Interactions between brain-resident and peripheral infiltrated immune cells are thought to contribute to neuroplasticity after cerebral ischemia. However, conventional bulk sequencing makes it challenging to depict this complex immune network. Using single-cell RNA sequencing, we mapped compositional and transcriptional features of peri-infarct immune cells. Microglia were the predominant cell type in the peri-infarct region, displaying a more diverse activation pattern than the typical pro- and anti-inflammatory state, with axon tract-associated microglia (ATMs) being associated with neuronal regeneration. Trajectory inference suggested that infiltrated monocyte-derived macrophages (MDMs) exhibited a gradual fate trajectory transition to activated MDMs. Inter-cellular crosstalk between MDMs and microglia orchestrated anti-inflammatory and repair-promoting microglia phenotypes and promoted post-stroke neurogenesis, with SOX2 and related Akt/CREB signaling as the underlying mechanisms. This description of the brain's immune landscape and its relationship with neurogenesis provides new insight into promoting neural repair by regulating neuroinflammatory responses.
Humans ; Ischemic Stroke ; Brain/metabolism* ; Macrophages ; Brain Ischemia/metabolism* ; Microglia/metabolism* ; Gene Expression Profiling ; Anti-Inflammatory Agents ; Neuronal Plasticity/physiology* ; Infarction/metabolism*

Triggering receptor expressed on myeloid cells 2 (TREM2) is a membrane receptor on myeloid cells and plays an important role in the body's immune defense. Recently, TREM2 has received extensive attention from researchers, and its activity has been found in Alzheimer's disease, neuroinflammation, and traumatic brain injury. The appearance of TREM2 is usually accompanied by changes in apolipoprotein E (ApoE), and there has been a lot of research into their structure, as well as the interaction mode and signal pathways involved in them. As two molecules with broad and important roles in the human body, understanding their correlation may provide therapeutic targets for certain diseases. In this article, we reviewed several diseases in which TREM2 and ApoE are synergistically involved in the development. We further discussed the positive or negative effects of the TREM2-ApoE pathway on nervous system immunity and inflammation.
Humans ; Alzheimer Disease/metabolism* ; Apolipoproteins E/genetics* ; Microglia/metabolism* ; Myeloid Cells/metabolism* ; Signal Transduction ; Neuroinflammatory Diseases

This study was aimed to investigate the effect of hypoxia on lipopolysaccharide (LPS)-induced CXC-chemokine ligand-10 (CXCL10) expression and the underlying mechanism. C57BL/6J mice were randomly divided into control, hypoxia, LPS, and hypoxia combined with LPS groups. The LPS group was intraperitoneally injected with 0.5 mg/kg LPS, and the hypoxia group was placed in a hypobaric hypoxia chamber (simulated altitude of 6 000 m). The serum and hippocampal tissue samples were collected after 6 h of the treatment. The levels of CXCL10 in the serum and hippocampal tissue of mice were detected by ELISA. The microglia cell line BV2 and primary microglia were stimulated with hypoxia (1% O₂) and/or LPS (100 ng/mL) for 6 h. The mRNA expression level of CXCL10 and its content in culture supernatant were detected by real-time quantitative PCR and ELISA, respectively. The phosphorylation levels of nuclear factor κB (NF-κB) signaling pathway-related proteins, p65 and IκBα, were detected by Western blot. Moreover, after NF-κB signaling pathway being blocked with a small molecular compound, PDTC, CXCL10 mRNA expression level was detected in the BV2 cells. The results showed that in the LPS-induced mouse inflammatory model, hypoxia treatment could promote LPS-induced up-regulation of CXCL10 in both serum and hippocampus. Compared with the cells treated with LPS alone, the expression of CXCL10 mRNA and the content of CXCL10 in the culture supernatant of BV2 cells treated with hypoxia combined with LPS were significantly increased. The CXCL10 mRNA level of primary microglial cells treated with hypoxia combined with LPS was significantly up-regulated. Compared with the cells treated with hypoxia or LPS alone, the phosphorylation levels of p65 and IκBα in the BV2 cells treated with hypoxia combined with LPS were significantly increased. PDTC blocked the induction of CXCL10 gene expression by LPS in the BV2 cells. These results suggest that hypoxia promotes LPS-induced expression of CXCL10 in both animal and cell models, and NF-κB signaling pathway plays an important role in this process.
Animals ; Mice ; Chemokines, CXC/pharmacology* ; Hypoxia ; Ligands ; Lipopolysaccharides/pharmacology* ; Mice, Inbred C57BL ; Microglia/metabolism* ; NF-kappa B/metabolism* ; NF-KappaB Inhibitor alpha/pharmacology* ; RNA, Messenger/metabolism*

