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BACKGROUND: Comorbid chronic neuropathic pain (NPP) and anxio-depressive disorders (ADD) have become a serious global public-health problem. The SLIT and NTRK-like 1 (SLITRK1) protein is important for synaptic remodeling and is highly expressed in the amygdala, an important brain region involved in various emotional behaviors. We examined whether SLITRK1 protein in the amygdala participates in NPP and comorbid ADD. METHODS: A chronic NPP mouse model was constructed by L5 spinal nerve ligation; changes in chronic pain and ADD-like behaviors were measured in behavioral tests. Changes in SLITRK1 protein and excitatory synaptic functional proteins in the amygdala were measured by immunofluorescence and Western blot. Adeno-associated virus was transfected into excitatory synaptic neurons in the amygdala to up-regulate the expression of SLITRK1. RESULTS: Chronic NPP-related ADD-like behavior was successfully produced in mice by L5 ligation. We found that chronic NPP and related ADD decreased amygdalar expression of SLITRK1 and proteins important for excitatory synaptic function, including Homer1, postsynaptic density protein 95 (PSD95), and synaptophysin. Virally-mediated SLITRK1 overexpression in the amygdala produced a significant easing of chronic NPP and ADD, and restored the expression levels of Homer1, PSD95, and synaptophysin. CONCLUSION: Our findings indicated that SLITRK1 in the amygdala plays an important role in chronic pain and related ADD, and may prove to be a potential therapeutic target for chronic NPP-ADD comorbidity.
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Amígdala del Cerebelo , Conducta Animal , Dolor Crónico , Homólogo 4 de la Proteína Discs Large , Proteínas del Tejido Nervioso , Neuralgia , Animales , Masculino , Ratones , Amígdala del Cerebelo/metabolismo , Ansiedad/metabolismo , Ansiedad/fisiopatología , Trastornos de Ansiedad/metabolismo , Trastornos de Ansiedad/fisiopatología , Conducta Animal/fisiología , Dolor Crónico/metabolismo , Dolor Crónico/fisiopatología , Depresión/metabolismo , Depresión/etiología , Depresión/fisiopatología , Trastorno Depresivo/metabolismo , Trastorno Depresivo/fisiopatología , Modelos Animales de Enfermedad , Homólogo 4 de la Proteína Discs Large/metabolismo , Proteínas de Andamiaje Homer/metabolismo , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Neuralgia/metabolismo , Sinaptofisina/metabolismoRESUMEN
Neuropathic pain (NPP) is a common type of chronic pain. Glial cells, including astrocytes (AS), are believed to play an important role in the progression of NPP. AS cells can be divided into various types based on their expression profiles, among which A1 and A2 types have clear functions. A1-type AS cells are neurotoxic, while A2-type AS cells exert neuroprotective functions. Some types of lysophosphatidic acid receptors (LPAR) have been shown to play a role in NPP. However, it remains unclear how AS cells and LPAR6 affect the occurrence and progression of NPP. In this study, we established a mouse model of chronic constriction injury (CCI) to simulate NPP. It was found that the expression of LPAR6 in AS cells of the spinal dorsal horn was increased in the CCI model, and the thresholds of mechanical and thermal pain were elevated after knocking out LPAR6, indicating that LPAR6 and AS cells participated in the occurrence of NPP. The experiment involved culturing primary AS cells and knocking down LPAR6 by Lentivirus. The results showed that the NF-κB signal pathway was activated and the number of A1-type AS cells increased in the CCI model. However, LPAR6 knockdown inhibited the NF-κB signal pathway and A1-type AS cells. The results of the mRNA sequencing and immunoprecipitation test indicate an interaction between LPAR6 and ROCK2. Inhibiting ROCK2 by Y-27632 increased mechanical and thermal pain thresholds and alleviated NPP at the molecular level. The study presents evidence that LPAR6 activates the NF-κB pathway through ROCK2 and contributes to the progression of NPP by increasing A1-type AS and decreasing A2-type AS. This suggests that LPAR6 could be a potential therapeutic target for alleviating NPP. Clinical applications that are successful can offer new therapeutic options, enhance the quality of life for patients, and potentially uncover new mechanisms for pain modulation.
