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1.
Arq. bras. oftalmol ; 84(1): 67-73, Jan.-Feb. 2021. graf
Article in English | LILACS | ID: biblio-1153097

ABSTRACT

ABSTRACT Purpose: Diabetic retinopathy is currently considered a chronic inflammatory disease involving NOD-like receptor family pyrin domain containing 3 inflammasome activation and retinal microglial pyroptosis. In this study, we aimed to investigate whether NOD-like receptor family pyrin domain containing 3 inflammasome signaling induces pyroptotic death of retinal microglia under high-glucose conditions. Methods: Retinal microglia were stimulated by high glucose levels for 24 h. Cell viability, lactate dehydrogenase release, and caspase-1 activity were detected in vitro. The expression of pro-inflammatory cytokine (interleukin-1β, activated microglia marker ionized calcium-binding adapter molecule-1), NOD-like receptor family pyrin domain containing 3, cleaved caspase-1, and cleaved gasdermin D were examined. Subsequently, retinal microglia were pretreated with the inhibitors of NOD-like receptor family pyrin domain containing 3 inflammasome signaling prior to stimulation with high glucose, and their molecular and functional changes were evaluated. Results: High-glucose (25, 50, or 100 mM) stimulation decreased cell viability, but enhanced lactate dehydrogenase release and caspase-1 activity in a dose-dependent manner. Moreover, high glucose upregulated the protein expression of interleukin-1β, ionized calcium-binding adapter molecule-1, NOD-like receptor family pyrin domain containing 3, cleaved caspase-1, and cleaved gasdermin D. However, pretreatment with the inhibitors of NOD-like receptor family pyrin domain containing 3 inflammasome signaling inhibited high glucose (25 mM)-induced cytotoxicity, NOD-like receptor family pyrin domain containing 3 inflammasome activation, and pyroptosis of retinal microglia. Conclusions: NOD-like receptor family pyrin domain containing 3 inflammasome signaling may modulate retinal microglia-related inflammation and pyroptosis under high-glucose conditions.


RESUMO Objetivo: Atualmente, a retinopatia diabética é considerada uma doença inflamatória crônica envolvendo a ativação de inflamassomas NLRP3 e piroptose da micróglia da retina. Neste estudo, objetivamos investigar se a sinalização de inflamassomas NLRP3 induz a morte da micróglia da retina sob condições de alta glicose. Métodos: A micróglia da retina foi estimulada por altos níveis de glicose durante 24 horas. A viabilidade celular, a liberação de LDH e a atividade da caspase1 foram analisadas in vitro. Avaliou-se a expressão de citocina pró-inflamatória (IL1β), de marcador de micróglia ativado (Iba1), de NLRP3, de caspase1 clivada e de GSDMD clivada. Subsequentemente, a micróglia da retina foi pré-tratada com inibidores da sinalização de inflamassomas NLRP3 antes da estimulação com altos níveis de glicose e suas alterações moleculares e funcionais foram avaliadas. Resultados: A estimulação com altos níveis de glicose (25 mM, 50 mM ou 100 mM) diminuiu a viabilidade celular, mas aumentou a liberação de LDH e a atividade da caspase1 de forma dependente da dose. Além disso, os altos níveis de glicose aumentaram a expressão das proteínas IL1β, Iba1, NLRP3, caspase1 clivada e GSDMD clivada. No entanto, o pré-tratamento com inibidores da sinalização de inflamassomas NLRP3 e a posterior estimulação com altos níveis de glicose (25 mM) induziu citotoxicidade, a ativação de inflamassomas NLRP3 e a piroptose da micróglia da retina. Conclusão: A sinalização de inflamassomas NLRP3 pode modular a inflamação e a piroptose da micróglia da retina na presença de altos níveis de glicose.


Subject(s)
Humans , Inflammasomes , Pyroptosis , Microglia , Interleukin-1beta , NLR Family, Pyrin Domain-Containing 3 Protein , Glucose
2.
Acta Physiologica Sinica ; (6): 10-16, 2021.
Article in Chinese | WPRIM | ID: wpr-878230

ABSTRACT

The aim of the present study was to observe the activation of microglia in the prefrontal cortex of type 1 diabetes mellitus (T1DM) mice, and the expression of the marker genes of the disease-associated microglia (DAM) associated with neurodegenerative diseases. Sixty healthy adult male C57BL/6J mice were randomly divided into two groups, normal control (CON) group and T1DM group. Streptozocin (STZ) was injected intraperitoneally to induce T1DM mice. The spatial learning and memory function of mice was detected by Morris water maze at the 8th week after the successful model establishment. The number and activation of microglia in the prefrontal cortex of mice were detected by immunofluorescence staining and Western blot. Changes in the mRNA level of several DAM molecular markers were detected by RT-FQ-PCR. The results showed that, compared with CON mice, the fasting blood glucose of T1DM mice increased significantly, while the body weight of T1DM mice decreased remarkably (P < 0.05). The escape latency of water maze in T1DM mice was longer than that in CON mice (P < 0.05). Compared with CON group, the Iba1 protein expression and the number of microglia in prefrontal cortex of T1DM group increased significantly (P < 0.05). In addition, the mRNA levels of several DAM markers in prefrontal cortex of T1DM group were increased significantly (P < 0.05). These results suggest that the microglia are activated and transformed to DAM type in the prefrontal cortex of T1DM mice.