Mechanical allodynia (MA), including punctate and dynamic forms, is a common and debilitating symptom suffered by millions of chronic pain patients. Some peripheral injuries result in the development of bilateral MA, while most injuries usually led to unilateral MA. To date, the control of such laterality remains poorly understood. Here, to study the role of microglia in the control of MA laterality, we used genetic strategies to deplete microglia and tested both dynamic and punctate forms of MA in mice. Surprisingly, the depletion of central microglia did not prevent the induction of bilateral dynamic and punctate MA. Moreover, in dorsal root ganglion-dorsal root-sagittal spinal cord slice preparations we recorded the low-threshold Aβ-fiber stimulation-evoked inputs and outputs of superficial dorsal horn neurons. Consistent with behavioral results, microglial depletion did not prevent the opening of bilateral gates for Aβ pathways in the superficial dorsal horn. This study challenges the role of microglia in the control of MA laterality in mice. Future studies are needed to further understand whether the role of microglia in the control of MA laterality is etiology-or species-specific.
Mice ; Animals ; Hyperalgesia/metabolism* ; Microglia/metabolism* ; Disease Models, Animal ; Spinal Cord/metabolism* ; Spinal Cord Dorsal Horn/metabolism* ; Ganglia, Spinal/metabolism*

Abnormal brain-gut interaction is considered the core pathological mechanism behind the disorders of gut-brain interaction (DGBI), in which the intestinal microbiota plays an important role. Microglia are the "sentinels" of the central nervous system (CNS), which participate in tissue damage caused by traumatic brain injury, resist central infection and participate in neurogenesis, and are involved in the occurrence of various neurological diseases. With in-depth research on DGBI, we could find an interaction between the intestinal microbiota and microglia and that they are jointly involved in the occurrence of DGBI, especially in individuals with comorbidities of mental disorders, such as irritable bowel syndrome (IBS). This bidirectional regulation of microbiota and microglia provides a new direction for the treatment of DGBI. In this review, we focus on the role and underlying mechanism of the interaction between gut microbiota and microglia in DGBI, especially IBS, and the corresponding clinical application prospects and highlight its potential to treat DGBI in individuals with psychiatric comorbidities.
Humans ; Gastrointestinal Microbiome ; Irritable Bowel Syndrome/therapy* ; Microglia ; Brain Diseases ; Brain

