Signalling mechanisms for ROS-induced TRPM2 mediated microglial cell activation

Taming of macrophage and microglial cell activation by microRNA-124

Copper (Cu2+) is vital for proper brain function, serving as a cofactor in essential biological processes. However, excessive levels of Cu2+ can disrupt redox homeostasis, resulting in oxidative stress. TRPM2 channel is a Ca2+-permeable channel that is highly sensitive to oxidative stress and is known to be critical in mediating cellular processes. The signaling mechanisms involved in TRPM2 channel activation, which lead to microglial cell activation, remain poorly elucidated. Therefore, in the present study, we investigated the mechanisms underlying TRPM2 channel and microglial activation induced by Cu2+. Treatment of Cu2+ for 8h significantly increased microglial intracellular Ca2+ concentration ([Ca2+]i), and such results were prevented by the presence of TRPM2 inhibitors, 2-APB and ACA. Extended exposure to Cu2+ (10-100μM) for 24h induced microglial cell activation, as exemplified by the morphological changes from elongated and small cell body to amoeboid-like shape. This Cu2+-induced microglial activation was remarkably inhibited by TRPM2 blockers. Poly(ADP-ribose) polymerase 1 (PARP-1) activates the TRPM2 channel by producing its primary activator, ADP-ribose (ADPR). Here, we found that the increases in [Ca2+]i and microglial morphological changes were attenuated by PJ-34 and DPQ, PARP-1 inhibitors. In addition, a noticeable increase in intracellular ROS level was observed in microglial cells induced by Cu2+, and such an increase was suppressed by the inhibition of protein kinase C (PKC) and NADPH oxidase (NOX). Interestingly, the increases in [Ca2+]i and microglial activation were also attenuated by the PKC and NOX inhibitors. Altogether, these data suggest that PKC and NOX mediate ROS production, leading to the PARP-dependent TRPM2 activation, which subsequently results in a Ca2+ response that is responsible for microglial cell activation.

Objective To observe the relationship between retinal microglial activations and ganglion cell (RGC) damages in early-stage diabetic rats. Methods A total of 20 Sprague-Dawley(SD)rats were randomly divided into 4 groups (each with 5 rats): 1 month control group, 1 month diabetes group, 3 month control group, 3 month diabetes group. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ). The RGCs of all rats were retrograde labeled by carbocyanine dye DiI injected at the superior colliculi. Microglial cells and RGCs in retinal flat-mounts and sections were stained immunohistochemically and recorded under confocal microscope. Results The diabetic microglial cells were amoeboid and ovoid with fewer processes on retinal flat-mounts. The density of microglial cells which phagocytosed Dil particles in the RGC layer significantly increased in the 3-month diabetes group(P<0.01). The density of microglial cells in the RGC layer significantly increased in the 1- and 3- month diabetes group (P<0. 05). However there were more microglial cells in the RGC layer in the 3-month diabetes group than the 1-month diabetes group (P < 0.0001 ). Significant correlation was found between the amount of microglial cells and that of RGCs in the early-stage of diabetes. Conclusions Microglial cell activation has close relationship with the RGC damages in early-stage diabetic rats. Key words: Diabetic retinopathy/Phisiopathology; Microglia/physiology; Retinal ganglion cells/physiology; Diabetes mellitus, experimental

Macrophage and Microglial Cell Response after Common Peroneal Nerve Cut and Crush in C57BL/6J Mice

Acute cerebral dysfunction is a pathological state common in severe infections and a pivotal determinant of long-term cognitive outcomes. Current evidence indicates that a loss of synaptic contacts orchestrated by microglial activation is central in sepsis-associated encephalopathy. However, the upstream signals that lead to microglial activation and the mechanism involved in microglial-mediated synapse dysfunction in sepsis are poorly understood. This study investigated the involvement of the NLRP3 inflammasome in microglial activation and synaptic loss related to sepsis. We demonstrated that septic insult using the cecal ligation and puncture (CLP) model induced the expression of NLRP3 inflammasome components in the brain, such as NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3), apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), caspase-1, and IL-1β. Immunostaining techniques revealed increased expression of the NLRP3 inflammasome in microglial cells in the hippocampus of septic mice. Meanwhile, an in vitro model of primary microglia stimulated with LPS exhibited an increase in mitochondrial reactive oxygen species (ROS) production, NLRP3 complex recruitment, and IL-1β release. Pharmacological inhibition of NLRP3, caspase-1, and mitochondrial ROS all decreased IL-1β secretion by microglial cells. Furthermore, we found that microglial NLRP3 activation is the main pathway for IL-1β-enriched microvesicle (MV) release, which is caspase-1-dependent. MV released from LPS-activated microglia induced neurite suppression and excitatory synaptic loss in neuronal cultures. Moreover, microglial caspase-1 inhibition prevented neurite damage and attenuated synaptic deficits induced by the activated microglial MV. These results suggest that microglial NLRP3 inflammasome activation is the mechanism of IL-1β-enriched MV release and potentially synaptic impairment in sepsis.

