Young microglia restore amyloid plaque clearance of aged microglia

Alzheimer′s disease (AD) is characterized by deposition of amyloid plaques, neurofibrillary tangles, and neuroinflammation. In order to study microglial contribution to amyloid plaque phagocytosis, we developed a novel ex vivo model by co‐culturing organotypic brain slices from up to 20‐month‐old, amyloid‐bearing AD mouse model (APPPS1) and young, neonatal wild‐type (WT) mice. Surprisingly, co‐culturing resulted in proliferation, recruitment, and clustering of old microglial cells around amyloid plaques and clearance of the plaque halo. Depletion of either old or young microglial cells prevented amyloid plaque clearance, indicating a synergistic effect of both populations. Exposing old microglial cells to conditioned media of young microglia or addition of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) was sufficient to induce microglial proliferation and reduce amyloid plaque size. Our data suggest that microglial dysfunction in AD may be reversible and their phagocytic ability can be modulated to limit amyloid accumulation. This novel ex vivo model provides a valuable system for identification, screening, and testing of compounds aimed to therapeutically reinforce microglial phagocytosis.     

Swelling activated Cl- channels in microglia

Microglia have a swelling-activated Cl- current (which we call IClswell), and while some of its biophysical properties and functional roles have been elucidated, its molecular identity is unknown. To relate this current to cell functions and determine whether it is regulated by mechanisms other than cell swelling, it is important to establish both biophysical and pharmacological fingerprints. Here, we used rat microglia and a cell line derived from them (MLS-9) to study biophysical, regulatory and pharmacological properties of IClswell. The whole-cell current was activated in response to a hypo-osmotic bath solution, but not by voltage, and was time-independent during long voltage steps. The halide selectivity sequence was I->Br->Cl- (Eisenman sequence I) and importantly, the excitatory amino acid, glutamate was permeant. Current activation required internal ATP, and was not affected by the guanine nucleotides, GTP?S or GDP?S, or physiological levels of internal Mg2+. The same current was activated by a low intracellular ionic strength solution without an osmotic gradient. IClswell was reversibly inhibited by known Cl- channel blockers (NPPB, flufenamic acid, glibenclamide, DCPIB), and by the glutamate release inhibitor, riluzole. Cell swelling evoked glutamate release from primary microglia and MLS-9 cells, and this was inhibited by the blockers (above), and by IAA-94, but not by tamoxifen or the Na+/K+/Cl- symport inhibitor, bumetanide. Together, these results confirm the similarity of IClswell in the two cell types, and point to a role for this channel in inflammation-mediated glutamate release in the CNS.     

Role of microglia in CNS inflammation

There is increasing confusion about the meaning of the terms inflammation, neuroinflammation, and microglial inflammation. We aim in this review to achieve greater clarity regarding these terms, which are essential for our understanding of the role of microglia in CNS inflammatory conditions. The important concept of sterile inflammation is explained against the backdrop of classical inflammation, and its key differences from what researchers refer to when they use the terms neuroinflammation and microglial inflammation are illustrated. We propose to replace the term "neuroinflammation" with "microglial activation" or "CNS pseudo-inflammation", if microglial activation does not suffice. In addition, we recommend abandoning the terms "microglial inflammation" and "inflamed microglia" because of the lack of a clear concept behind them.     

Kinin receptors in cultured rat microglia

Kinins are produced and act at the site of injury and inflammation in various tissues. They are likely to initiate a particular cascade of inflammatory events, which evokes physiological and pathophysiological responses including an increase in blood flow and plasma leakage. In the central nervous system (CNS), kinins are potent stimulators of the production and release of pro-inflammatory mediators represented by prostanoids and cytotoxins. They are known to induce neural tissue damage. Many of the cytotoxins such as cytokines and free radicals and prostanoids are released from glial cells. Among glial cells, astrocytes and oligodendrocytes are known to possess bradykinin (BK) B(2) receptors that phosphoinositide (PI) turnover and raise intracellular Ca(2+) concentration. The presence of bradykinin receptors in microglia has been of great significance. We recently showed that rat primary microglia express kinin receptors. In resting microglia, B(2) receptors but not B(1) receptors are expressed. When the microglia are activated by bradykinin, B(1) receptors are up-regulated, while B(2) receptors are down-regulated. As observed in other glial cells, electrophysiological measurements suggest that B(2) receptors in phosphoinositide turnover and intracellular Ca(2+) concentration in microglia. Release of cytotoxins is likely consequent upon the activation of BK receptors. Our study provides the first evidence that microglia express functional kinin receptors and suggests that microglia play an important role in CNS inflammatory responses.     