Parkinson's disease (PD) is a common neurodegenerative disease in middle-aged and elderly people. In particular, increasing evidence has showed that astrocyte-mediated neuroinflammation is involved in the pathogenesis of PD. As a precious traditional Chinese medicine, bear bile powder (BBP) has a long history of use in clinical practice. It has numerous activities, such as clearing heat, calming the liver wind and anti-inflammation, and also exhibits good therapeutic effect on convulsive epilepsy. However, whether BBP can prevent the development of PD has not been elucidated. Hence, this study was designed to explore the effect and mechanism of BBP on suppressing astrocyte-mediated neuroinflammation in a mouse model of PD. PD-like behavior was induced in the mice by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg·kg^-1) for five days, followed by BBP (50, 100, and 200 mg·kg^-1) treatment daily for ten days. LPS stimulated rat C6 astrocytic cells were used as a cell model of neuroinflammation. THe results indicated that BBP treatment significantly ameliorated dyskinesia, increased the levels of tyrosine hydroxylase (TH) and inhibited astrocyte hyperactivation in the substantia nigra (SN) of PD mice. Furthermore, BBP decreased the protein levels of glial fibrillary acidic protein (GFAP), cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS), and up-regulated the protein levels of takeda G protein-coupled receptor 5 (TGR5) in the SN. Moreover, BBP significantly activated TGR5 in a dose-dependent manner, and decreased the protein levels of GFAP, iNOS and COX2, as well as the mRNA levels of GFAP, iNOS, COX2, interleukin (IL) -1β, IL-6 and tumor necrosis factor-α (TNF-α) in LPS-stimulated C6 cells. Notably, BBP suppressed the phosphorylation of protein kinase B (AKT), inhibitor of NF-κB (IκBα) and nuclear factor-κB (NF-κB) proteins in vivo and in vitro. We also observed that TGR5 inhibitor triamterene attenuated the anti-neuroinflammatory effect of BBP on LPS-stimulated C6 cells. Taken together, BBP alleviates the progression of PD mice by suppressing astrocyte-mediated inflammation via TGR5.
Humans ; Mice ; Rats ; Animals ; Aged ; Middle Aged ; Parkinson Disease/pathology* ; Astrocytes/pathology* ; Powders/therapeutic use* ; Ursidae/metabolism* ; NF-kappa B/metabolism* ; Neuroinflammatory Diseases ; Neurodegenerative Diseases/metabolism* ; Cyclooxygenase 2/metabolism* ; Lipopolysaccharides/pharmacology* ; Bile ; Mice, Inbred C57BL ; Microglia ; Disease Models, Animal

OBJECTIVE: To observe the effects of electro-scalp acupuncture （ESA） on the expression of microglial markers CD206 and CD32, as well as interleukin (IL)-6, IL-1β, and IL-10 in the ischemic cortex of rats with ischemic stroke, and to explore the mechanisms of ESA on alleviating inflammatory damage of ischemic stroke. METHODS: Sixty 7-week-old male SD rats were randomly selected, with 15 rats assigned to a sham surgery group. The remaining rats were treated with suture method to establish rat model of middle cerebral artery occlusion (MCAO). The rats with successful model were randomly divided into a model group, a VitD₃ group, and an ESA group, with 15 rats in each group. In the ESA group, ESA was performed bilaterally at the "top-temporal anterior oblique line" with disperse-dense wave, a frequency of 2 Hz/100 Hz, and an intensity of 1 mA. Each session lasted for 30 min, once daily, for a total of 7 days. The VitD₃ group were treated with intragastric administration of 1,25-dihydroxyvitamin D₃ (1,25-VitD₃) solution (3 ng/100 g), once daily for 7 days. The neurological deficit scores and neurobehavioral scores were assessed before and after the intervention. After the intervention, the brain infarct volume was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Immunofluorescence double staining was performed to detect the protein expression of CD32 and CD206 in the ischemic cortex. Western blot analysis was conducted to measure the protein expression of IL-6, IL-1β, and IL-10 in the ischemic cortex. RESULTS: Compared with the sham surgery group, the model group showed increased neurological deficit scores and neurobehavioral scores (P<0.01), increased brain infarct volume (P<0.01), increased protein expression of CD32, IL-6, and IL-1β in the ischemic cortex (P<0.01), and decreased protein expression of CD206 and IL-10 in the ischemic cortex (P<0.01). Compared with the model group, both the ESA group and the VitD₃ group showed decreased neurological deficit scores and neurobehavioral scores (P<0.01), reduced brain infarct volume (P<0.01), decreased protein expression of CD32, IL-6, and IL-1β in the ischemic cortex (P<0.01), and increased protein expression of CD206 and IL-10 in the ischemic cortex (P<0.01). Compared with the VitD₃ group, the ESA group had lower neurological deficit score (P<0.05), larger brain infarct volume (P< 0.05), and lower protein expression of CD32, CD206, IL-1β, and IL-10 in the ischemic cortex (P<0.01, P<0.05). CONCLUSION ESA could improve neurological function in MCAO rats, and its mechanism may be related to promoting microglial M1-to-M2 polarization and alleviating inflammatory damage.
Male ; Animals ; Rats ; Rats, Sprague-Dawley ; Ischemic Stroke ; Interleukin-10 ; Interleukin-6/genetics* ; Microglia ; Scalp ; Acupuncture Therapy ; Vitamins ; Infarction, Middle Cerebral Artery

