Microglia In Cortex Research Articles

Linderane Attenuates Complete Freund's Adjuvant-Induced Pain and Anxiety in Mice by Restoring Anterior Cingulate Cortex Microglia M2 Polarization through Activating Cannabinoid 2 Receptor.

Chronic pain is a common condition that causes negative emotions as the disease progresses. The anterior cingulate cortex (ACC) is a key region in the integration of nociceptive perception and emotional response in chronic pain. Linderane (LDR) is an active ingredient from Linderae radix, a traditional Chinese medicine with anti-inflammatory, analgesic, and antibacterial properties. In this study, the analgesic and antianxiety effects of LDR were evaluated using a complete Freund's adjuvant (CFA)-induced inflammatory pain model in C57BL/6 male mice. Mechanical and thermal pain sensitivity were measured through plantar mechanical analgesia and hot plate apparatus, and anxiety-like behavior was evaluated by open field and elevated plus maze tests. The results showed that LDR-alleviated CFA-induced pain and anxiety, reduced the release of inflammatory cytokines, and inhibited ACC microglial activation. Target prediction, molecular docking, and cellular thermal shift assay demonstrated that LDR could bind to the cannabinoid 2 receptor (CB2R), a key component of the endocannabinoid system with an important role in regulating pain and related emotions. Moreover, both the analgesic effect of LDR and its regulation of microglia polarization were reversed by a CB2R antagonist (SR144528) treatment. Therefore, our results suggested that LDR exerted analgesic effects by regulating microglial polarization in ACC via CB2R activation.

Brain microglia serve as a persistent HIV reservoir despite durable antiretroviral therapy.

Brain microglia (MG) may serve as a human immunodeficiency virus 1 (HIV) reservoir and ignite rebound viremia following cessation of antiretroviral therapy (ART), but they have yet to be proven to harbor replication-competent HIV. Here, we isolated brain myeloid cells (BrMCs) from nonhuman primates and rapid autopsy of people with HIV (PWH) on ART and sought evidence of persistent viral infection. BrMCs predominantly displayed microglial markers, in which up to 99.9% of the BrMCs were TMEM119+ MG. Total and integrated SIV or HIV DNA was detectable in the MG, with low levels of cell-associated viral RNA. Provirus in MG was highly sensitive to epigenetic inhibition. Outgrowth virus from parietal cortex MG in an individual with HIV productively infected both MG and PBMCs. This inducible, replication-competent virus and virus from basal ganglia proviral DNA were closely related but highly divergent from variants in peripheral compartments. Phenotyping studies characterized brain-derived virus as macrophage tropic based on the ability of the virus to infect cells expressing low levels of CD4. The lack of genetic diversity in virus from the brain suggests that this macrophage-tropic lineage quickly colonized brain regions. These data demonstrate that MG harbor replication-competent HIV and serve as a persistent reservoir in the brain.

Glycogen synthesis kinase-3β involves in the analgesic effect of liraglutide on diabetic neuropathic pain

AimsExplore whether Glycogen synthesis kinase-3β (GSK3β) involved in the analgesic effect of liraglutide on diabetic neuropathic pain (DNP). MethodsDNP was induced by streptozocin (STZ) in WT and GSK3β(S9A) mice, which carried a constitutively active form of GSK3β. DNP mice were intracerebroventricularly injected with liraglutide 5 weeks after STZ injection. The behavior of neuropathic pain was evaluated 2 h after drugs administration. The microglial activation and the expression of NOD-like receptor protein 3 (NLRP3) in microglia in cortex were evaluated. The role of GSK3β in the inhibitory effect of liraglutide on the NLRP3 inflammasome was explored in BV2 microglia. ResultsIntracerebroventricular administration of liraglutide significantly relieved neuropathic pain and inhibited the activation of cortical microglia in WT mice with DNP. But the effect of liraglutide disappeared in GSK3β(S9A) mice. In BV2 microglia, GSK3β inhibitor significantly suppressed NLRP3 inflammasome activation. And activating GSK3β through GSK3β(S9A) lentivirus significantly blocked the inhibitory effect of liraglutide on NLRP3 inflammasome in BV2 microglia. Intracerebroventricular administration of liraglutide significantly inhibited the expression of NLRP3 in cortex microglia of DNP group in WT mice but failed in GSK3β(S9A) mice. ConclusionGSK3β involves in the analgesic effect of liraglutide on DNP through NLRP3 inflammasome in microglia.