老年大鼠脊髓小胶质细胞分选新方法及功能特征观察*

目的: 建立分离纯化老年大鼠小胶质细胞的改良方法,并初步观察老年大鼠脊髓小胶质细胞的生物学特性。方法: 以年轻SD大鼠(2月龄)为对照组,采用胰酶、胰酶替代物和机械网搓法等不同的制备方法,制备大鼠小胶质细胞的单细胞悬液,通过检测细胞纯度、存活率,观察细胞形态特征,分析细胞的炎性功能特征等,确定老年大鼠(20月龄)小胶质细胞的分离纯化方法,观察老年大鼠脊髓小胶质细胞功能特征。结果: 胰酶消化所得细胞的存活率低(年轻大鼠83%,老年大鼠60%);机械网搓法虽得到的存活率较高(95%),但是细胞获取率最低(年轻大鼠((0.207±0.020)×10⁶,老年大鼠(0.243±0.023)×10⁶);采用胰酶替代物解离、密度梯度离心方法分选出的老年大鼠脊髓小胶质细胞数量多、活性好、存活率高,细胞纯度可达85%以上,我们采用此方法分选纯化不同年龄大鼠脊髓小胶质细胞,与年轻大鼠相比,老年鼠脊髓组织量大,所需消化液多,但消化时间缩短;与年轻大鼠小胶质细胞相比,老年大鼠脊髓小胶质细胞其胞体较大较圆,突起少且粗短,形态上偏向于激活状态,老年大鼠小胶质细胞促炎因子IL-1β表达降低(P＜0.05),而抗炎因子IL-10(P＜0.01)表达升高。结论: 成功建立胰酶替代物解离结合密度梯度离心法从大鼠脊髓组织中分离纯化小胶质细胞,老年大鼠脊髓内小胶质细胞整体表现出抗炎表型。     

Physiological function of microglia

Broad interest in the rapidly advancing field of microglial involvement in forming neural circuits is evident from the fresh findings published in leading journals. This special issue of Neuron Glia Biology contains a special collection of research articles and reviews concerning the new appreciation of microglial function in the normal physiology of the brain that extends beyond their traditionally understood role in pathology.     

Monitoring in vivo function of cortical microglia

Microglia, the innate immune cells of the brain, are becoming increasingly recognized as an important player both in the context of physiological brain function and brain pathology. To fulfill their executive functions microglia can modify their morphology, migrate or move their processes in a directed fashion, and modify the intracellular Ca²⁺ dynamics leading to modifications in gene expression, phagocytosis, release of cytokines and other inflammation markers, etc. Here we describe the recently developed tools enabling in vivo monitoring of morphology and Ca²⁺ signaling of microglia and show how these techniques may be used for examining microglial function in healthy and diseased brain.     

小胶质细胞在阿尔茨海默病中的作用

近年来,小胶质细胞在阿尔茨海默病发生和发展中的作用成为一个新的研究热点.由于它在该病中的作用具有"两面性"的性质,因此如何平衡两方面之间的关系成为摆在研究者面前的一个重要课题.而突破这一课题的关键在于对β淀粉样蛋白与小胶质细胞相互作用机理的研究.本文主要讨论了各种β淀粉样蛋白与小胶质细胞的相互作用和它们的机理.综述了到目前为止为平衡这两方面作用所作的工作,主要是抗炎性药物和细胞因子作用机理的研究.     

Acetate reduces microglia inflammatory signaling in vitro

Acetate supplementation increases brain acetyl‐CoA and histone acetylation and reduces lipopolysaccharide (LPS)‐induced neuroglial activation and interleukin (IL)‐1β expression in vivo. To determine how acetate imparts these properties, we tested the hypothesis that acetate metabolism reduces inflammatory signaling in microglia. To test this, we measured the effect acetate treatment had on cytokine expression, mitogen‐activated protein kinase (MAPK) signaling, histone H3 at lysine 9 acetylation, and alterations of nuclear factor‐kappa B (NF‐κB) in primary and BV‐2 cultured microglia. We found that treatment induced H3K9 hyperacetylation and reversed LPS‐induced H3K9 hypoacetylation similar to that found in vivo. LPS also increased IL‐1β, IL‐6, and tumor necrosis factor‐alpha (TNF‐α) mRNA and protein, whereas treatment returned the protein to control levels and only partially attenuated IL‐6 mRNA. In contrast, treatment increased mRNA levels of transforming growth factor‐β1 (TGF‐β1) and both IL‐4 mRNA and protein. LPS increased p38 MAPK and JNK phosphorylation at 4 and 2–4 h, respectively, whereas treatment reduced p38 MAPK and JNK phosphorylation only at 2 h. In addition, treatment reversed the LPS‐induced elevation of NF‐κB p65 protein and phosphorylation at serine 468 and induced acetylation at lysine 310. These data suggest that acetate metabolism reduces inflammatory signaling and alters histone and non‐histone protein acetylation.     

Lectin staining of sheep microglia

The B₄-isolectin from Griffonia simplicifolia is known to stain microglial cells in a variety of species. The present report describes a lectin staining method that has been modified to facilitate staining of resting microglia, as well as perivascular cells, in vibratome sections of normal sheep brain. This modified method employs tissue fixed in formaldehyde or paraformaldehyde and requires incubating sections with Triton X-100 prior to staining.     

