and tissues. Characterizations of conditional Met knockout mice have indicated that the HGF–Met pathway plays important roles in regeneration, protection, and homeostasis in various cells and tissues, which includes hepatocytes, renal tubular cells, and neurons. Preclinical studies designed to address the therapeutic significance of HGF have been performed on injury/disease models, including acute tissue injury, chronic fibrosis, and cardiovascular and neurodegenerative diseases. The promotion of cell growth, survival, migration, and morphogenesis that is associated with extracellular matrix proteolysis are the biological activities that underlie the therapeutic actions of HGF. Recombinant HGF protein and the expression vectors for HGF are biological drug candidates for the treatment of patients with diseases and injuries that are associated with impaired tissue function. The intravenous/systemic administration of recombinant HGF protein has been well tolerated in phase I/II clinical trials. The phase-I and phase-I/II clinical trials of the intrathecal administration of HGF protein for the treatment of patients with amyotrophic lateral sclerosis and spinal cord injury, respectively, are ongoing.

Anti-inflammatory effects of noradrenaline on LPS-treated microglial cells: Suppression of NFκB nuclear translocation and subsequent STAT1 phosphorylation.

Noradrenaline (NA) has marked anti-inflammatory effects on activated microglial cells. The present study was conducted to elucidate the mechanisms underlying the NA effects using rat primary cultured microglial cells. NA, an α1 agonist, phenylephrine (Phe) and a β2 agonist, terbutaline (Ter) suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) release by microglia and prevented neuronal degeneration in LPS-treated neuron-microglia coculture. The agents suppressed expression of mRNA encoding proinflammatory mediators. Both an α1-selective blocker terazocine and a β2-selective blocker butoxamine overcame the suppressive effects of NA. cAMP-dependent kinase (PKA) inhibitors did not abolish the suppressive NA effects. LPS decreased IκB leading to NFκB translocation into nuclei, then induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) and expression of interferon regulatory factor 1 (IRF1). NA inhibited LPS-induced these changes. When NFκB expression was knocked down with siRNA, LPS-induced STAT1 phosphorylation and IRF1 expression was abolished. NA did not suppress IL-6 induced STAT1 phosphorylation and IRF1 expression. These results suggest that one of the critical mechanisms underlying the anti-inflammatory effects of NA is the inhibition of NFκB translocation. Although inhibitory effects of NA on STAT1 phosphorylation and IRF1 expression may contribute to the overall suppressive effects of NA, these may be the downstream events of inhibitory effects on NFκB. Since NA, Phe and Ter exerted almost the same effects and PKA inhibitors did not show significant antagonistic effects, the suppression by NA might not be dependent on specific adrenergic receptors and cAMP-dependent signaling pathway.