Enhanced meningeal lymphatic drainage ameliorates lipopolysaccharide-induced brain injury in aged mice.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is an acute cerebral dysfunction caused by sepsis. Neuroinflammation induced by sepsis is considered a potential mechanism of SAE; however, very little is known about the role of the meningeal lymphatic system in SAE. METHODS: Sepsis was established in male C57BL/6J mice by intraperitoneal injection of 5 mg/kg lipopolysaccharide, and the function of meningeal lymphatic drainage was assessed. Adeno-associated virus 1-vascular endothelial growth factor C (AAV1-VEGF-C) was injected into the cisterna magna to induce meningeal lymphangiogenesis. Ligation of deep cervical lymph nodes (dCLNs) was performed to induce pre-existing meningeal lymphatic dysfunction. Cognitive function was evaluated by a fear conditioning test, and inflammatory factors were detected by enzyme-linked immunosorbent assay. RESULTS: The aged mice with SAE showed a significant decrease in the drainage of OVA-647 into the dCLNs and the coverage of the Lyve-1 in the meningeal lymphatic, indicating that sepsis impaired meningeal lymphatic drainage and morphology. The meningeal lymphatic function of aged mice was more vulnerable to sepsis in comparison to young mice. Sepsis also decreased the protein levels of caspase-3 and PSD95, which was accompanied by reductions in the activity of hippocampal neurons. Microglia were significantly activated in the hippocampus of SAE mice, which was accompanied by an increase in neuroinflammation, as indicated by increases in interleukin-1 beta, interleukin-6 and Iba1 expression. Cognitive function was impaired in aged mice with SAE. However, the injection of AAV1-VEGF-C significantly increased coverage in the lymphatic system and tracer dye uptake in dCLNs, suggesting that AAV1-VEGF-C promotes meningeal lymphangiogenesis and drainage. Furthermore, AAV1-VEGF-C reduced microglial activation and neuroinflammation and improved cognitive dysfunction. Improvement of meningeal lymphatics also reduced sepsis-induced expression of disease-associated genes in aged mice. Pre-existing lymphatic dysfunction by ligating bilateral dCLNs aggravated sepsis-induced neuroinflammation and cognitive impairment. CONCLUSION: The meningeal lymphatic drainage is damaged in sepsis, and pre-existing defects in this drainage system exacerbate SAE-induced neuroinflammation and cognitive dysfunction. Promoting meningeal lymphatic drainage improves SAE. Manipulation of meningeal lymphangiogenesis could be a new strategy for the treatment of SAE.

The Sustained Antidepressant Effects of Ketamine Are Independent of the Lateral Habenula.

Ketamine is known to have a rapid and lasting antidepressant effect. Recent studies have shown that ketamine exerts it rapid antidepressant effect by blocking burst firing in the lateral habenula (LHb). Whether the sustained antidepressant effect of ketamine occurs through the same mechanism has not been explored. Here, using male rats, we found that local infusion of (R,S)-ketamine into the LHb resulted in a rapid antidepressant-like effect 1 h after infusion, which almost returned to baseline levels after 24 h. Intra-LHb injection of (S)-ketamine also showed a significant antidepressant-like effect 1 h after injection, which recovered at 24 h. No significant antidepressant-like effect was found at 1 or 24 h after the administration of (R)-ketamine into the LHb. Injection of (2R,6R)-hydroxynorketamine, a ketamine metabolite, into the LHb did not result in any obvious antidepressant-like effect 1 or 24 h after injection. Systemic administration of (R,S)-ketamine (intraperitoneally) significantly suppressed LHb bursting activity at 1 h, but the inhibitory effect was reversed 24 h after injection. No significant effect of (R,S)-ketamine on miniature excitatory postsynaptic potentials of LHb neurons was found at 1 or 24 h after systemic application. Our study demonstrated that the sustained antidepressant-like effect of ketamine may not depend on burst firing of LHb neurons.SIGNIFICANCE STATEMENT Ketamine exerts it rapid antidepressant effect by blocking burst firing in the lateral habenula (LHb). However, whether the sustained antidepressant effect of ketamine occurs through the same mechanism has not been explored. In the present study, we demonstrated that the sustained antidepressant effect of ketamine may not depend on the burst firing of LHb neurons. This finding may lead to a novel perspective on LHb in the antidepressant effect of ketamine.