P2X7 receptor-activated microglia in cortex is critical for sleep disorder under neuropathic pain.

Neuropathic pain (NP) is associated with sleep disturbances, which may substantially influence the quality of life. Clinical and animal studies demonstrated that neurotransmitter is one of the main contributors to cause sleep disturbances induced by NP. Recently, it was reported that P2X7 receptors (P2X7R) are widely expressed in microglia, which serves crucial role in neuronal activity in the pain and sleep-awake cycle. In this study, we adopted the chronic constriction injury (CCI) model to establish the progress of chronic pain and investigated whether P2X7R of microglia in cortex played a critical role in sleep disturbance induced by NP. At electroencephalogram (EEG) level, sleep disturbance was observed in mice treated with CCI as they exhibited mechanical and thermal hypersensitivity, and inhibition of P2X7R ameliorated these changes. We showed a dramatic high level of P2X7R and Iba-1 co-expression in the cortical region, and the inhibition of P2X7R also adversely affected it. Furthermore, the power of LFPs in ventral posterior nucleus (VP) and primary somatosensory cortex (S1) which changed in the CCI group was adverse after the inhibition of P2X7R. Furthermore, inhibition of P2X7R also decreased the VP-S1 coherence which increased in CCI group. Nuclear magnetic resonance demonstrated inhibition of P2X7R decreased glutamate (Glu) levels in thalamic and cortical regions which were significantly increased in the CCI mice. Our findings provide evidence that NP has a critical effect on neuronal activity linked to sleep and may built up a new target for the development of sleep disturbances under chronic pain conditions.

Density of TMEM119-positive microglial cells in postmortem cerebrospinal fluid as a surrogate marker for assessing complex neuropathological processes in the CNS

Routine coronal paraffin-sections through the dorsal frontal and parieto-occipital cortex of a total of sixty cases with divergent causes of death were immunohistochemically (IHC) stained with an antibody against TMEM119. Samples of cerebrospinal fluid (CSF) of the same cases were collected by suboccipital needle-puncture, subjected to centrifugation and processed as cytospin preparations stained with TMEM119. Both, cytospin preparations and sections were subjected to computer-assisted density measurements. The density of microglial TMEM119-positive cortical profiles correlated with that of cytospin results and with the density of TMEM119-positive microglial profiles in the medullary layer. There was no statistically significant correlation between the density of medullary TMEM119-positive profiles and the cytospin data. Cortical microglial cells were primarily encountered in supragranular layers I, II, and IIIa and in infragranular layers V and VI, the region of U-fibers and in circumscribed foci or spread in a diffuse manner and high density over the white matter. We have evidence that cortical microglia directly migrate into CSF without using the glympathic pathway. Microglia in the medullary layer shows a strong affinity to the adventitia of deep vessels in the myelin layer. Selected rapidly fatal cases including myocardial infarcts and drowning let us conclude that microglia in cortex and myelin layer can react rapidly and its reaction and migration is subject to pre-existing external and internal factors. Cytospin preparations proved to be a simple tool to analyze and assess complex changes in the CNS after rapid fatal damage. There is no statistically significant correlation between cytospin and postmortem interval. Therefore, the quantitative analyses of postmortem cytospins obviously reflect the neuropathology of the complete central nervous system. Cytospins provide forensic pathologists a rather simple and easy to perform method for the global assessment of CNS affliction.

Brain-specific TRAF7 deletion ameliorates traumatic brain injury by suppressing MEKK3-regulated glial inflammation and neuronal death

