OBJECTIVES: Pain sensitization, as a core feature of neuropathic pain (NP), is closely associated with inflammatory imbalance within the central nervous system. To investigate the effects of intrathecal injection of noggin (NOG) on mechanical hypersensitivity, microglial (MG) activation and polarization, and iron metabolism in a spinal nerve ligation (SNL)-induced rat model of NP, and to explore the role of signal transducer and activator of transcription 3 (STAT3) in MG phenotypic transformation. METHODS: Sixty-six Sprague-Dawley (SD) rats were randomly divided into 3 groups: Sham, SNL, and SNL+NOG. Paw withdrawal threshold (PWT) was assessed using von Frey filaments. Western blotting and real-time polymerase chain reaction (RT-PCR) were used to detect spinal cord expression of MG activation marker CD11b, STAT3, phosphorylated STAT3 (p-STAT3), M1 polarization markers [CD86, CD32, interleukin (IL)-1β], tumor necrosis factor-alpha (TNF-α), and CC chemokine receptor 2 (CCR2), M2 markers [CD204, CD163, CX3C chemokine receptor 1 (CX3CR1), IL-10, and arginase-1 (ARG-1)], and iron metabolism-related proteins including ferroportin (FPN, gene: SLC40A1), hepcidin (gene: HAMP), transferrin receptor (gene: TFRC), and divalent metal transporter 1 (DMT-1, gene: SLC11A2). p-STAT3 localization in MGs was visualized via immunofluorescence. In vitro, primary MGs were divided into Control, bone morphogenetic protein-4 (BMP4), and BMP4+Stattic (STAT3 inhibitor) groups to examine the effects of STAT3 inhibition on MG activation, polarization, and iron regulation. RESULTS: In vivo, compared with the Sham group, the SNL and SNL+NOG groups exhibited significantly decreased PWT (P<0.05), elevated spinal CD11b and p-STAT3 protein levels (all P<0.05), increased M1 markers (CD86, CD32, IL-1β, TNF-α, and CCR2) (all P<0.05), and decreased M2 markers (CD204 protein; mRNA of CD204, ARG-1) (all P<0.05). Hepcidin protein and mRNA levels of HAMP, SLC11A2, and TFRC were significantly elevated, while FPN protein and SLC40A1 mRNA were reduced (all P<0.05). Compared to SNL alone, the SNL+NOG group showed increased PWT, decreased CD11b, p-STAT3, and M1 marker expression (except TNF-α), increased M2 marker expression, reduced hepcidin and HAMP levels, and increased FPN and SLC40A1 expression (all P<0.05). In vitro, BMP4 treatment increased CD11b, STAT3, p-STAT3, CD86, and hepcidin levels, while reducing CD204 and FPN (all P<0.05). Inhibition STAT3 with Stattic reversed these changes (all P<0.05). CONCLUSIONS NOG alleviates SNL-induced NP by antagonizing the STAT3 signaling pathway, thereby rebalancing microglial polarization and restoring iron metabolism.
Animals ; Neuralgia/drug therapy* ; Rats, Sprague-Dawley ; Microglia/cytology* ; STAT3 Transcription Factor/metabolism* ; Rats ; Iron/metabolism* ; Male ; Signal Transduction/drug effects* ; Carrier Proteins/therapeutic use* ; Homeostasis/drug effects* ; Spinal Cord/metabolism*