Interleukin-13 enhances cyclooxygenase-2 expression in activated rat brain microglia: implications for death of activated microglia

Brain inflammation has recently attracted widespread interest because it is a risk factor for the onset and progression of brain diseases. In this study, we report that cyclooxygenase-2 (COX-2) plays a key role in the resolution of brain inflammation by inducing the death of microglia. We previously reported that IL-13, an anti-inflammatory cytokine, induced the death of activated microglia. These results revealed that IL-13 significantly enhanced COX-2 expression and production of PGE(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in LPS-treated microglia. Two other anti-inflammatory cytokines, IL-10 and TGF-beta, neither induced microglial death nor enhanced COX-2 expression or PGE(2) or 15d-PGJ(2) production. Therefore, we hypothesized that the effect of IL-13 on COX-2 expression may be linked to death of activated microglia. We found that COX-2 inhibitors (celecoxib and NS398) suppressed the death of microglia induced by a combination of LPS and IL-13 and that exogenous addition of PGE(2) and 15d-PGJ(2) induced microglial death. Agonists of EP2 (butaprost) and peroxisome proliferator-activated receptor gamma (ciglitazone) mimicked the effect of PGE(2) and 15d-PGJ(2), and an EP2 antagonist (AH6809) and a peroxisome proliferator-activated receptor gamma antagonist (GW9662) suppressed microglial death induced by LPS in combination with IL-13. In addition, IL-13 potentiated LPS-induced activation of JNK, and the JNK inhibitor SP600125 suppressed the enhancement of COX-2 expression and attenuated microglial death. Taken together, these results suggest that IL-13 enhanced COX-2 expression in LPS-treated microglia through the enhancement of JNK activation. Furthermore, COX-2 products, PGE(2) and 15d-PGJ(2), caused microglial death, which terminates brain inflammation.     

Thiaminepyrophosphatase activity in the plasma membrane of microglia

An intense thiaminepyrophosphatase (TPPase) activity was demonstrated in glial cells and blood vessels in the central nervous system (CNS), when incubation was carried out with thiaminepyrophosphte (TPP, cocarboxylase), using the method of Novikoff and Goldfischer (1961). Glial cells with TPPSase activity were identified as microglia because they were morphologically similar to microglial cells in the sections stained with silver impregnation. TPPase activity was localized in the microglial perikaryon and in the processes, as viewed under a light microscope. Electron microscopically, enzyme activity was localized in the plasma membrane of microglia. We consider this activity to be a true TPPase activity hydrolyzing TPP, and we then went on to examine the substrate specificity, optimum pH, effect of chemical inhibitors and activators, and the effect of glutaraldehyde fixation. Our data are reported herein.     

Ion channels in microglia (brain macrophages)

Microglia are immunocompetent cells in the brain that have manysimilarities with macrophages of peripheral tissues. In normal adultbrain, microglial cells are in a resting state, but they becomeactivated during inflammation of the central nervous system, afterneuronal injury, and in several neurological diseases. Patch-clamp studies of microglial cells in cell culture and in tissue slices demonstrate that microglia express a wide variety of ion channels. Sixdifferent types of K⁺ channelshave been identified in microglia, namely, inward rectifier, delayedrectifier, HERG-like, G protein-activated, as well as voltage-dependentand voltage-independentCa²⁺-activatedK⁺ channels. Moreover, microgliaexpress H⁺ channels,Na⁺ channels, voltage-gatedCa²⁺ channels,Ca²⁺-release activatedCa²⁺ channels, andvoltage-dependent and voltage-independentCl channels. With respectto their kinetic and pharmacological properties, most microglial ionchannels closely resemble ion channels characterized in othermacrophage preparations. Expression patterns of ion channels inmicroglia depend on the functional state of the cells. Microglial ionchannels can be modulated by exposure to lipopolysaccharide or variouscytokines, by activation of protein kinase C or G proteins, by factorsreleased from astrocytes, by changes in the concentration of internalfree Ca²⁺, and by variations ofthe internal or external pH. There is evidence suggesting that ionchannels in microglia are involved in maintaining the membranepotential and are also involved in proliferation, ramification, and therespiratory burst. Further possible functional roles of microglial ionchannels are discussed.

     

Thiaminepyrophosphatase activity in the plasma membrane of microglia

Summary An intense thiaminepyrophosphatase (TPPase) activity was demonstrated in glial cells and blood vessels in the central nervous system (CNS), when incubation was carried out with thiaminepyrophosphate (TPP, cocarboxylase), using the method of Novikoff and Goldfischer (1961). Glial cells with TPPase activity were identified as microglia because they were morphologically similar to microglial cells in the sections stained with silver impregnation. TPPase activity was localized in the microglial perikaryon and in the processes, as viewed under a light microscope. Electron microscopically, enzyme activity was localized in the plasma membrane of microglia. We consider this activity to be a true TPPase activity hydrolyzing TPP, and we then went on to examine the substrate specificity, optimum pH, effect of chemical inhibitors and activators, and the effect of glutaraldehyde fixation. Our data are reported herein.This work was supported by grant (No. 437002) from the Ministry of Education, Science and Culture, Japan