Genetic Rescue Reverses Microglial Activation in Preclinical Models of Retinitis Pigmentosa.

Neurons induce the activation of microglial cells in vitro.

Microglial cells are resident macrophages in the brain and their activation is an important part of the brain immune response and the pathology of the major CNS diseases. Microglial activation is triggered by pathological signals and is characterized by morphological changes, proliferation, phagocytosis and the secretion of various cytokines and inflammatory mediators, which could be both destructive and protective for the nervous tissue. Purines are one of the most important mediators which regulate different aspects of microglial function. They could be released to the extracellular space from neurons, astrocytes and from the microglia itself, upon physiological neuronal activity and in response to pathological stimuli and cellular damage. Microglial activation is regulated by various subtypes of nucleotide (P2X, P2Y) and adenosine (A1, A2A and A3) receptors, which control ionic conductances, membrane potential, gene transcription, the production of inflammatory mediators and cell survival. Among them, the role of P2X7 receptors is especially well delineated, but P2X4, various P2Y, A1, A2A and A3 receptors also powerfully participate in the microglial response. The pathological role of microglial purine receptors has also been demonstrated in disease models; e.g., in ischemia, sclerosis multiplex and neuropathic pain. Due to their upregulation and selective activation under pathological conditions, they provide new avenues in the treatment of neurodegenerative and neuroinflammatory illnesses.

Clozapine reduces Toll-like receptor 4/NF-κB-mediated inflammatory responses through inhibition of calcium/calmodulin-dependent Akt activation in microglia

Botulinum toxin A dampened inflammatory response in BV-2 microglial cells

Motoneurons with naturally elevated calcium binding protein content, such as parvalbumin, are more resistant against injury. Furthermore, increase of intracellular calcium, which plays a pivotal role in injury of neurons, could be moderated by elevating their calcium binding proteins. To test whether by elevating parvalbumin content of motoneurons, activation of neighboring microglial cells, a robust component of the inflammatory reaction after injury, could be influenced. Mice overexpressing neuronal parvalbumin were derived and the spinal motoneurons were challenged by cutting the sciatic nerve. At postoperative days 1, 4, 7, 14 and 21 the change of the chemokine ligand 2 immunostaining in the motoneurons and the activation of microglial cells, measured as alterations in CD11b immunostaining were determined. Calcium level of motoneurons was tested electron microscopically at postoperative day 7. After axotomy, increased level of chemokine ligand 2 was detected in the lumbar motoneurons. The staining intensity reached its maximum at day 7 and decayed faster in transgenic mice compared to controls. Microglial activation around motoneurons attenuated faster in parvalbumin overexpressing mice, too, but the decrease of microglial activation was delayed compared to the decline of the chemokine ligand 2 signal. At the time when the microglial reaction peaked, no intracellular calcium increase was detected in the motoneurons of transgenic mice, in contrast to the twofold increase in wild type animals. Increased calcium buffering capacity, which augments resistance of motoneurons against calcium-mediated injury, leads to earlier termination of motoneuronal emission of CCL2 followed by a reduction of neighboring microglial activation after axotomy.

We observed highly aggressively proliferating immortalized (HAPI) cells growing in cultures that had been enriched for microglia. The cells were initially obtained from mixed glial cultures prepared from 3-day-old rat brains. HAPI cells are typically round with few or no processes when cultured in 10% serum containing medium. As the percentage of serum in the medium is decreased, the HAPI cells have more processes. HAPI cells stain for the isolectin B4, OX-42, and GLUT5, which are markers for microglial cells, but the cells do not immunolabel with A2B5, a marker of cells in the oligodendroglial cell lineage, or with the astrocyte-specific marker, glial fibrillary aciidic protein (GFAP). In addition, HAPI cells are capable of phagocytosis. We conclude that HAPI cells are of microglia/macrophage lineage. Exposing HAPI cells to lipopolysaccharide (LPS) induces the mRNAs for tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). LPS exposure also induces secretion of TNF-alpha and production of nitric oxide (NO) in HAPI cells. Because activation of microglia is associated with an increase in iron accumulation and ferritin expression, we tested the hypothesis that iron status affects the production of TNF-alpha and NO. Our studies demonstrate that both iron chelation and iron loading diminished the LPS-induced effect of TNF-alpha and NO. The results of this study indicate that HAPI cells possess the characteristics of microglia/brain macrophages, providing an alternative cell culture model for the study of microglia. In addition, we demonstrate that the activation of microglial cells could be modified by iron.