OBJECTIVE: : To determine the effects of cysteinyl leukotriene receptors (CysLTR and CysLTR) on phagocytosis of mouse BV2 microglial cells. METHODS: : BV2 cells were stimulated with microglial activators lipopolysaccharide (LPS) or CysLT receptor agonists LTD. The phagocytosis of BV2 cells was observed by immunofluorescence analysis and flow cytometry. The intracellular distributions of CysLTR and CysLTR in BV2 cells were examined with immunofluorescence staining. RESULTS: : Both LPS and LTD could significantly enhance the phagocytosis of BV2 cells, and such effect could be inhibited by CysLTR selective antagonist Montelukast and CysLTR selective antagonist HAMI 3379. The activation of BV2 cells induced by LTD or LPS resulted in changes in intracellular distributions of CysLTR and CysLTR. CysLTR and CysLTR was co-localization with a similar distribution. CONCLUSIONS : CysLTR and CysLTR regulate the phagocytosis of mouse BV2 microglial cells with a synergistic effect.
Acetates ; pharmacology ; Animals ; Cell Line ; Cyclohexanecarboxylic Acids ; pharmacology ; Lipopolysaccharides ; pharmacology ; Mice ; Microglia ; cytology ; Phagocytosis ; drug effects ; Phthalic Acids ; pharmacology ; Protein Binding ; drug effects ; Quinolines ; pharmacology ; Receptors, Leukotriene ; agonists ; metabolism

The possible role of minocycline in microglial activation and neuronal death after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) in mice was investigated in this study. The mice were given potassium chloride to stop the heart beating for 8 min to achieve CA, and they were subsequently resuscitated with epinephrine and chest compressions. Forty adult C57BL/6 male mice were divided into 4 groups (n=10 each): sham-operated group, CA/CPR group, CA/CPR+minocycline group, and CA/CPR+vehicle group. Animals in the latter two groups were intraperitoneally injected with minocycline (50 mg/kg) or vehicle (normal saline) 30 min after recovery of spontaneous circulation (ROSC). Twenty-four h after CA/CPR, the brains were removed for histological evaluation of the hippocampus. Microglial activation was evaluated by detecting the expression of ionized calcium-binding adapter molecule-1 (Iba1) by immunohistochemistry. Neuronal death was analyzed by hematoxylin and eosin (H&E) staining and the levels of tumor necrosis factor-alpha (TNF-α) in the hippocampus were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that the neuronal death was aggravated, most microglia were activated and TNF-α levels were enhanced in the hippocampus CA1 region of mice subjected to CA/CPR as compared with those in the sham-operated group (P<0.05). Administration with minocycline 30 min after ROSC could significantly decrease the microglial response, TNF-α levels and neuronal death (P<0.05). It was concluded that early administration with minocycline has a strong therapeutic potential for CA/CPR-induced brain injury.
Animals ; Cardiopulmonary Resuscitation ; Cell Death ; drug effects ; Enzyme-Linked Immunosorbent Assay ; Heart Arrest ; pathology ; Hippocampus ; cytology ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Microglia ; cytology ; drug effects ; Minocycline ; pharmacology ; Neurons ; drug effects ; Tumor Necrosis Factor-alpha ; metabolism

Animals ; Animals, Newborn ; Cells, Cultured ; Corticotropin-Releasing Hormone ; pharmacology ; Interleukin-6 ; metabolism ; Microglia ; cytology ; drug effects ; metabolism ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; metabolism

Microglia are the principal immune effectors in brain and participate in a series ofneurodegenerative diseases. The microglial shapes are highly plastic. The morphology is closely related with their activation status and biological functions. Cerebral ischemia could induce microglial activation, and microglial activation is subjected to precise regulation. Microglia could play either protective or neurotoxic roles in cerebral ischemia. Therefore, regulating the expression of receptors or protein molecules on microglia, inhibiting the excessive activation of microglia and production of pro-inflammatory factors, promoting the release of neuroprotective substances might be beneficial to the treatment of cerebral ischemia. The study about relationship between microglia and cerebral ischemia will shed a light on the treatment of cerebral ischemia. This paper is a review of microglial activation and regulation during cerebral ischemia as well as related therapeutic methods.
Animals ; Brain Ischemia ; metabolism ; pathology ; Class Ib Phosphatidylinositol 3-Kinase ; metabolism ; Humans ; Inflammation ; metabolism ; Microglia ; cytology ; drug effects ; metabolism ; physiology ; Neuroprotective Agents ; pharmacology ; Nitric Oxide Synthase ; metabolism ; Receptors, Purinergic P2X7 ; metabolism ; Regeneration ; TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Toll-Like Receptors ; metabolism

OBJECTIVETo observe the effect of normobaric hyperoxia exposure on the functions of N9 microglia and explore the underlying mechanism of hyperoxia-induced immature brain injury.