Subject(s)
Animals , Diabetes Mellitus, Experimental , Diabetes Mellitus, Type 1 , Hippocampus , Male , Mice , Mice, Inbred C57BL , Microglia , Prefrontal Cortex
3.
Autops. Case Rep ; 10(4): e2020208, 2020. graf
Article in English | LILACS | ID: biblio-1131855

ABSTRACT

Multicystic encephalomalacia is varying sized cystic lesions in the brain encountered in developing fetuses or infants. These cysts start at the periventricular area and may extend onto the cortex. The cause of the formation of these cystic lesions is secondary to an ischemic or hypoxic insult, which leads to liquefactive necrosis and subsequent formation of gliotic cyst walls having an admixture of microglia. We discuss four autopsy cases that had multicystic encephalomalacia to highlight the scenarios in which these lesions are encountered.


Subject(s)
Humans , Male , Infant, Newborn , Encephalomalacia/complications , Autopsy , Microglia , Gliosis , Hypoxia
4.
Braz. j. med. biol. res ; 53(4): e8604, 2020. graf
Article in English | LILACS | ID: biblio-1100926

ABSTRACT

Maraba virus is a member of the genus Vesiculovirus of the Rhabdoviridae family that was isolated in 1983 from sandflies captured in the municipality of Maraba, state of Pará, Amazônia, Brazil. Despite 30 years having passed since its isolation, little is known about the neuropathology induced by the Maraba virus. Accordingly, in this study the histopathological features, inflammatory glial changes, cytokine concentrations, and nitric oxide activity in the encephalon of adult mice subjected to Maraba virus nostril infection were evaluated. The results showed that 6 days after intranasal inoculation, severe neuropathological-associated disease signs appeared, including edema, necrosis and pyknosis of neurons, generalized congestion of encephalic vessels, and intra- and perivascular meningeal lymphocytic infiltrates in several brain regions. Immunolabeling of viral antigens was observed in almost all central nervous system (CNS) areas and this was associated with intense microglial activation and astrogliosis. Compared to control animals, infected mice showed significant increases in interleukin (IL)-6, tumor necrosis factor (TNF)-α, interferon (INF)-γ, MCP-1, nitric oxide, and encephalic cytokine levels. We suggest that an exacerbated inflammatory response in several regions of the CNS of adult BALB/c mice might be responsible for their deaths.


Subject(s)
Animals , Male , Rabbits , Vesicular Stomatitis/complications , Meningoencephalitis/complications , Brazil , Astrocytes/metabolism , Cytokines/analysis , Vesiculovirus , Microglia/metabolism , Disease Models, Animal , Vesicular Stomatitis/pathology , Flow Cytometry , Meningoencephalitis/pathology , Mice, Inbred BALB C , Nitric Oxide/analysis
5.
Article in Chinese | WPRIM | ID: wpr-879929

ABSTRACT

OBJECTIVE@#To investigate the effects of astragaloside Ⅳ (AS-Ⅳ) on microglia/macrophage M1/M2 polarization and inflammatory response after cerebral ischemia in rats.@*METHODS@#Forty eight male SD rats were randomly divided into sham operation control group, model control group and AS-Ⅳ group with 16 rats in each. Focal cerebral ischemia model was induced by occlusion of the right middle cerebral artery (MCAO) using the intraluminal filament. After ischemia induced, the rats in AS-Ⅳ group were intraperitoneally injected with 40 mg/kg AS-Ⅳ once a day for 3 days. The neurological functions were evaluated by the modified neurological severity score (mNSS) and the corner test on d1 and d3 after modelling. The infarct volume was measured by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining on d3 after ischemia. The expression of M1 microglia/macrophage markers CD86, inducible nitric oxide synthase (iNOS) and pro-inflammatory factors TNF-α, IL-1β, IL-6, M2 microglia/macrophages markers CD206, arginase-1 (Arg-1), chitinase-like protein (YM1/2) and anti-inflammatory factors interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) was detected by real-time RT-PCR. The expression of CD16/32/Iba1 and CD206/Iba1 was determined by double labeling immunefluorescence method in the peripheral area of cerebral ischemia.@*RESULTS@#Compared with model control group, AS-Ⅳ treatment improved neurological function recovery and reduced infarct volume after ischemia (@*CONCLUSIONS@#The findings suggest that AS-Ⅳ ameliorates brain injury after cerebral ischemia in rats, which may be related to inhibiting inflammation through promoting the polarization of the microglia/macrophage from M1 to M2 phenotype in the ischemic brain.