Exosomal miR-409-3p secreted from activated mast cells promotes microglial migration, activation and neuroinflammation by targeting Nr4a2 to activate the NF-κB pathway.

OBJECTIVE: Neuroinflammation plays a critical role in central nervous system diseases. Exosomal miRNAs released from various cells are implicated in cell-to-cell communication. Prior studies have placed substantial emphasis on the role of cytokines in mast cell-microglia interactions during neuroinflammation. However, it has never been clearly determined whether exosomal miRNAs participate in the interaction between mast cells and microglia and thus mediate neuroinflammation. METHODS: The characteristics of exosomes isolated from cell culture supernatants were confirmed by transmission electron microscopy (TEM), nanoparticle-tracking analysis (NTA) and Western blot. The transfer of PKH67-labelled exosomes and Cy3-labelled miR-409-3p was observed by fluorescence microscopy. Migration and activation of murine BV-2 microglial cells were evaluated through Transwell assays and immunofluorescence staining for Iba1 and CD68. CD86, IL-1ß, IL-6 and TNF-α were assessed via qRT-PCR and ELISA. MiR-409-3p was detected by qRT-PCR. Nr4a2 and NF-κB levels were measured by western blot. Regulatory effects were identified by luciferase reporter assays. RESULTS: Lipopolysaccharide (LPS)-stimulated murine P815 mast cells secreted exosomes that were efficiently taken up by murine BV-2 cells, which promoted murine BV-2 cell migration and activation. LPS-P815 exosomes increased the CD86, IL-1ß, IL-6 and TNF-α levels in murine BV-2 microglia. Furthermore, activated mast cells delivered exosomal miR-409-3p to murine BV-2 microglia. Upregulated miR-409-3p promoted murine BV-2 microglial migration, activation and neuroinflammation by targeting Nr4a2 to activate the NF-κB pathway. CONCLUSION: Exosomal miR-409-3p secreted from activated mast cells promotes microglial migration, activation and neuroinflammation by targeting Nr4a2 to activate the NF-κB pathway, which provides evidence that not only cytokines but also exosomal miRNAs participate in neuroinflammation. In the future, targeting exosomal miRNAs may provide new insights into neuroinflammation.

The effect of anesthesia depth on radiofrequency catheter ablation of ventricular tachycardia: a retrospective study.

BACKGROUND: Radiofrequency catheter ablation (RFCA) as a safe and effective method has been widely used in ventricular tachycardia (VT) patients, and with which anesthesiologists frequently manage their perioperative care. The aim of this study was to investigate the effects of different anesthetic depths on perioperative RFCA and recurrence in patients who with intractable VT and could not tolerate an awake procedure. METHODS: We reviewed electronic medical records of patients with VT who underwent RFCA by general anesthesia from January 2014 to March 2019. According to intraoperative VT induction, they were divided into two groups: non-inducible group (group N) and inducible group (group I). We constructed several multivariable regression models, in which covariates included patient characteristics, comorbidities, protopathy and bispectral index (BIS) value. RESULTS: One hundred one patients were analyzed. Twenty-nine patients (28.7%) experienced VT no induction, and 26 patients (25.7%) relapsed within 1 year. Compared with group I, the proportion of patients with arrhythmogenic right ventricular cardiomyopathy in group N were higher (P < 0.05), and the recurrence rate of VT was significantly higher (51.7% vs 15.3%) (P < 0.05). The BIS value in group N was significantly lower (P < 0.01), in addition, the BIS < 40 was associated with elevated odds of VT no induction compared with a BIS > 50 (odds ratio, 6.92; 95% confidence interval, 1.47-32.56; P = 0.01). VT no induction was an independent predictor of recurrence after RFCA (odds ratio, 5.01; 95% confidence interval, 1.88-13.83; P < 0.01). CONCLUSION: Lower BIS value during VT induction in RFCA operation was associated with high risk of VT no induction, which affects postoperative outcomes. We proposed that appropriate depth of anesthesia should be maintained during the process of VT induction.