Neuronal death and neuroinflammation play critical roles in regulating the progression of traumatic brain injury (TBI). However, associated pathogenesis has not been fully understood. Tumor necrosis factor receptor-associated factor 7 (TRAF7), as the unique noncanonical member of the TRAF family, mediates various essential biological processes. Nevertheless, the effects of TRAF7 on TBI are still unclear. In this study, we showed that TRAF7 expression was markedly up-regulated in cortex and hippocampus of mice after TBI. Brain-specific TRAF7 deletion markedly ameliorated neuronal death in cortical and hippocampal samples of TBI mice, accompanied with cognitive impairments and motor dysfunction. Moreover, the aberrant activation of astrocyte and microglia in cortex and hippocampus of TBI mice was significantly restrained by TRAF7 conditional knockout in brain, as indicated by the increased expression of GFAP and Iba1. In addition, the releases of pro-inflammatory factors caused by TBI were also considerably diminished by brain-specific TRAF7 knockout, which were largely through the blockage of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways. Importantly, mitogen-activated protein kinase kinase kinase 3 (MEKK3) expression levels were greatly enhanced in cortex and hippocampus of mice with TBI, while being dramatically ameliorated by TRAF7 knockout in brain. Mechanistically, we showed that TRAF7 directly interacted with MEKK3. Of note, MEKK3 over-expression almost abrogated the capacity of TRAF7 knockout to mitigate neuronal death and neuroinflammation in the isolated primary cortical neurons and glial cells upon oxygen-glucose-deprivation/reperfusion (OGD/R) stimulation. Collectively, TRAF7 may be an important molecular switch that leads to TBI in a MEKK3-dependent manner, and can be served as a therapeutic target for TBI treatment.

Delivery of Periodontopathogenic Extracellular Vesicles to Brain Monocytes and Microglial IL-6 Promotion by RNA Cargo

Gram-negative bacterial extracellular vesicles (EVs), also known as outer membrane vesicles (OMVs), are secreted from bacterial cells and have attracted research attention due to their role in cell-to-cell communication. During OMV secretion, a variety of cargo such as extracellular RNA (exRNA) is loaded into the OMV. The involvement of exRNAs from a range of bacteria has been identified in several diseases, however, their mechanism of action has not been elucidated. We have recently demonstrated that OMVs secreted by the periodontopathogen Aggregatibacter actinomycetemcomitans can cross the blood–brain barrier (BBB) and that its exRNA cargo could promote the secretion of proinflammatory cytokines in the brain. However, it was unclear whether the brain immune cells could actually take up bacterial OMVs, which originate outside of the brain, in an appropriate immune response. In the present study, using monocyte-specific live CX3CR1-GFP mice, we visualized OMV-colocalized meningeal macrophages and microglial cells into which bacterial OMVs had been loaded and intravenously injected through tail veins. Our results suggested that meningeal macrophages uptake BBB-crossed OMVs earlier than do cortex microglia. BV2 cells (a murine microglia cell line) and exRNAs were also visualized after OMV treatment and their proinflammatory cytokine levels were observed. Interleukin (IL)-6 and NF-κB of BV2 cells were activated by A. actinomycetemcomitans exRNAs but not by OMV DNA cargo. Altogether, these findings indicate that OMVs can successfully deliver exRNAs into brain monocyte/microglial cells and cause neuroinflammation, implicating a novel pathogenic mechanism in neuroinflammatory diseases.

Impaired Autophagy Flux is Associated with Proinflammatory Microglia Activation Following Japanese Encephalitis Virus Infection.

Role of autophagy in Japanese encephalitis viral (JEV) infection is not well known. In the present study, we reported the role of autophagy flux in microglia activation, neurobehavioral function and neuronal death using a mouse model of JEV. Markers for autophagy (LC3-II/I, SQSTM1/P62, phos-Akt, phos-AMPK), and neuronal death (cleaved caspase 12, H2Ax, polyubiquitin) were investigated by western blot at 1, 3 and 7days post inoculation. Cathepsin D was measured in cerebral cotex of JEV infected mice spectrophotometrically. Microglia activation and pro-inflammatory cytokines (IL1β, TNF-α, IFNγ, IL6) were measured by immunohistochemistry, western blot and qPCR analysis. In order to determine the neuroinflammatory changes and autophagy mediated neuronal cell death, BV2-microgliaand N2a-neuronal cells were used. Autophagy activation marker LC3-II/I and its substrate SQSTM1/P62 were significantly increased while cathepsin D activity was decreased on day 7 post inoculation in cerebral cortex. Microglia in cortex were activated and showed higher expression of proinflammatory mRNA of IL1β, TNF-α, IFNγ and IL6, with increased DNA damage (H2AX) and neuronal cell death pathways in hippocampus and neurobehavioral dysfunction. Similar observations on JEV infection mediated autophagy flux inhibition and neuronal cell death was found in N2a neuronal cell. Collectively, our study provides evidence on the role of autophagy regulation, microglial activation and neurodegeneration following JEV infection.

Increased EZH2 Levels in Anterior Cingulate Cortex Microglia Aggravate Neuropathic Pain by Inhibiting Autophagy Following Brachial Plexus Avulsion in Rats.