Subject(s)
Animals , Anti-Inflammatory Agents/therapeutic use , Brain Ischemia/drug therapy , Cell Polarity/drug effects , Inflammation/drug therapy , Macrophages/drug effects , Male , Microglia/drug effects , Random Allocation , Rats , Rats, Sprague-Dawley , Saponins/therapeutic use , Triterpenes/therapeutic use
6.
Article in Chinese | WPRIM | ID: wpr-879781

ABSTRACT

OBJECTIVE@#To investigate the role of microglial pyroptosis in hypoxic-ischemic brain damage.@*METHODS@#An oxygen-glucose deprivation/reoxygenation (OGD/R) model of rat microglial cells were cultured in vitro. Western blot was used to measure the expression of the pyroptosis-related proteins caspase-1, interleukin-1β (IL-1β), and N-terminal gasdermin D (GSDMD-N) at 0, 1, 3, 6, 12, and 24 hours after OGD/R. After the microglial cells were transfected with lentivirus-mediated silenced gasdermin D (GSDMD), immunofluorescence assay and Western blot were used to measure the transfection rate of GSDMD. Microglial cell lines were divided into three groups: normal control, negative control, and LV-sh_GSDMD (lentivirus-mediated GSDMD silencing). CCK-8 assay and LDH kit were used to observe the effect of GSDMD silencing on the viability and toxicity of microglial cells at 24 hours after OGD/R. Western blot was used to observe the effect of GSDMD silencing on the levels of caspase-1, GSDMD-N, and IL-1β in the microglial cells at 24 hours after OGD/R.@*RESULTS@#The expression levels of the pyroptosis-related proteins caspase-1, GSDMD-N, and IL-1β in microglial cells were upregulated since 0 hour after OGD/R and reached the peak levels at 24 hours. A microglial cell model of lentivirus-mediated GSDMD silencing was successfully constructed. At 24 hours after OGD/R, compared with the normal control group, the GSDMD silencing group had a significant increase in the cell viability and a significant reduction in the cytotoxicity (P<0.05), as well as significant reductions in the protein expression levels of caspase-1, GSDMD-N, and IL-1β in microglial cells (P<0.05).@*CONCLUSIONS@#Lentivirus silencing of the key substrate protein for pyroptosis GSDMD can alleviate hypoxic-ischemic brain damage, suggesting that microglial pyroptosis aggravates hypoxic-ischemic brain damage.


Subject(s)
Animals , Brain/metabolism , Intracellular Signaling Peptides and Proteins , Microglia/metabolism , Pyroptosis , Rats
7.
Article in Chinese | WPRIM | ID: wpr-828115

ABSTRACT

Postoperative cognitive dysfunction (POCD) is one of the most common complications after surgery under general anesthesia and usually manifests as newly presented cognitive impairment. However, the mechanism of POCD is still unclear. In addition to neurons, glial cells including microglia, astrocytes and oligodendrocytes, represent a large cell population in the nervous system. The bi-directional communication between neurons and glia provides basis for neural circuit function. Recent studies suggest that glial dysfunctions may contribute to the occurrence and progress of POCD. In this paper, we review the relevant work on POCD, which may provide new insights into the mechanism and therapeutic strategy for POCD.


Subject(s)
Anesthesia, General , Humans , Microglia , Postoperative Cognitive Complications , Postoperative Complications
8.
Article in English | WPRIM | ID: wpr-787136

ABSTRACT

Regulator of calcineurin 1 (RCAN1) can be induced by an intracellular calcium increase and oxidative stress, which are characteristic features of temporal lobe epilepsy. Thus, we investigated the spatiotemporal expression and cellular localization of RCAN1 protein and mRNA in the mouse hippocampus after pilocarpine-induced status epilepticus (SE). Male C57BL/6 mice were given pilocarpine hydrochloride (280 mg/kg, i.p.) and allowed to develop 2 h of SE. Then the animals were given diazepam (10 mg/kg, i.p.) to stop the seizures and sacrificed at 1, 3, 7, 14, or 28 day after SE. Cresyl violet staining showed that pilocarpine-induced SE resulted in cell death in the CA1 and CA3 subfields of the hippocampus from 3 day after SE. RCAN1 immunoreactivity showed that RCAN1 was mainly expressed in neurons in the shammanipulated hippocampi. At 1 day after SE, RCAN1 expression became detected in hippocampal neuropils. However, RCAN1 signals were markedly enhanced in cells with stellate morphology at 3 and 7 day after SE, which were confirmed to be reactive astrocytes, but not microglia by double immunofluorescence. In addition, real-time reverse transcriptase–polymerase chain reaction showed a significant upregulation of RCAN1 isoform 4 (RCAN1-4) mRNA in the SE-induced hippocampi. Finally, in situ hybridization with immunohistochemistry revealed astrocytic expression of RCAN1-4 after SE. These results demonstrate astrocytic upregulation of RCAN1 and RCAN1-4 in the mouse hippocampus in the acute and subacute phases of epileptogenesis, providing foundational information for the potential role of RCAN1 in reactive astrocytes during epileptogenesis.