Histamine 2/3 receptor agonists alleviate perioperative neurocognitive disorders by inhibiting microglia activation through the PI3K/AKT/FoxO1 pathway in aged rats.

BACKGROUND: Microglia, the principal sentinel immune cells of the central nervous system (CNS), play an extensively vital role in neuroinflammation and perioperative neurocognitive disorders (PND). Histamine, a potent mediator of inflammation, can both promote and prevent microglia-related neuroinflammation by activating different histamine receptors. Rat microglia express four histamine receptors (H1R, H2R, H3R, and H4R), among which the histamine 1 and 4 receptors can promote microglia activation, whereas the role and cellular mechanism of the histamine 2 and 3 receptors have not been elucidated. Therefore, we evaluated the effects and potential cellular mechanisms of histamine 2/3 receptors in microglia-mediated inflammation and PND. METHODS: This study investigated the role of histamine 2/3 receptors in microglia-induced inflammation and PND both in vivo and in vitro. In the in vivo experiments, rats were injected with histamine 2/3 receptor agonists in the right lateral ventricle and were then subjected to exploratory laparotomy. In the in vitro experiments, primary microglia were pretreated with histamine 2/3 receptor agonists before stimulation with lipopolysaccharide (LPS). Cognitive function, microglia activation, proinflammatory cytokine production, NF-κb expression, M1/M2 phenotypes, cell migration, and Toll-like receptor-4 (TLR4) expression were assessed. RESULTS: In our study, the histamine 2/3 receptor agonists inhibited exploratory laparotomy- or LPS-induced cognitive decline, microglia activation, proinflammatory cytokine production, NF-κb expression, M1/M2 phenotype transformation, cell migration, and TLR4 expression through the PI3K/AKT/FoxO1 pathway. CONCLUSION: Based on our findings, we conclude that histamine 2/3 receptors ameliorate PND by inhibiting microglia activation through the PI3K/AKT/FoxO1 pathway. Our results highlight histamine 2/3 receptors as potential therapeutic targets to treat neurological conditions associated with PND.

Mast Cell Deficiency Protects Mice from Surgery-Induced Neuroinflammation.

Neuroinflammation plays a key role in the occurrence and development of neurodegenerative diseases. Microglia, the resident immune cells in the brain, have been recognized to contribute to neuroinflammation. Previous studies have shown that activated mast cells may be involved in surgery-induced neuroinflammation and neuronal apoptosis by using pharmacological methods. This study is aimed at ascertaining the exactly role of mast cells on neuroinflammation with the mast cell-deficient mice. Adult male C57BL6/J wild-type (WT) and mast cell-deficient (C57BL6/J KitWsh/Wsh (Wsh)) mice underwent tibial fracture surgery. Blood-brain barrier (BBB) breakdown, microglial activation, and neuroinflammatory levels were examined at 1 day after surgery. Surgery-induced BBB breakdown, microglial activation, and neuroinflammatory levels were significantly, pharmacologically reduced using a mast cell stabilizer, cromolyn sodium in WT mice (P < 0.05). These results were reproduced with mast cell deficiency. WT mice administered intraventricularly with cromolyn exhibited reduced BBB breakdown, microglial activation, and neuroinflammatory levels versus vehicle (P < 0.05). But there was no effect of cromolyn versus vehicle in Wsh mice, clarifying the specificity of cromolyn on brain mast cells. These findings demonstrated that activated mast cells promote surgery-induced BBB breakdown and neuroinflammation in mice, and open up a new therapeutic target for neuroinflammation-related diseases.

The Mast Cell Is an Early Activator of Lipopolysaccharide-Induced Neuroinflammation and Blood-Brain Barrier Dysfunction in the Hippocampus.