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Major depressive disorder ranks as a major burden of disease worldwide, yet the current antidepressant medications are limited by frequent non-responsiveness and significant side effects. The lateral septum (LS) is thought to control of depression, however, the cellular and circuit substrates are largely unknown. Here, we identified a subpopulation of LS GABAergic adenosine A2A receptors (A2AR)-positive neurons mediating depressive symptoms via direct projects to the lateral habenula (LHb) and the dorsomedial hypothalamus (DMH). Activation of A2AR in the LS augmented the spiking frequency of A2AR-positive neurons leading to a decreased activation of surrounding neurons and the bi-directional manipulation of LS-A2AR activity demonstrated that LS-A2ARs are necessary and sufficient to trigger depressive phenotypes. Thus, the optogenetic modulation (stimulation or inhibition) of LS-A2AR-positive neuronal activity or LS-A2AR-positive neurons projection terminals to the LHb or DMH, phenocopied depressive behaviors. Moreover, A2AR are upregulated in the LS in two male mouse models of repeated stress-induced depression. This identification that aberrantly increased A2AR signaling in the LS is a critical upstream regulator of repeated stress-induced depressive-like behaviors provides a neurophysiological and circuit-based justification of the antidepressant potential of A2AR antagonists, prompting their clinical translation.
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Trastorno Depresivo Mayor , Habénula , Ratones , Animales , Masculino , Habénula/fisiología , Adenosina/farmacología , Neuronas/metabolismo , Hipotálamo/metabolismo , Receptor de Adenosina A2A/metabolismoRESUMEN
There are an increasing number of experiments to study programmed cell death/apoptosis, one of the characteristics of which is DNA fragmentation. The only current method for in situ detection of DNA fragmentation is Terminal deoxynucleotidyl transferase mediated-dUTP Nick End Labeling, TUNEL. In this study, a new method for in situ detection of apoptotic DNA fragments, namely In Situ Hybridization Chain Reaction, isHCR, was established. The principle of the assay is that the sticky end sequence of the apoptotic cell DNA fragment non-specifically initiates a hybridization chain reaction that specifically detects the apoptotic cell. The results of the combined TUNEL and isHCR method demonstrated that the majority of isHCR-positive cells were also labeled by TUNEL. In situ HCR often detect DNA fragments in the cytoplasm that the classical TUNEL method couldnot, and these cells may be in the early stages of apoptosis. It also indicates that DNA fragments are transferred to the cytoplasm during apoptosis. Because the staining process does not require terminal deoxynucleotidyl transferase as TUNEL staining does, isHCR staining cost low and can be performed on a large number of tissue specimens. It is believed that isHCR has the potential to detect DNA fragmentation of apoptotic cells in situ.
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Apoptosis , ADN Nucleotidilexotransferasa , Apoptosis/genética , ADN Nucleotidilexotransferasa/genética , Etiquetado Corte-Fin in Situ , Fragmentación del ADN , ADN , Hibridación in SituRESUMEN
Blood-brain barrier (BBB) impairment is an early prevalent feature of multiple sclerosis (MS), and remains vital for MS progression. Microglial activation precedes BBB disruption and cellular infiltrates in the brain of MS patients. However, little is known about the function of microglia in BBB impairment. Here, microglia acts as an important modulator of BBB integrity in inflammatory demyelination. Microglial depletion profoundly ameliorates BBB impairment in experimental autoimmune encephalomyelitis (EAE). Specifically, miR-126a-5p in microglia is positively correlated with BBB integrity in four types of MS plaques. Mechanistically, microglial deletion of miR-126a-5p exacerbates BBB leakage and EAE severity. The protective effect of miR-126a-5p is mimicked and restored by specific inhibition of MMP9 in microglia. Importantly, Auranofin, an FDA-approved drug, is identified to protect BBB integrity and mitigate EAE progression via a microglial miR-126a-5p dependent mechanism. Taken together, microglia can be manipulated to protect BBB integrity and ameliorate inflammatory demyelination. Targeting microglia to regulate BBB permeability merits consideration in therapeutic interventions in MS.