Subject(s)
Animals , Astrocytes , Calcineurin , Calcium , Cell Death , Diazepam , Epilepsy , Epilepsy, Temporal Lobe , Fluorescent Antibody Technique , Hippocampus , Humans , Immunohistochemistry , In Situ Hybridization , Male , Mice , Microglia , Neurons , Neuropil , Oxidative Stress , Pilocarpine , RNA, Messenger , Seizures , Status Epilepticus , Up-Regulation , Viola
9.
Rev. chil. pediatr ; 90(5): 555-558, oct. 2019. graf
Article in Spanish | LILACS | ID: biblio-1058183

ABSTRACT

Resumen: En los últimos años se ha intentado comprender la etiología del Trastorno del Espectro Autista (TEA), evidenciandose que existe una compleja interacción entre factores genéticos y ambientales. Estudios epidemiológicos y en modelos animales sugieren que la activación inmune de la madre durante el embarazo puede asociarse un mayor riesgo de desarrollar TEA en los hijos, destacando el rol de las citoquinas proinflamatorias, los auto-anticuerpos y el rol de la microglia activada en la poda sináptica durante el desarrollo embrionario. Comprender mejor los factores asociados con los Trastornos del Neurodesarrollo permitirá en el futuro desarrollar estrategias de manejo y detección precoz en población de riesgo.


Abstract: Autism Spectrum Disorder (ASD) etiology has been related whit complex interaction between ge netic and environmental factors. In the last years, numerous studies have suggested that maternal immune activation during pregnancy could be related to ASD in the offspring. This relation could be explained by the effects of pro-inflammatory cytokines, autoantibodies and microglial synap tic pruning during early embryonic development. Better understanding of Neurodevelopmental Disorders risk factors will support appropriate strategies of screening and management of risk population.


Subject(s)
Humans , Female , Pregnancy , Pregnancy Complications/immunology , Prenatal Exposure Delayed Effects/immunology , Autism Spectrum Disorder/immunology , Autoantibodies/immunology , Risk Factors , Cytokines/immunology , Microglia/immunology , Autism Spectrum Disorder/etiology
10.
Rev. med. (Säo Paulo) ; 98(4): 241-253, jul.-ago. 2019.
Article in English | LILACS | ID: biblio-1023526

ABSTRACT

Introduction: Neural development is an enormously complex and dynamic process. From very early in brain development 'immune cells' play a key role in a number of processes including the formation and refinement of neural circuits, as well as sexual differentiation. There is a growing body of evidence that the immune system also plays an important role in the pathobiology of several neurodevelopmental and neuropsychiatric disorders. Objective: The goal of this article is to review the currently available data concerning the role of the 'immune system' in normal brain development, as well as its role in the pathobiology of neurodevelopmental and neuropsychiatric disorders. Methodology: We conducted a traditional literature search using PubMed and recent special issues of journals to locate relevant review articles. Results: The cellular and molecular processes that make up our 'immune system' are crucial to normal brain development and the formation and maintenance of neural circuits. It is also increasingly evident that the immune system and neuroinflammation play important roles in the pathobiology of at least a subset of individuals with Autism Spectrum Disorder (ASD), schizophrenia, obsessive-compulsive disorder, Tourette syndrome and mood disorders, such as depression, as well as autoimmune and neurodegenerative disorders. Emerging evidence also points to the importance of the 'gut-brain axis' and an individual's microbiome, which can impact an individual's somatic and mental well-being. Conclusions: There are multidirectional interconnections across multiple biological systems in our brains and bodies that are mediated in part by the immune system. At present, however, the 'promise' of this field remains greater than the 'deliverables'. Time will tell whether novel interventions will be developed that will make a positive difference in the care of our patients. It is also possible that valid biomarkers will emerge that will guide a more personalized approach to treatment.


Introdução: O desenvolvimento neural é um processo extremamente complexo e dinâmico. Tao pronto se inicia o desenvolvimento do cérebro, as "células imunológicas" desempenham um papel fundamental em vários processos, incluindo a formação e aperfeiçoamento de circuitos neurais, bem como a diferenciação sexual. Há um crescente corpo de evidências de que o sistema imunológico também desempenha um papel importante na fisiopatologia de diversos transtornos neurodesenvolvimentais e neuropsiquiátricos. Objetivo: O objetivo deste artigo é revisar os dados atualmente disponíveis sobre o papel do "sistema imunológico" em relação ao desenvolvimento normal do cérebro, bem como a fisiopatogenia dos transtornos de neurodesenvolvimento e neuropsiquiátricos. Metodologia: Foi realizada uma pesquisa bibliográfica tradicional para localizar artigos de revisão relevantes. Resultados: Os processos celulares e moleculares que compõem o nosso "sistema imunológico" são cruciais para o desenvolvimento normal do cérebro e a formação e manutenção de circuitos neurais. É cada vez mais evidente que o sistema imunológico e neuroinflamação desempenham papéis importantes na etiopatogenia de pelo menos um subconjunto de indivíduos com autismo, esquizofrenia, transtorno obsessivo-compulsivo, síndrome de Tourette, depressão e transtornos do humor, bem como distúrbios autoimunes e neurodegenerativos. Evidências emergentes também apontam para a importância do eixo intestino-cerebral e do microbioma de um indivíduo em relação à sua saúde e bem-estar somático e mental. Conclusões: Existem interconexões multidirecionais entre múltiplos sistemas biológicos em nossos cérebros e corpos que são mediados em parte pelo sistema imunológico. No momento, no entanto, a "promessa" desse campo continua sendo maior do que os "resultados finais". O tempo dirá se novas intervenções serão desenvolvidas que farão uma diferença positiva no cuidado de nossos pacientes. Também é possível que surjam biomarcadores válidos que orientarão uma abordagem mais personalizada ao tratamento.