Exposure to short-lasting impulse noise causes microglial and astroglial cell activation in the adult rat brain

Objective To investigate the regulatory effect of dehydroepiandrosterone (DHEA) on the microglial activation after subarachnoid hemorrhage (SAH) in vivo and in vitro. Methods C57BL/6 mice were used for in vivo experiments. A SAH model was induced by intravascular puncture. They were randomly divided into solvent group, model group, and DHEA pretreatment group. TUNEL staining was used to detect neuronal apoptosis level at 24 h after modeling. Iba-1/CD86 fluorescence double staining was used to detect the activation of microglia. Quantitative fluorescent polymerase chain reaction and Western blot analysis were used to detect the expression of inflammatory factors, including interleukin (IL) -1β, IL-6, tumor necrosis factor (TNF) -α, and inducible nitric oxide synthase (iNOS). The primary cultured microglia was used for in vitro experiments and it was simulated SAH by hemoglobin stimulation. They were randomly divided into control group, model group, and DHEA pretreatment group. Iba-1/CD86 fluorescence double staining was used to detect the microglial activation, and fluorescence quantitative polymerase chain reaction and Western blot analysis were used to detect the expression of inflammatory factors. Results In vivo model experiments showed that DHEA significantly reduced neuronal apoptosis (P<0.01) and microglial activation (P<0.01) after SAH modeling, and IL-6 expression level significantly decreased (P<0.01), while IL-1β, TNF-α and iNOS showed a downward trend, but there were no statistical differences. In vitro model experiments showed that DHEA could significantly inhibit microglial activation (P<0.001) and the expression levels of various inflammatory factors (P<0.001). Conclusions DHEA pretreatment can reduce neuronal apoptosis and microglia activation after SAH, and it has neuroprotective effect. Key words: Subarachnoid haemorrhage; Microglia; Dehydroepiandrosterone; Inflammation; Neuroprotective agents; Disease models, animal; Mice

Objective To investigate the effects of melatonin on activation of microglia and the changes of superoxide dismutase (SOD) and malondialdehyde (MDA) after intracerebral hemorrhage (ICH) in rats. Methods One hundred and thirty male SD rats were randomly divided into four groups: normal group,sham-operated group,intracerebral hemorrhage model group (Model group) and melatonin intervention group (MT group). Each group was further divided into 5 subgroups respectively at 12 h,1 d, 2 d,4 d and 7 d after modeling. The ICH models were made in SD rats by using Rosenberg methods. Melatonin in dose of 10 mg/ kg in solution of 1 mg/ mL was given intraperitoneally to rats of MT group. The morphology of microglia was observed under electron microscope. OX42-positive cells were detected by immunohistochemical (ABC) methods. The contents of MDA or the activity of SOD was measured respectively by thiobarbituric acid or xanthine oxidation method. Results Electron microscope showed that the activation of microglia cell displayed in ameboid shape and swelling of neuron in hemisphere cortex in model group at 2 d after ICH,the activation of microglia cell of the cortex was insignificant in MT group. OX42-positive microglia cell in large amount surrounded the hematoma at 12 h after ICH,peaked at 1 d and lasted for 7 d. OX42-positive microglia cells in MT group were significantly fewer than that in model group at different intervals after ICH (P <0.05). The content of MDA in model group increased significantly after ICH,and higher than that in normal group at 7 d,[(0.875 ±0.098) nmol/ mg prot vs. (0.725 ±0.061) nmol/ mg prot,P <0.05,whereas the activity of SOD changes in the opposite direction,(70.46 ±3.12) U/ mg prot vs. (85.86 ±4.95) /U/ mg prot,P <0.05. Compared with model group,the content of MDA were lower and the activity of SOD were higher in MT group after ICH (P <0.05). Conclusion Melatonin provides a protective effect on the damage of nerve cell after ICH. The mechanism might be associated with melatonin by reducing the level of oxidative stress in brain tissue and attenuating the activation of microglia after ICH. Key words: Melatonin; Intracerebral hemorrhage; Microglia; Oxidative stress; Superoxide dismutase; Rats