METHODSN9 microglial cells were exposed to 900 ml/L O(2) for 2, 6, 12, 24 or 48 h, and the cell apoptotic rate was assessed using flow cytometry. The intracellular oxidative stress was measured using a fluorescent DCFH-DA probe, and the expression of Toll-like receptor 4 (TLR4) mRNA was detected using RT-PCR. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) concentrations in the supernatant of the cell cultures were tested with ELISA following the exposures. TLR4 protein expression was observed using immunofluorescence staining.

RESULTSSignificant cell apoptosis was detected after oxygen exposures for 12-24 h. Accumulation of reactive oxygen species (ROS) were detected after a 2-h exposure. After prolonged hyperoxia exposure, TLR4 expression and IL-1β and TNF-α levels significantly increased in the cells.

CONCLUSIONHyperoxia exposure activates TLR4 signaling pathway in N9 microglial cells in vitro, leading to massive production of ROS, IL-1β, and TNF-α and thus triggering cell apoptosis.

Animals ; Apoptosis ; drug effects ; Cell Hypoxia ; Cells, Cultured ; Interleukin-1beta ; metabolism ; Mice ; Mice, Inbred ICR ; Microglia ; cytology ; drug effects ; physiology ; Oxygen ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism

This study examined the neuroprotective effect of cluster of differentiation molecule 200 (CD200) against methamphetamine (METH)-induced neurotoxicity. In the in vitro experiment, neuron-microglia cultures were treated with METH (20 μmol/L), METH (20 μmol/L)+CD200-Fc (10 μg/mL) or CD200-Fc (10 μg/mL). Those untreated served as control. Microglia activation expressed as the ratio of MHC-II/CD11b was assessed by flow cytometry. The cytokines (IL-1β, TNF-α) secreted by activated microglia were detected by enzyme-linked immunosorbent assay (ELISA). In the in vivo experiment, 40 SD rats were divided into control, METH, METH+CD200-Fc and CD200-Fc groups at random. Rats were intraperitoneally injected with METH (15 mg/kg 8 times at 12 h interval) in METH group, with METH (administered as the same dose and time as the METH group) and CD200-Fc (1 mg/kg at day 0, 2, 4 after METH injection) in METH+CD200-Fc group, with CD200-Fc (1 mg/kg injected as the same time as the METH+CD200-Fc group) or with physiological saline solution in the control group. The level of striatal dopamine (DA) in rats was measured by high-performance liquid chromatography (HPLC). The microglial cells were immunohistochemically detected for the expression of Iba-1, a marker for microglial activation. The results showed that METH could increase the microglia activation in the neuron-microglia cultures and elevate the secretion of IL-1β and TNF-α, which could be attenuated by CD200-Fc. Moreover, CD200-Fc could partially reverse the striatal DA depletion induced by METH and reduce the number of activated microglia, i.e. Iba-1-positive cells. It was concluded that CD200 may have neuroprotective effects against METH-induced neurotoxicity by inhibiting microglial activation and reversing DA depletion in striatum.
Animals ; Animals, Newborn ; Antigens, CD ; administration & dosage ; Cells, Cultured ; Coculture Techniques ; Corpus Striatum ; cytology ; drug effects ; immunology ; Cytokines ; immunology ; Dopamine ; immunology ; Drug Interactions ; Male ; Methamphetamine ; toxicity ; Microglia ; drug effects ; immunology ; Neurons ; metabolism ; Rats ; Rats, Sprague-Dawley

Cell Line ; Cyclin-Dependent Kinase 5 ; metabolism ; Humans ; Lipopolysaccharides ; Microglia ; cytology ; drug effects ; metabolism ; Stilbenes ; pharmacology