Subject(s)
Autistic Disorder , Neuroimmunomodulation , Tourette Syndrome , Microglia , Mood Disorders , Neurodevelopmental Disorders , Autism Spectrum Disorder , Immune System , Immunity, Maternally-Acquired , Obsessive-Compulsive Disorder , Schizophrenia , Stress, Psychological , Cytokines , Depression , Allergy and Immunology
11.
Article in English | WPRIM | ID: wpr-776646

ABSTRACT

OBJECTIVE@#To investigate the anti-neuroinflammation effect of extract of Fructus Schisandrae chinensis (EFSC) on lipopolysaccharide (LPS)-induced BV-2 cells and the possible involved mechanisms.@*METHODS@#Primary cortical neurons were isolated from embryonic (E17-18) cortices of Institute of Cancer Research (ICR) mouse fetuses. Primary microglia and astroglia were isolated from the frontal cortices of newborn ICR mouse. Different cells were cultured in specific culture medium. Cells were divided into 5 groups: control group, LPS group (treated with 1 μg/mL LPS only) and EFSC groups (treated with 1 μg/mL LPS and 100, 200 or 400 mg/mL EFSC, respectively). The effect of EFSC on cells viability was tested by methylthiazolyldiphenyltetrazolium bromide (MTT) colorimetric assay. EFSC-mediated inhibition of LPS-induced production of pro-inflammatory mediators, such as nitrite oxide (NO) and interleukin-6 (IL-6) were quantified and neuron-protection effect against microglia-mediated inflammation injury was tested by hoechst 33258 apoptosis assay and crystal violet staining assay. The expression of pro-inflammatory marker proteins was evaluated by Western blot analysis or immunofluorescence.@*RESULTS@#EFSC (200 and 400 mg/mL) reduced NO, IL-6, inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression in LPS-induced BV-2 cells (P<0.01 or P<0.05). EFSC (200 and 400 mg/mL) reduced the expression of NO in LPS-induced primary microglia and astroglia (P<0.01). In addition, EFSC alleviated cell apoptosis and inflammation injury in neurons exposed to microglia-conditioned medium (P<0.01). The mechanistic studies indicated EFSC could suppress nuclear factor (NF)-?B phosphorylation and its nuclear translocation (P<0.01). The anti-inflammatory effect of EFSC occurred through suppressed activation of mitogen-activated protein kinase (MAPK) pathway (P<0.01 or P<0.05).@*CONCLUSION@#EFSC acted as an anti-inflammatory agent in LPS-induced glia cells. These effects might be realized through blocking of NF-κB activity and inhibition of MAPK signaling pathways.


Subject(s)
Animals , Astrocytes , Metabolism , Pathology , Cell Line , Cell Nucleus , Metabolism , Chromatography, High Pressure Liquid , Down-Regulation , Inflammation , Pathology , Inflammation Mediators , Metabolism , Lipopolysaccharides , MAP Kinase Signaling System , Mice, Inbred ICR , Microglia , Metabolism , Pathology , NF-kappa B , Metabolism , Nervous System , Pathology , Neurons , Metabolism , Pathology , Neuroprotective Agents , Pharmacology , Plant Extracts , Pharmacology , Schisandra , Chemistry , Spectrometry, Mass, Electrospray Ionization
13.
Acta Physiologica Sinica ; (6): 883-893, 2019.
Article in Chinese | WPRIM | ID: wpr-781386

ABSTRACT

In this study, we improved the culture method of mouse hippocampal primary microglia to obtain hippocampal ramified microglia with high activity and purity, which were resemble to the resting status of normal microglia in healthy brain in vivo. Hippocampal tissue was excised from 2-4-week-old SPF C57BL/6J mice and cut into pieces after PBS perfusion, and then manually dissociated into the single-cell suspension by using Miltenyi Biotec's Adult Brain Dissociation Kit. The tissue fragments such as myelin in the supernatant were removed by debris removal solution in the kit. The cell suspension was incubated with CD11b immunomagnetic beads for 15 min at 4 °C. To obtain high-purity microglia, we used two consecutive cell-sorting steps by magnetic activated cell sorting (MACS). After centrifugation, the cells were resuspended and seeded in a 24-well culture plate. The primary microglia were cultured with complete medium (CM) or TIC medium (a serum-free medium with TGF-β, IL-34 and cholesterol as the main nutritional components) for 4 days, and then were used for further experiments. The results showed that: (1) The cell viability was (56.03 ± 2.10)% by manual dissociation of hippocampus; (2) Compared with immunopanning, two-step MACS sorting allowed for efficient enrichment of microglia with higher purity of (86.20 ± 0.68)%; (3) After being incubated in TIC medium for 4 d, microglia exhibited branching, quiescent morphology; (4) The results from qRT-PCR assay showed that the levels of TNF-α, IL-1β and CCL2 mRNA in TIC cultured-microglia were similar to freshly isolated microglia, while those were much higher in CM cultured-microglia after incubation for 4 d and 7 d (P < 0.05). Taken together, compared to the conventional approaches, this modified protocol of mouse hippocampal primary microglia culture by using MACS and TIC medium enables the increased yield and purity of microglia in the quiescent state, which is similar to normal ramified microglia in healthy brain in vivo.