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Encefalomielitis Autoinmune Experimental , MicroARNs , Esclerosis Múltiple , Animales , Barrera Hematoencefálica , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Humanos , Metaloproteinasa 9 de la Matriz/farmacología , Metaloproteinasa 9 de la Matriz/uso terapéutico , Ratones , MicroARNs/genética , MicroglíaRESUMEN
OBJECTIVE: To assess the effect of serine protein inhibitor A3N (serpinA3N) in ischemic stroke and to explore its mechanism of action. METHODS: Mouse ischemic stroke model was induced by transient middle cerebral artery occlusion followed by reperfusion. The expression pattern of serpinA3N was assessed using immunofluorescence, Western blot analysis, and real-time quantitative PCR. An adeno-associated virus (AAV) and recombinant serpinA3N were administered. Additionally, co-immunoprecipitation-mass spectrometry and immunofluorescence co-staining were used to identify protein interactions. RESULTS: SerpinA3N was upregulated in astrocytes and neurons within the ischemic penumbra after stroke in the acute phase. The expression of serpinA3N gradually increased 6 h after reperfusion, peaked on the day 2-3, and then decreased by day 7. Overexpression of serpinA3N by AAV significantly reduced the infarct size and improved motor function, associated with alleviated inflammation and oxidative stress. SerpinA3N treatment also reduced apoptosis both in vivo and in vitro. Co-immunoprecipitation-mass spectrometry and Western blotting revealed that clusterin interacts with serpinA3N, and Akt-mTOR pathway members were upregulated by serpinA3N both in vivo and in vitro. CONCLUSIONS: SerpinA3N is expressed in astrocytes and penumbra neurons after stroke in mice. It reduces brain damage possibly via interacting with clusterin and inhibiting neuronal apoptosis and neuroinflammation.
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Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Daño por Reperfusión , Serpinas , Accidente Cerebrovascular , Proteínas de Fase Aguda , Animales , Apoptosis , Isquemia Encefálica/metabolismo , Clusterina , Ratones , Enfermedades Neuroinflamatorias , Daño por Reperfusión/metabolismoRESUMEN
INTRODUCTION: The role of extra-hypothalamic thyrotropin-releasing hormone (TRH) has been investigated by pharmacological studies using TRH or its analogues and found to produce a wide array of effects in the central nervous system. METHODS: Immunofluorescence, In situ labeling of DNA (TUNEL), in situ hybridization chain reaction and quantitative real-time polymerase chain reaction were used in this study. RESULTS: We found that the granular cells of the dentate gyrus expressed transiently a significant amount of TRH-like immunoreactivity and TRH mRNA during the 6-24 h period following global cerebral ischemia/reperfusion injury. TUNEL showed that apoptosis of neurons in the CA1 region occurred from 48 h and almost disappeared at 7 days. TRH administration 30 min before or 24 h after the injury could partially inhibit neuronal loss, and improve the survival of neurons in the CA1 region. CONCLUSION: These data suggest that endogenous TRH expressed transiently in the dentate gyrus of the hippocampus may play an important role in the survival of neurons during the early stage of ischemia/reperfusion injury and that delayed application of TRH still produced neuroprotection. This delayed application of TRH has a promising therapeutic significance for clinical situations.