After brachial plexus avulsion (BPA), microglia induce inflammation, initiating and maintaining neuropathic pain. EZH2 (enhancer of zeste homolog 2) has been implicated in inflammation and neuropathic pain, but the mechanisms by which it regulates neuropathic pain remain unclear. Here, we found that EZH2 levels were markedly upregulated during BPA-induced neuropathic pain in vivo and in vitro, stimulating pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) secretion in vivo. In rats with BPA-induced neuropathic pain, mechanical and cold hypersensitivities were induced by EZH2 upregulation and inhibited by EZH2 downregulation in the anterior cingulate cortex. Microglial autophagy was also significantly inhibited, with EZH2 inhibition activating autophagy and reducing neuroinflammation in vivo. However, this effect was impaired by inhibiting autophagy with 3-methyladenine, suggesting that the MTOR signaling pathway is a functional target of EZH2. These data suggest that EZH2 regulates neuroinflammation and neuropathic pain via a novel MTOR-mediated autophagy signaling pathway, providing a promising approach for managing neuropathic pain.

Aβ-HBc virus-like particles immunization without additional adjuvant ameliorates the learning and memory and reduces Aβ deposit in PDAPP mice

β-Amyloid peptide (Aβ) immunization is regarded as a promising therapy to Alzheimer's disease. The full length Aβ as antigen might induce meningoencephalitis adverse effect since the middle and C-terminal fragments of Aβ contain T cell epitopes. While N-terminal fragment of Aβ, containing B cell epitope, has weak or no immunogenicity. To improve the immunogenicity, in the previous study, we used HBv core antigen as carrier to make fusion protein containing 2 Aβ1–15. The fusion protein could form virus-like particles (VLPs) and had strong immunogenicity. The antisera prevented Aβ fiber formation and protected the PC12 cells against toxicity of Aβ. In the present study, we immunized 12-month old AD transgenic mice, PDAPP mice, to observe the therapeutic effect of immunization on behaviour and pathology. During immunization, the titer of anti-Aβ antibody reached to nearly 1:106 after 4th inoculation, and then maintained that level to the end of the experiment. After 6-month immunization, the behavioral changes of mice were tested by Morris Water Maze (MWM). The escape latency of immunized mice was shorter than control, and these mice entered platform quadrant more times. Immunohistochemistry results showed that Aβ-HBc VLPs immunized mice had less amyloid deposit with less microglia in cortex and hippocampus. In conclusion, Aβ-HBc VLPs ameliorated the learning and memory and reduced cerebral Aβ deposit in PDAPP mice.

A single mild fluid percussion injury induces short-term behavioral and neuropathological changes in the Long–Evans rat: Support for an animal model of concussion

Brain concussion is a serious public health concern and is associated with short-term cognitive impairments and behavioral disturbances that typically occur in the absence of significant brain damage. The current study addresses the need to better understand the effects of a mild lateral fluid percussion injury on rat behavior and neuropathology in an animal model of concussion. Male Long–Evans rats received either a single mild fluid percussion injury or a sham-injury, and either a short (24 h) or long (4 weeks) post-injury recovery period. After recovery, rats underwent a detailed behavioral analysis consisting of tests for rodent anxiety, cognition, social behavior, sensorimotor function, and depression-like behavior. After testing all rats were sacrificed and brains were examined immunohistochemically with markers for microglia/macrophage activation, reactive astrocytosis, and axonal injury. Injured rats (mean injury force: 1.20 ± .03 atm) displayed significant short-term cognitive impairments in the water maze and significantly more anxiolytic-like behavior in the elevated-plus maze compared to sham controls. Neuropathological analysis of the brains of injured rats showed an acute increase in reactive astrogliosis and activated microglia in cortex and evidence of axonal injury in the corpus callosum. There were no significant long-term effects on any behavioral or neuropathological measure 4 weeks after injury. These short-term behavioral and neuropathological changes are consistent with findings in human patients suffering a brain concussion, and provide further evidence for the use of a single mild lateral fluid percussion injury to study concussion in the rat.