Subject(s)
Animals , Cell Culture Techniques , Methods , Cell Separation , Methods , Cells, Cultured , Hippocampus , Magnetics , Mice , Mice, Inbred C57BL , Microglia , Cell Biology
14.
Acta Physiologica Sinica ; (6): 732-740, 2019.
Article in Chinese | WPRIM | ID: wpr-777137

ABSTRACT

Parkinson's disease (PD) is the second most common neurodegenerative disease, characterized by loss of dopaminergic (DA) neurons in the dense part of the substantia nigra (SNpc). Postmortem analysis of PD patients and experimental animal studies found that microglial cell activation and increased levels of pro-inflammatory factors were common features of PD brain tissue. At the same time, the invasion and accumulation of peripheric immune cells were detected in the brain of PD patients. In this paper, peripheral inflammation across the blood-brain barrier (BBB), the misfolded α-synuclein (α-syn)-induced microglial cell activation and intracerebral inflammation in PD are summarized, providing potential therapeutic measures for delaying the onset of PD.


Subject(s)
Animals , Blood-Brain Barrier , Dopaminergic Neurons , Pathology , Humans , Inflammation , Pathology , Microglia , Parkinson Disease , Pathology , Substantia Nigra , Pathology , alpha-Synuclein
15.
Chinese Journal of Traumatology ; (6): 161-165, 2019.
Article in English | WPRIM | ID: wpr-771617

ABSTRACT

PURPOSE@#To investigate whether dexmedetomidine (Dex) can reduce the production of inflammatory factor IL-1β by inhibiting the activation of NLRP3 inflammasome in hippocampal microglia, thereby alleviating the inflammatory response of the central nervous system induced by surgical injury.@*METHODS@#Exploratory laparotomy was used in experimental models in this study. Totally 48 Sprague Dawley male rats were randomly divided into 4 groups (n = 12 for each), respectively sham control (group A), laparotomy only (group B); and Dex treatment with different doses of 5 μg/kg (group D1) or 10 μg/kg (group D2). Rats in groups D1 and D2 were intraperitoneally injected with corresponding doses of Dex every 6 h. The rats were sacrificed 12 h after operation; the hippocampus tissues were isolated, and frozen sections were made. The microglia activation was estimated by immunohistochemistry. The protein expression of NLRP3, caspase-1, ASC and IL-1β were detected by immunoblotting. All data were presented as mean ± standard deviation, and independent sample t test was used to analyze the statistical difference between groups.@*RESULTS@#The activated microglia in the hippocampus of the rats significantly increased after laparotomy (group B vs. sham control, p < 0.01). After Dex treatment, the number was decreased in a dose-dependent way (group D1 vs. D2, p < 0.05), however the activated microglia in both groups were still higher than that of sham controls (both p < 0.05). Further Western blot analysis showed that the protein expression levels of NLRP3, caspase-1, ASC and downstream cytokine IL-1β in the hippocampus from the laparotomy group were significantly higher than those of the sham control group (all p < 0.01). The elevated expression of these proteins was relieved after Dex treatment, also in a dose-dependent way (D2 vs. D1 group, p < 0.05).@*CONCLUSION@#Dex can inhibit the activation of microglia and NLRP3 inflammasome in the hippocampus of rats after operation, and the synthesis and secretion of IL-1β are also reduced in a dose-dependent manner by using Dex. Hence, Dex can alleviate inflammation activation on the central nervous system induced by surgical injury.


Subject(s)
Animals , Dexmedetomidine , Pharmacology , Dose-Response Relationship, Drug , Hippocampus , Metabolism , Immunohistochemistry , Inflammasomes , Metabolism , Inflammation Mediators , Metabolism , Injections, Intraperitoneal , Interleukin-1beta , Metabolism , Laparotomy , Male , Microglia , Metabolism , NLR Family, Pyrin Domain-Containing 3 Protein , Metabolism , Rats, Sprague-Dawley , Time Factors
16.
Article in Chinese | WPRIM | ID: wpr-771504