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Isquemia Encefálica , Daño por Reperfusión , Animales , Isquemia Encefálica/metabolismo , Hipocampo/metabolismo , Péptidos/metabolismo , ARN Mensajero/metabolismo , Ratas , Daño por Reperfusión/metabolismo , Hormona Liberadora de Tirotropina/genética , Hormona Liberadora de Tirotropina/metabolismoRESUMEN
Ischemic stroke is the most serious disease that harms human beings. In principle, its treatment is to restore blood flow supply as soon as possible. However, after the blood flow is restored, it will lead to secondary brain injury, that is, ischemia-reperfusion injury. The mechanism of ischemia-reperfusion injury is very complicated. This study showed that P2X4 receptors in the pyramidal neurons of rat hippocampus were significantly upregulated in the early stage of ischemia-reperfusion injury. Neurons with high expression of P2X4 receptors are neurons that are undergoing apoptosis. Intraventricular injection of the P2X4 receptor antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) and PSB-12062 can partially block neuronal apoptosis, to promote the survival of neurons, indicating that ATP through P2X4 receptors is involved in the process of cerebral ischemia-reperfusion injury. Therefore, identifying the mechanism of neuronal degeneration induced by extracellular ATP via P2X4 receptors after ischemia-reperfusion will likely find new targets for the treatment of ischemia-reperfusion injury, and will provide a useful theoretical basis for the treatment of ischemia-reperfusion injury.
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Isquemia Encefálica/metabolismo , Región CA1 Hipocampal/metabolismo , Células Piramidales/metabolismo , Receptores Purinérgicos P2X4/biosíntesis , Daño por Reperfusión/metabolismo , Animales , Benzodiazepinonas/farmacología , Isquemia Encefálica/genética , Isquemia Encefálica/patología , Región CA1 Hipocampal/efectos de los fármacos , Expresión Génica , Masculino , Antagonistas del Receptor Purinérgico P2X/farmacología , Células Piramidales/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2X4/genética , Daño por Reperfusión/genética , Daño por Reperfusión/patologíaRESUMEN
PURPOSE: To detect the spatio-temporal expression of S100A4 in a spinal nerve ligation (SNL) rat model. Also to figure out which other molecules directly interact with S100A4 to explore the possible mechanisms which might be involved in neuropathic pain. METHODS: Seven-week-old male SD rats were used for the SNL model construction. Immunofluorescence and Western blotting were used to detect the spatio-temporal expression of S100A4 in the model. S100A4 was co-labeled with a number of related molecules and marker molecules that can distinguish between cell types. After intrathecal injection of S100A4 neutralizing antibody, the behavioral changes of SNL rats were recorded, and molecular changes compared. The direct interaction between S100A4 and other related molecules was verified by co-immunoprecipitation (co-IP) to explore its possible mechanism. RESULTS: After spinal nerve ligation, the content of S100A4 in the dorsal root ganglion (DRG) and spinal dorsal horn increased significantly. Intrathecal injection of S100A4 neutralizing antibody could effectively relieve the mechanical pain in rats. co-IP revealed a direct interaction between S100A4 and RAGE. CONCLUSION: The content of S100A4 in the DRG and spinal dorsal horn of SNL rats increased, compared with that of the control group. Intrathecal injection of S100A4 neutralizing antibody could effectively relieve the mechanical pain in SNL rats. S100A4 may be involved in the production of neuropathic pain through RAGE or other ways, but the specific mechanism needs to be further studied.
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In this study, the distribution patterns of P2X1 to P2X7 receptors in the anterior pituitary cells of rat were studied with single-, double-, and triple-labeling immunofluorescence, combined method of immunofluorescence and in situ hybridization, and Western blot. The results showed that the expression level of the P2X4 receptor protein was highest, followed by P2X5, P2X3, P2X2, P2X6, and P2X7 receptor proteins, but no P2X1 receptor protein was detected. Strong P2X4 receptor-immunoreactivity was detected in almost all the anterior pituitary cells. Different combinations of P2X receptors were detected in each individual cell type of the rat anterior pituitary. Gonadotrophs express P2X4, P2X5, and P2X6 receptors. Corticotrophs express P2X3 and P2X4 receptors. Folliculo-stellate cells express P2X2 and P2X4 receptors, and somatotrophs, lactotrophs, and thyrotrophs express only P2X4 receptors. The macrophages with Iba-1-ir expressed P2X7 receptors. The possible functions of these P2X receptors in each individual cell type of the rat anterior pituitary are discussed.