Sleep is an extremely important physiological state to maintain human life. Sleep disorders can not only cause anxiety and depression, but also induce multi-system diseases that seriously affect brain function and physical health. The neuroinflammation is a key pathological process after sleep disorders, which can induce a series of nervous system diseases. In recent years, the role of microglia activation in neuroinflammation has been paid more and more attention and become a research hotspot in this field. The imbalance of the central microenvironment after sleep disorders leads to changes in the activation and polarization of microglia, which triggers neuroinflammatory response. The activation and polarization of microglia in the sleep disorders are regulated by multiple signaling pathways and complex molecular mechanisms. This paper summarizes five signaling pathways of microglia activation in central inflammation induced by sleep disorders, including P2X7 receptor (P2X7R), p38MAPK, Toll-like receptor 4 (TLR4)/NF-κB, JAK/STAT, and α7 nicotinic acetylcholine receptor (α7-nAChR) pathways, in order to provide reference for further research and clinical treatment targets selection of sleep disorders.
Humans ; Neuroinflammatory Diseases ; Microglia/metabolism* ; Signal Transduction/physiology* ; NF-kappa B/metabolism* ; Inflammation/metabolism* ; Sleep Wake Disorders/metabolism*

The aim of this study was to investigate the growth characteristics of primarily cultured astrocytes and microglia of different generations and then optimize the method for obtaining primary astrocytes and microglia effectively. Primarily cultured microglia were isolated and purified from the cortices of neonatal mice. The proliferation curve of mixed glia cells was measured by Cell Counting Kit-8 (CCK-8) assay, the proportion of astrocytes and microglia was detected by flow cytometry, and the polarization of the two types of glia cells was identified by immunofluorescence staining. Cell growth results showed that the mixed glia cells of P0 and P1 generation had the best proliferative activity; 97.3% of the high purity microglia could be obtained by mechanical shaking at 170 r/min for 30 min, and there was no significant difference in the morphology of ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia and the proportion of M1 and M2 phenotype among the P0, P1 and P2 generations of microglia isolated by the above methods. Moreover, 95.7 % of the high purity astrocytes could be obtained by astrocyte cell surface antigen-2 (ACSA-2) magnetic beads separation, and there was no significant difference in the morphology of glial fibrillary acidic protein (GFAP) positive astrocyte and the proportion of A1 and A2 phenotype among the P0, P1 and P2 generations of astrocyte isolated by the above methods. Taken together, this study observed the growth characteristics of primarily cultured microglia and astrocyte in vitro, and then proved the best generations for purifying microglia and astrocytes. Finally, we optimized the methods of obtaining microglia and astrocyte, and verified that continuous culture within 2 generations will not affect the functional phenotypes of glia cells. These results provide technical support for studying the molecular mechanism of inflammation-associated diseases in nervous system.
Mice ; Animals ; Astrocytes/metabolism* ; Microglia/metabolism* ; Cell Count ; Flow Cytometry/methods* ; Cell Proliferation ; Cells, Cultured