ABSTRACT

Ten fractions(A-J) were prepared by separation of Longxue Tongluo Capsules(LTC) by using silica gel column chromatography and orthogonal experimental design,showing similar chemical profiles with different abundances of peaks.These ten samples were assessed with UHPLC-QE OrbitrapHRMS for 97 common peaks.For the pharmacological activity experiment,three kinds of in vitro cell models including lipopolysaccharide(LPS)-induced BV-2 microglial cells NO release model,oxygen-glucose deprivation/reoxygenation(OGD/R)-treated HUVEC vascular endothelial cells injury model,and OGD/R-treated PC-12 nerve cells injury model were employed to evaluated the bioactivity of each fraction.Based on the contribution of each identified component,grey relation analysis and partial least squares(PLS) analysis were performed to establish component-activity relationship of LTC,identify the potential active components.After that,validation of the potential active components in LTC was carried out by using the same models.The results indicated that 4 phenolic compounds including 7,4'-dihydroxyhomoisoflavanone,loureirin C,4,4'-dihydroxy-2,6-dimethoxydihydrochalcone,and homoisosocotrin-4'-ol,might be the active components for anti-neuroinflammation effect;five phenolic compounds such as 3,5,7,4'-tetrahydroxyhomoisoflavanone,loureirin D,7,4'-dihydroxyhomoisoflavane,and 5,7-dihydroxy-4'-methoxy-8-methyflavane,might have positive effects on the vascular endothelial injury;three phenolic compounds including 5,7,4'-trihydroxyflavanone,7,4'-dihydroxy-5-methoxyhomoisoflavane,and loureirin D,might be the active components in LTC against neuronal injury.


Subject(s)
Brain Ischemia , Drug Therapy , Capsules , Cell Line , Drugs, Chinese Herbal , Pharmacology , Glucose , Human Umbilical Vein Endothelial Cells , Humans , Microglia , Oxygen
17.
Article in Chinese | WPRIM | ID: wpr-776048

ABSTRACT

Objective To explore the effects of cathepsin B(CTSB)on the activation of nucleotide-binding domain and leucine-rich-repeat-containing family and pyrin domain-containing 3(NLRP3)inflammasome via transient receptor potential mucolipin-1(TRPML1)in cell oxidative stress model and specific gene silencing cell model. Methods BV2 cells cultured in vivo were treated separately or simultaneously with hydrogen peroxide(HO),calcium-sensitive receptor agonist gadolinium trichloride(GdCl),and CTSB inhibitor CA-074Me,and interleukin-1(IL-1)beta and caspase-1 protein were detected by enzyme-linked immunosorbent assay.The growth activity of BV2 cells in each group was measured by MTT.BV2 cells were treated with different concentrations of HO.Cystatin C mRNA and TRPML1 mRNA in BV2 cells were detected by real-time quantitative polymerase chain reaction and the proteins of TRPML1,CTSB,cathepsin D(CTSD),cathepsin L(CTSL)and cathepsin V(CTSV)were detected by Western blot.Specific small interfering RNA was designed for TRPML1 gene target sequence.TRPML1 gene silencing cell lines(named Tr-si-Bv2 cells)were established in BV2 cells and treated with or without HO.TRPML1,CTSB and transcription factor EB(TFEB)proteins in Tr-si-Bv2 cells or control cells were detected by Western blot. Results After treatment with HO,the expression of caspase-1 protein and NLRP3 mRNA in BV2 cells was increased,and IL-1beta protein in BV2 cells was significantly increased after treatment with GdCl(P=0.0036).After treatment with CA-074Me,the doses of NLRP3 mRNA(P=0.037),caspase-1(P=0.021),and IL-1β(P= 0.036)were significantly reduced.Cells in the HO group and HO+GdCl group grew more slowly.The expressions of CTSB mRNA and TRPML1 mRNA,or CTSB and TRPML1 proteins in BV2 cells in the treatment group with 200 μmol/L of HO concentration were similar.HO-induced CTSB protein expression was inhibited after silencing TRPML1 gene.The changes of other cathepsins were not affected for the different concentration of HO.In the BV2 cells treated with TRPML1 gene silencing,the expression of CTSB protein was significantly reduced and the difference was statistically significant(P=0.021)between the HO +siRNA treatment group and the HO treatment group.Conclusion CTSB regulates the activation of NLRP3 inflammasome in the oxidative stress model of microglia cells,probably mediated by calcium channel protein TRPML1.


Subject(s)
Animals , Cathepsin B , Metabolism , Cell Line , Gene Silencing , Hydrogen Peroxide , Inflammasomes , Metabolism , Interleukin-1beta , Mice , Microglia , NLR Family, Pyrin Domain-Containing 3 Protein , Metabolism , Oxidative Stress , Pyrin Domain , Transient Receptor Potential Channels , Metabolism
18.
Neuroscience Bulletin ; (6): 347-361, 2019.
Article in English | WPRIM | ID: wpr-775441