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Adenohipófisis/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Animales , Ratas , Ratas Sprague-DawleyRESUMEN
Intronic polymorphisms of the GABAA receptor ß2 subunit gene (GABRB2) under adaptive evolution were associated with schizophrenia and reduced expression, especially of the long isoform which differs in electrophysiological properties from the short isoform. The present study was directed to examining the gene dosage effects of Gabrb2 in knockout mice of both heterozygous (HT) and homozygous (KO) genotypes with respect to possible schizophrenia-like and comorbid phenotypes. The KO mice, and HT mice to a lesser extent, were found to display prepulse inhibition (PPI) deficit, locomotor hyperactivity, stereotypy, sociability impairments, spatial-working and spatial-reference memory deficits, reduced depression and anxiety, and accelerated pentylenetetrazol (PTZ)-induced seizure. In addition, the KO mice were highly susceptible to audiogenic epilepsy. Some of the behavioral phenotypes showed evidence of imprinting, gender effect and amelioration by the antipsychotic risperidone, and the audiogenic epilepsy was inhibited by the antiepileptic diazepam. GABAergic parvalbumin (PV)-positive interneuron dystrophy, astrocyte dystrophy, and extensive microglia activation were observed in the frontotemporal corticolimbic regions, and reduction of newborn neurons was observed in the hippocampus by immunohistochemical staining. The neuroinflammation indicated by microglial activation was accompanied by elevated brain levels of oxidative stress marker malondialdehyde (MDA) and the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). These extensive schizophrenia-like and comorbid phenotypes brought about by Gabrb2 knockout, in conjunction with our previous findings on GABRB2 association with schizophrenia, support a pivotal role of GABRB2 in schizophrenia etiology.
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Astrocitos/patología , Interneuronas/patología , Microglía/patología , Receptores de GABA/genética , Esquizofrenia/genética , Animales , Antipsicóticos/farmacología , Escala de Evaluación de la Conducta , Femenino , Predisposición Genética a la Enfermedad , Hipocampo/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Polimorfismo de Nucleótido Simple , Inhibición Prepulso/efectos de los fármacos , Receptores de GABA-A , Risperidona/farmacología , Esquizofrenia/tratamiento farmacológicoRESUMEN
With immunohistochemical and Western blot techniques, P2X1 receptors were detected in the whole mouse gastrointestinal tract and pancreatic islets of mouse and human. (1) δ Cells containing somatostatin (SOM) in the stomach corpus, small intestines, distal colon, pancreatic islets of both mouse and human express P2X1 receptors; (2) strong immunofluorescence of P2X1 receptors was detected in smooth muscle fibers and capillary networks of the villus core of mouse intestine; and (3) P2X1 receptor-immunoreactive neurons were also detected widely in both mouse myenteric and submucosal plexuses, all of which express SOM. The present data implies that ATP via P2X1 receptors is involved in SOM release from pancreatic δ cells, enteric neurons, and capillary networks in villi.