The effect and mechanism of Heixiaoyao Powder on the polarization of microglia(MG) in APP/PS1 double transgenic mice were explored based on NADPH oxidase 2(NOX2)/reactive oxygen species(ROS)/nuclear factor kappaB(NF-κB) signaling pathway. Fifty 4-month-old male APP/PS1 mice were randomly divided into a model group, an MCC950 group(10 mg·kg~(-1)), and low-, medium-, and high-dose Heixiaoyao Powder groups(6.45, 12.89, and 25.78 g·kg~(-1)). Thirty male C57BL/6J mice of the same age and strain were randomly divided into a blank group, a blank + intragastric intervention group, and a blank + intraperitoneal injection group. Drug intervention lasted 90 days. Morris water maze test was used to detect learning and cognitive ability. Nissl staining and transmission electron microscopy were used to observe the pathological morphology and ultrastructure of hippocampal neurons. Immunofluorescence was used to detect the positive expression of M1-type marker CD16/32~+/Iba-1~+, M2-type marker CD206~+/Iba-1~+ of MG and the expression of hippocampal ROS. The colorimetric method was used to detect the content of malondialdehyde(MDA) and superoxide dismutase(SOD) in the hippocampus. Enzyme linked immunosorbent assay(ELISA) was used to detect the levels of inflammatory factors, including interleukin-6(IL-6), interleukin-8(IL-8), and tumor necrosis factor-α(TNF-α), in the hippocampus. Western blot was used to detect the protein expression of β-amyloid protein(Aβ), Iba-1, CD16/32, CD206, NOX2, NF-κB, p-NF-κB, NF-κB inhibitor alpha(IκBα), and p-IKBα in the hippocampus. The results showed that as compared with the blank group, the model group showed prolonged target quadrant movement distance and escape latency(P<0.01), shortened target quadrant retention time and percentage(P<0.01), disorganized neuronal cells with swelling, nuclear disappearance or bias, reduced number of cells, dissolved or absent Nissl bodies, and a clear area in the cytoplasm, damaged and shrunk cell membrane with abnormal cell morphology, few organelles in the cytoplasm, reduced and swollen mitochondria, increased MG M1-type marker CD16/32~+/Iba-1~+(P<0.01), decreased M2-type marker CD206~+/Iba-1~+(P<0.01), increased ROS activity and MDA content(P<0.01), decreased SOD level(P<0.01), elevated inflammatory factors IL-6, IL-8, and TNF-α(P<0.01), up-regulated protein expression and phosphorylation of Aβ, CD16/32, Iba-1, NOX2, NF-κB, and IKBα(P<0.01), and down-regulated CD206(P<0.01). There was no statistically significant difference between the blank group, the blank + intragastric intervention group, and the blank + intraperitoneal injection group. After the intervention of Heixiaoyao Powder, the Heixiaoyao Powder groups showed shortened target quadrant movement distance and escape latency(P<0.01), prolonged target quadrant retention time and percentage(P<0.01), increased and neatly arranged cells with relieved swelling, increased Nissl bodies, regular cell morphology, and intact cell membrane, relieved swelling of mitochondria, slightly expanded endoplasmic reticulum, decreased CD16/32~+/Iba-1~+(P<0.05 or P<0.01), increased CD206~+/Iba-1~+(P<0.01), decreased ROS activity and MDA content(P<0.01), increased SOD level(P<0.01), decreased content of inflammatory factors IL-6, IL-8, and TNF-α(P<0.01), down-regulated protein expression and phosphorylation of Aβ, CD16/32, Iba-1, NOX2, NF-κB, and IKBα(P<0.01), and up-regulated CD206(P<0.01). In conclusion, Heixiaoyao Powder can alleviate neuronal damage and improve the learning and memory abilities of APP/PS1 mice. The mechanism of action may be related to the inhibition of NOX2/ROS/NF-κB signaling pathway, regulating the polarization of MG, increasing the expression of M2 type, inhibiting the expression of M1 type, and reducing the release of inflammatory factor.
Mice ; Male ; Animals ; NF-kappa B/genetics* ; Microglia ; Reactive Oxygen Species ; Interleukin-8 ; Powders ; Tumor Necrosis Factor-alpha ; Interleukin-6 ; Mice, Inbred C57BL ; Signal Transduction ; Mice, Transgenic ; Superoxide Dismutase