Abundant evidence has suggested that neuroinflammation participates in the pathogenesis of Parkinson's disease (PD). The emerging evidence has supported that microglia may play key roles in the progressive neurodegeneration in PD and might be a promising therapeutic target. Ganoderma lucidum (GL), a traditional Chinese medicinal herb, has been shown potential neuroprotective effect in our clinical trials that lead us to speculate that it might possess potent anti-inflammatory and immunomodulating properties. To test this hypothesis, the present study investigated the potential neuroprotective effect of GL and underlying mechanism through inhibiting microglial activation using co-cultures of dopaminergic neurons and microglia. The cultures of microglia or MES23.5 cells alone or together were treated for 24 h with lipopolysaccharide (LPS, 0.25 μg/mL) as a positive control, GL extracts (50-400 μg/mL) or MES23.5 cell membrane fragments (150 μg/mL) were used in treatment groups. Microglia activation, microglia-derived harmful factors and [(3)H]dopamine ([(3)H]DA) uptake of MES23.5 cells were analyzed. The results showed that microglia were activated by LPS and MPP(+)-treated MES23.5 cell membrane fragments, respectively. Meanwhile, GL extracts significantly prevented the production of microglia-derived proinflammatory and cytotoxic factors, including nitric oxide, tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β), in a dose-dependent manner and down-regulated the TNF-α and IL-1β expressions on mRNA level. In addition, GL extracts antagonized the reduction of [(3)H]DA uptake induced by MPP(+) and microglial activation. In conclusion, these results suggest that GL may be a promising agent for the treatment of PD through anti-inflammation.
Cell Line ; Dopaminergic Neurons ; cytology ; drug effects ; Down-Regulation ; drug effects ; Humans ; Interleukin-1beta ; metabolism ; Materia Medica ; pharmacology ; Microglia ; cytology ; metabolism ; Neuroprotective Agents ; pharmacology ; Nitric Oxide ; metabolism ; Parkinson Disease ; physiopathology ; Reishi ; chemistry ; Tumor Necrosis Factor-alpha ; metabolism

Recent studies have reported that the "cholinergic anti-inflammatory pathway" regulates peripheral inflammatory responses via alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) and that acetylcholine and nicotine regulate the expression of proinflammatory mediators such as TNF-alpha and prostaglandin E2 in microglial cultures. In a previous study we showed that ATP released by beta-amyloid-stimulated microglia induced reactive oxygen species (ROS) production, in a process involving the P2X7 receptor (P2X7R), in an autocrine fashion. These observations led us to investigate whether stimulation by nicotine could regulate fibrillar beta amyloid peptide (1-42) (fA beta(1-42))-induced ROS production by modulating ATP efflux-mediated Ca2+ influx through P2X7R. Nicotine inhibited ROS generation in fA beta(1-42)-stimulated microglial cells, and this inhibition was blocked by mecamylamine, a non-selective nAChR antagonist, and a-bungarotoxin, a selective alpha7 nAChR antagonist. Nicotine inhibited NADPH oxidase activation and completely blocked Ca2+ influx in fA beta(1-42)-stimulated microglia. Moreover, ATP release from fA beta(1-42)-stimulated microglia was significantly suppressed by nicotine treatment. In contrast, nicotine did not inhibit 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP)-induced Ca2+ influx, but inhibited ROS generation in BzATP-stimulated microglia, indicating an inhibitory effect of nicotine on a signaling process downstream of P2X7R. Taken together, these results suggest that the inhibitory effect of nicotine on ROS production in fA beta(1-42)-stimulated microglia is mediated by indirect blockage of ATP release and by directly altering the signaling process downstream from P2X7R.
Adenosine Triphosphate/analogs & derivatives/metabolism/pharmacology ; Amyloid/*metabolism ; Amyloid beta-Protein/*pharmacology ; Animals ; Calcium/metabolism ; Enzyme Activation/drug effects ; Microglia/cytology/*drug effects/enzymology/*metabolism ; NADPH Oxidase/metabolism ; Nicotine/pharmacology ; Nicotinic Antagonists/pharmacology ; Peptide Fragments/*pharmacology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/*metabolism ; Receptors, Nicotinic/*metabolism ; Receptors, Purinergic P2/metabolism