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Tracto Gastrointestinal/metabolismo , Islotes Pancreáticos/metabolismo , Receptores Purinérgicos P2X1/metabolismo , Células Secretoras de Somatostatina/metabolismo , Animales , Tracto Gastrointestinal/citología , Humanos , Islotes Pancreáticos/citología , Ratones , Plexo Mientérico/citología , Plexo Mientérico/metabolismo , Células Secretoras de Somatostatina/citologíaRESUMEN
The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen leads to ER stress, which is related to cellular reactive oxygen species production. Neuropathic pain may result from spinal dorsal horn (SDH) ER stress. In this study, we examined the cause-effect relationship between ER stress and neuropathic pain using the spinal nerve ligation (SNL) rat model. We showed that ER stress was mutually promotive with oxidative stress during the process. We also tested the hypothesis that spinal sensitization arose from reduced activities of GABA-ergic interneurons and that spinal sensitization was mediated by SDH ER stress. Other important findings in this study including the following: (1) nociceptive behavior was alleviated in SNL rat as long as tauroursodeoxycholic acid injections were repeated to inhibit ER stress; (2) inducing SDH ER stress in healthy rat resulted in mechanical hyperalgesia; (3) blocking protein disulfide isomerase pharmacologically reduced ER stress and nociceptive behavior in SNL rat; (4) cells in the dorsal horn with elevated ER stress were mainly neurons; and (5) whole-cell recordings made in slide preparations revealed significant inhibition of GABA-ergic interneuron activity in the dorsal horn with ER stress vs in the healthy dorsal horn. Taken together, results of the current study demonstrate that coregulation of ER stress and oxidative stress played an important role in neuropathic pain process. Inhibiting SDH ER stress could be a potential novel strategy to manage neuropathic pain.
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Estrés del Retículo Endoplásmico/fisiología , Neuralgia/metabolismo , Estrés Oxidativo/fisiología , Enfermedades del Sistema Nervioso Periférico/metabolismo , Médula Espinal/metabolismo , Animales , Masculino , Neuralgia/etiología , Neuronas/metabolismo , Técnicas de Placa-Clamp , Enfermedades del Sistema Nervioso Periférico/complicaciones , Ratas , Ratas WistarRESUMEN
The regulation of inflammation is pivotal for preventing the development or reoccurrence of multiple sclerosis (MS). A biased ratio of high-M1 versus low-M2 polarized microglia is a major pathological feature of MS Here, using microarray screening, we identify the long noncoding RNA (lncRNA) GAS5 as an epigenetic regulator of microglial polarization. Gain- and loss-of-function studies reveal that GAS5 suppresses microglial M2 polarization. Interference with GAS5 in transplanted microglia attenuates the progression of experimental autoimmune encephalomyelitis (EAE) and promotes remyelination in a lysolecithin-induced demyelination model. In agreement, higher levels of GAS5 are found in amoeboid-shaped microglia in MS patients. Further, functional studies demonstrate that GAS5 suppresses transcription of TRF4, a key factor controlling M2 macrophage polarization, by recruiting the polycomb repressive complex 2 (PRC2), thereby inhibiting M2 polarization. Thus, GAS5 may be a promising target for the treatment of demyelinating diseases.
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Microglía/fisiología , Esclerosis Múltiple/fisiopatología , ARN Largo no Codificante/genética , Diferenciación Celular , Enfermedades Desmielinizantes/fisiopatología , Encefalomielitis Autoinmune Experimental , Epigénesis Genética , Regulación de la Expresión Génica , Humanos , Inflamación , Macrófagos , Esclerosis Múltiple/genética , ARN Largo no Codificante/metabolismoRESUMEN
Traumatic brain injury (TBI) is the leading cause of death and disability for people under the age of 45 years worldwide. Neuropathology after TBI is the result of both the immediate impact injury and secondary injury mechanisms. Secondary injury is the result of cascade events, including glutamate excitotoxicity, calcium overloading, free radical generation, and neuroinflammation, ultimately leading to brain cell death. In this study, the P2X7 receptor (P2X7R) was detected predominately in microglia of the cerebral cortex and was up-regulated on microglial cells after TBI. The microglia transformed into amoeba-like and discharged many microvesicle (MV)-like particles in the injured and adjacent regions. A P2X7R antagonist (A804598) and an immune inhibitor (FTY720) reduced significantly the number of MV-like particles in the injured/adjacent regions and in cerebrospinal fluid, reduced the number of neurons undergoing apoptotic cell death, and increased the survival of neurons in the cerebral cortex injured and adjacent regions. Blockade of the P2X7R and FTY720 reduced interleukin-1ßexpression, P38 phosphorylation, and glial activation in the cerebral cortex and improved neurobehavioral outcomes after TBI. These data indicate that MV-like particles discharged by microglia after TBI may be involved in the development of local inflammation and secondary nerve cell injury.