Cell type-specific activation of p38 MAPK in the brain regions of hypoxic preconditioned mice

Activation of p38 mitogen-activated protein kinase (p38 MAPK) has been implicated as a mechanism of ischemia/hypoxia-induced cerebral injury. The current study was designed to explore the involvement of p38 MAPK in the development of cerebral hypoxic preconditioning (HPC) by observing the changes in dual phosphorylation (p-p38 MAPK) at threonine180 and tyrosine182 sites, protein expression, and cellular distribution of p-p38 MAPK in the brain of HPC mice. We found that the p-p38 MAPK levels, not protein expression, increased significantly ( p < 0.05) in the regions of frontal cortex, hippocampus, and hypothalamus of mice in response to repetitive hypoxic exposure (H1–H6, n = 6 for each group) when compared to values of the control normoxic group (H0, n = 6) using Western blot analysis. Similar results were also confirmed by an immunostaining study of the p-p38 MAPK location in the frontal cortex, hippocampus, and hypothalamus of mice from HPC groups. To further define the cell type of p-p38 MAPK positive cells, we used a double-labeled immunofluorescent staining method to co-localize p-p38 MAPK with neurofilaments heavy chain (NF-H, neuron-specific marker), S100 (astrocyte-specific marker), and CD11b (microglia-specific maker), respectively. We found that the increased p-p38 MAPK occurred in microglia of cortex and hippocampus, as well as in neurons of hypothalamus of HPC mice. These results suggest that the cell type-specific activation of p38 MAPK in the specific brain regions might contribute to the development of cerebral HPC mechanism in mice.

Central Nervous System-directed AAV2/5-Mediated Gene Therapy Synergizes with Bone Marrow Transplantation in the Murine Model of Globoid-cell Leukodystrophy

Globoid-cell leukodystrophy (GLD) is a rapidly progressing inherited neurodegenerative disorder caused by a deficiency in galactosylceramidase activity. Previous studies in the murine model of GLD (Twitcher mouse) have shown that both bone marrow transplantation (BMT) and central nervous system (CNS)-directed gene therapy can be moderately effective at ameliorating certain aspects of GLD. As BMT and CNS-directed gene therapy target fundamentally different tissues, we tested the hypothesis that combining these disparate therapies would be more efficacious than either therapy alone. Mice receiving myeloreductive conditioning at birth followed by syngeneic BMT had approximately 25-35% donor chimerism. Untreated Twitcher mice, Twitcher mice treated with BMT alone, AAV2/5 alone, or a combination of BMT and AAV2/5 had mean lifespans of 39, 44, 49, and 104 days, respectively. Twitcher mice treated with a combination of BMT and AAV2/5 also had significantly improved performance in several behavioral tests and greater reduction in demyelination, astrocytosis, and macrophage infiltration compared to untreated Twitcher mice or mice that received either therapy alone. These data suggest that CNS-directed gene therapy synergizes with BMT. The combination of these disparate therapeutic approaches may form the basis for more effective treatment of this inherited neurodegenerative disorder.

Activated microglia in cortex of mouse models of mucopolysaccharidoses I and IIIB.

Alpha-N-acetylglucosaminidase deficiency (mucopolysaccharidosis IIIB, MPS IIIB) and alpha-l-iduronidase deficiency (MPS I) are heritable lysosomal storage diseases; neurodegeneration is prominent in MPS IIIB and in severe cases of MPS I. We have obtained morphologic and molecular evidence for the involvement of microglia in brain pathology of mouse models of the two diseases. In the cortex, a subset of microglia (sometimes perineuronal) consists of cells that are probably phagocytic; they have large storage vacuoles, react with MOMA-2 (monoclonal antibody against macrophages) and Griffonia simplicifolia isolectin IB(4), and stain intensely for the lysosomal proteins Lamp-1, Lamp-2, and cathepsin D as well as for G(M3) ganglioside. MOMA-2-positive cells appear at 1 and 6 months in MPS IIIB and MPS I mice, respectively, but though their number increases with age, they remain sparse. However, a profusion of cells carrying the macrophage CD68/macrosialin antigen appear in the cortex of both mouse models at 1 month. mRNA encoding CD68/macrosialin also increases at that time, as shown by microarray and Northern blot analyses. Ten other transcripts elevated in both mouse models are associated with macrophage functions, including complement C4, the three subunits of complement C1q, lysozyme M, cathepsins S and Z, cytochrome b558 small subunit, macrophage-specific protein 1, and DAP12. An increase in IFN-gamma and IFN-gamma receptor was observed by immunohistochemistry. These functional increases may represent activation of resident microglia, an influx and activation of blood monocytes, or both. They show an inflammatory component of brain disease in the two MPS, as is known for many neurodegenerative disorders.