ABSTRACT

Occupational exposure to 1-bromopropane (1-BP) induces learning and memory deficits. However, no therapeutic strategies are currently available. Accumulating evidence has suggested that N-methyl-D-aspartate receptors (NMDARs) and neuroinflammation are involved in the cognitive impairments in neurodegenerative diseases. In this study we aimed to investigate whether the noncompetitive NMDAR antagonist MK801 protects against 1-BP-induced cognitive dysfunction. Male Wistar rats were administered with MK801 (0.1 mg/kg) prior to 1-BP intoxication (800 mg/kg). Their cognitive performance was evaluated by the Morris water maze test. The brains of rats were dissected for biochemical, neuropathological, and immunological analyses. We found that the spatial learning and memory were significantly impaired in the 1-BP group, and this was associated with neurodegeneration in both the hippocampus (especially CA1 and CA3) and cortex. Besides, the protein levels of phosphorylated NMDARs were increased after 1-BP exposure. MK801 ameliorated the 1-BP-induced cognitive impairments and degeneration of neurons in the hippocampus and cortex. Mechanistically, MK801 abrogated the 1-BP-induced disruption of excitatory and inhibitory amino-acid balance and NMDAR abnormalities. Subsequently, MK801 inhibited the microglial activation and release of pro-inflammatory cytokines in 1-BP-treated rats. Our findings, for the first time, revealed that MK801 protected against 1-BP-induced cognitive dysfunction by ameliorating NMDAR function and blocking microglial activation, which might provide a potential target for the treatment of 1-BP poisoning.


Subject(s)
Animals , Brain , Metabolism , Pathology , Cognitive Dysfunction , Drug Therapy , Metabolism , Pathology , Disease Models, Animal , Dizocilpine Maleate , Pharmacology , Excitatory Amino Acid Antagonists , Pharmacology , Hydrocarbons, Brominated , Inflammasomes , Metabolism , Male , Maze Learning , Physiology , Microglia , Metabolism , Pathology , NLR Family, Pyrin Domain-Containing 3 Protein , Metabolism , Neurons , Metabolism , Pathology , Nootropic Agents , Pharmacology , Random Allocation , Rats, Wistar , Receptors, N-Methyl-D-Aspartate , Metabolism , Spatial Memory , Physiology , Specific Pathogen-Free Organisms
19.
Neuroscience Bulletin ; (6): 461-470, 2019.
Article in English | WPRIM | ID: wpr-775428

ABSTRACT

Gastrodin is a phenolic glycoside that has been demonstrated to provide neuroprotection in preclinical models of central nervous system disease, but its effect in subarachnoid hemorrhage (SAH) remains unclear. In this study, we showed that intraperitoneal administration of gastrodin (100 mg/kg per day) significantly attenuated the SAH-induced neurological deficit, brain edema, and increased blood-brain barrier permeability in rats. Meanwhile, gastrodin treatment significantly reduced the SAH-induced elevation of glutamate concentration in the cerebrospinal fluid and the intracellular Ca overload. Moreover, gastrodin suppressed the SAH-induced microglial activation, astrocyte activation, and neuronal apoptosis. Mechanistically, gastrodin significantly reduced the oxidative stress and inflammatory response, up-regulated the expression of nuclear factor erythroid 2-related factor 2, heme oxygenase-1, phospho-Akt and B-cell lymphoma 2, and down-regulated the expression of BCL2-associated X protein and cleaved caspase-3. Our results suggested that the administration of gastrodin provides neuroprotection against early brain injury after experimental SAH.


Subject(s)
Animals , Apoptosis , Astrocytes , Metabolism , Benzyl Alcohols , Blood-Brain Barrier , Metabolism , Brain , Metabolism , Brain Edema , Calcium , Metabolism , Glucosides , Glutamic Acid , Metabolism , Male , Microglia , Metabolism , Neurons , Neuroprotective Agents , Oxidative Stress , Rats, Sprague-Dawley , Subarachnoid Hemorrhage , Metabolism
20.
Article in English | WPRIM | ID: wpr-739331

ABSTRACT

Recovery from central nervous system (CNS) injury, such as stroke or spinal cord injury (SCI), largely depends on axonal regeneration, and the neuronal and glial cells plasticity in the lesioned tissue. The lesioned tissue following CNS injury forms a scar that is composed of astrocytes and mixed with connective tissues. At the glial scar, the regenerating axon forms dystrophic endbulbs which do not regenerate and grow beyond the glial scar without a suitable environment. Along with the astrocytes, microglia are also suspected of being involved in necrotic and apoptotic neuronal cell death and the early response to axonal damage in CNS injury. The inflammatory response, a major component of secondary injury and controlled by the microglia, plays a pivotal role in nerve injury and control the regenerative response. As a result, it is very important to control the glial cell function in order to assure the recovery of the CNS injury. Studies have suggested that agmatine, a L-arginine derived primary amine, is a potential modulator of glial cell function after CNS injuries. Agmatine was found to possess anti-inflammatory and neuroprotective characteristics that benefited the rehabilitation process following CNS injury. In this review, we will discuss the effect of agmatine on glial cells in the process of recovery after CNS injury.


Subject(s)
Agmatine , Arginine , Astrocytes , Axons , Cell Death , Central Nervous System , Cicatrix , Connective Tissue , Microglia , Neuroglia , Neurons , Plastics , Regeneration , Rehabilitation , Spinal Cord Injuries , Stroke
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