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Lesiones Traumáticas del Encéfalo/patología , Guanidinas/farmacología , Microglía/patología , Antagonistas del Receptor Purinérgico P2X/farmacología , Quinolinas/farmacología , Receptores Purinérgicos P2X7/metabolismo , Animales , Lesiones Traumáticas del Encéfalo/metabolismo , Micropartículas Derivadas de Células/patología , Masculino , Microglía/efectos de los fármacos , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
Multiple sclerosis (MS) is a classical inflammatory demyelinating disease of the central nervous system (CNS). Microglia are the main resident immune cells in the CNS and are closely associated with the pathogenesis of MS. In the present study, we found that miR-30a was highly expressed in jellyfish-like microglia in chronic active lesions of MS patients, as well as in the microglia of mice with experimental autoimmune encephalomyelitis (EAE) at the chronic phase. In vitro, the conditioned supernatant of mouse microglia overexpressing miR-30a promoted the apoptosis of oligodendrocyte precursor cells (OPCs), and inhibited OPC differentiation. In vivo, overexpressing miR-30a in transplanted microglia exacerbated the progression of EAE. Overexpression and knock-down experiments in primary cultured mouse microglia showed that miR-30a increased the expression of IL-1ß and iNOS, which are pro-inflammatory, while inhibiting the expression of Ym-1 and CD206. Mechanistically, miR-30a inhibited the expression of Ppargc1b, which is the co-activator of peroxisome proliferator-activated receptor gamma, resulting in pro-inflammatory effects. Our work shows that miR-30a is an important regulator of the inflammatory response in microglia, and may be a promising therapeutic target for inflammatory diseases like MS in the CNS.
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Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , MicroARNs/metabolismo , Microglía/metabolismo , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Células Precursoras de Oligodendrocitos/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Increasing evidence suggests the potential involvement of metalloproteinase family proteins in the pathogenesis of neuropathic pain, although the underlying mechanisms remain elusive. METHODS: Using the spinal nerve ligation model, we investigated whether ADAM10 proteins participate in pain regulation. By implementing invitro methods, we produced a purified culture of satellite glial cells to study the underlying mechanisms of ADAM10 in regulating neuropathic pain. RESULTS: Results showed that the ADAM10 protein was expressed in calcitonin gene-related peptide (CGRP)-containing neurons of the dorsal root ganglia, and expression was upregulated following spinal nerve ligation surgery invivo. Intrathecal administration of GI254023X, an ADAM10 selective inhibitor, to the rats one to three days after spinal nerve ligation surgery attenuated the spinal nerve ligation-induced mechanical allodynia and thermal hyperalgesia. Intrathecal injection of ADAM10 recombinant protein simulated pain behavior in normal rats to a similar extent as those treated by spinal nerve ligation surgery. These results raised a question about the relative contribution of ADAM10 in pain regulation. Further results showed that ADAM10 might act by cleaving E-cadherin, which is mainly expressed in satellite glial cells. GI254023X reversed spinal nerve ligation-induced downregulation of E-cadherin and activation of cyclooxygenase 2 after spinal nerve ligation. ß-catenin, which creates a complex with E-cadherin in the membranes of satellite glial cells, was also downregulated by spinal nerve ligation surgery in satellite glial cells. Finally, knockdown expression of ß-catenin by lentiviral infection in purified satellite glial cells increased expression of inducible nitric oxide synthase and cyclooxygenase 2. CONCLUSION: Our findings indicate that neuron-derived ADAM10 production stimulates peripheral nerve injury-induced neuropathic pain by cleaving E-cadherin in satellite glial cells.
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Proteína ADAM10/biosíntesis , Cadherinas/metabolismo , Neuralgia/metabolismo , Neuronas/metabolismo , Células Satélites Perineuronales/metabolismo , Animales , Ganglios Espinales/metabolismo , Ligadura , Masculino , Traumatismos de los Nervios Periféricos/metabolismo , Ratas , Ratas Sprague-Dawley , Nervios EspinalesRESUMEN
Microglia are critical in the pathogenesis of neuropathic pain. In this study, we investigated the role of microvesicles (MVs) in neuropathic pain induced by spinal nerve ligation (SNL) in rats. First, we found that MVs shed from microglia were increased in the cerebrospinal fluid and dorsal horn of the spinal cord after SNL. Next, MVs significantly reduced paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). In addition, the P2X7-p38 pathway was related to the bleb of MVs after SNL. Interleukin (IL)-1ß was found to be significantly upregulated in the package of MVs, and PWT and PWL increased following inhibition with shRNA-IL-1ß. Finally, the amplitude and frequency of spontaneous excitatory postsynaptic currents increased following stimulation with MVs. Our results indicate that the P2X7-p38 pathway is closely correlated with the shedding of MVs from microglia in neuropathic pain, and MVs had a significant effect on neuropathic pain by participating in the interaction between microglia and neurons.
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Micropartículas Derivadas de Células/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Microglía/metabolismo , Neuralgia/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Nervios Espinales/lesiones , Animales , Modelos Animales de Enfermedad , Neuralgia/etiología , Traumatismos de los Nervios Periféricos/complicaciones , Ratas , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
A new multiple fluorescence in situ hybridization method based on hybridization chain reaction was recently reported, enabling simultaneous mapping of multiple target mRNAs within intact zebrafish and mouse embryos. With this approach, DNA probes complementary to target mRNAs trigger chain reactions in which metastable fluorophore-labeled DNA hairpins self-assemble into fluorescent amplification polymers. The formation of the specific polymers enhances greatly the sensitivity of multiple fluorescence in situ hybridization. In this study we describe the optimal parameters (hybridization chain reaction time and temperature, hairpin and salt concentration) for multiple fluorescence in situ hybridization via amplification of hybridization chain reaction for frozen tissue sections. The combined use of fluorescence in situ hybridization and immunofluorescence, together with other control experiments (sense probe, neutralization and competition, RNase treatment, and anti-sense probe without initiator) confirmed the high specificity of the fluorescence in situ hybridization used in this study. Two sets of three different mRNAs for oxytocin, vasopressin and somatostatin or oxytocin, vasopressin and thyrotropin releasing hormone were successfully visualized via this new method. We believe that this modified protocol for multiple fluorescence in situ hybridization via hybridization chain reaction would allow researchers to visualize multiple target nucleic acids in the future.
Asunto(s)
Hibridación Fluorescente in Situ/métodos , ARN Mensajero/genética , Animales , Encéfalo/metabolismo , Secciones por Congelación , Microscopía Fluorescente , RatasRESUMEN
Antidepressants including paroxetine, fluoxetine and desipramine are commonly used for treating depression. P2×7 receptors are member of the P2X family. Recent studies indicate that these receptors may constitute a novel potential target for the treatment of depression. In the present study, we examined the action of these antidepressants on cloned rat P2×7 receptors that were stably expressed in human embryonic kidney (HEK) 293 cells by using the whole-cell patch-clamp technique, and found that paroxetine at a dose of 10µM could significantly reduce the inward currents evoked by the P2×7 receptors agonist BzATP by pre-incubation for 6-12 but not by acute application (10µM) or pre-incubation for 2-6h at a dose of 1µM, 3µM or 10µM paroxetine. Neither fluoxetine nor desipramine had significant effects on currents evoked by BzATP either applied acutely or by pre-incubation at various concentrations. These results suggest that the sensitivity of rat P2×7 receptors to antidepressants is different, which may represent an unknown mechanism by which these drugs exert their therapeutic effects and side effects.