Neuroinflammation contributes to or even causes central nervous system (CNS) diseases, and its regulation is thus crucial for brain disorders. Mast cells (MCs) and microglia, two resident immune cells in the brain, together with astrocytes, play critical roles in the progression of neuroinflammation-related diseases. MCs have been demonstrated as one of the fastest responders, and they release prestored and newly synthesized mediators including histamine, ß-tryptase, and heparin. However, temporal changes in MC activation in this inflammation process remain unclear. This study demonstrated that MC activation began at 2 h and peaked at 4 h after lipopolysaccharide (LPS) administration. The number of activated MCs remained elevated until 24 h after LPS administration. In addition, the levels of histamine and ß-tryptase in the hippocampus markedly and rapidly increased within 6 h and remained higher than the baseline level within 24 h after LPS challenge. Furthermore, mast cell-deficient KitW-sh/W-sh mice were used to investigate the effects of MCs on microglial and astrocytic activation and blood-brain barrier (BBB) permeability at 4 h after LPS stimulation. Notably, LPS-induced proinflammatory cytokine secretion, microglial activation, and BBB damage were inhibited in KitW-sh/W-sh mice. However, no detectable astrocytic changes were found in WT and KitW-sh/W-sh mice at 4 h after LPS stimulation. Our findings indicate that MC activation precedes CNS inflammation and suggest that MCs are among the earliest participants in the neuroinflammation-initiating events.

Pro-inflammatory role of high-mobility group box-1 on brain mast cells via the RAGE/NF-κB pathway.

High-mobility group box-1 (HMGB-1) acts as a pro-inflammatory cytokine contributing to the occurrence of many central inflammatory and infectious disorders. Brain mast cells (MCs) are the first responders to peripheral inflammatory stimulation because of their rapid response to external stimuli coupled with their release of preformed and newly synthesized reactive chemicals. Little is known about the involvement of brain MCs in the pro-inflammatory effects of HMGB-1 on the central nervous system (CNS). Thus, we investigated the activation process of MCs by HMGB-1 and explored whether this process is involved in the pro-inflammatory effects of HMGB-1 on the CNS. In this study, we used P815 cells to study the activating role of HMGB-1 on MCs and to explore its potential mechanism in vitro. In an in vivo study, adult male Sprague-Dawley rats received i.c.v. injection of sterile saline or cromoglycate (stabilizer of MCs) 30 min prior to i.p. injection of HMGB-1. Increased levels of tumor necrosis factor and IL-1ß were observed in the P815 cells, as well as in the rats' brains, after HMGB-1 treatment. Pretreatment with the receptor of advanced glycation endproducts (RAGE)-siRNA inhibited the HMGB-1-induced inflammatory process in the P815 cells. Activation of the RAGE/nuclear factor-κB (NF-κB) pathway was observed in both the P815 cells and rats' brains. In addition, HMGB-1 induced the accumulation of brain MCs in the hippocampal CA1 region, and the blood-brain barrier was disrupted. Pretreatment with cromoglycate, a stabilizer of MCs, mitigated these HMGB-1-induced pro-inflammatory processes in rats. These findings indicate that brain MCs are involved in the pro-inflammatory effect of HMGB-1 on the CNS, probably via activating the RAGE/NF-κB pathway.

Preoperative Sleep Disturbance Exaggerates Surgery-Induced Neuroinflammation and Neuronal Damage in Aged Mice.

Postoperative cognitive dysfunction (POCD) is defined as new cognitive impairment (memory impairment and impaired performance) after surgery, especially in aged patients. Sleep disturbance is a common phenomenon before surgery that has been increasingly thought to affect patient recovery. However, little is known about the functional impact of preoperative sleep disturbance on POCD. Here, we showed that tibial fracture surgery induced cognitive deficit and production of proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß, along with microglia and astrocyte activation, neuronal damage, and blood-brain barrier (BBB) disruption. Preoperative sleep disturbance enhanced the surgery-induced neuroinflammation, neuronal damage, BBB disruption, and memory impairment 24 h after surgery. Taken together, these results demonstrated that preoperative sleep disturbance aggravated postoperative cognitive function in aged mice and the mechanism may be related to central nervous system (CNS) inflammation and neuronal damage.

IL-17A contributes to perioperative neurocognitive disorders through blood-brain barrier disruption in aged mice.

BACKGROUND: Perioperative neurocognitive disorders (PND) occur frequently after surgery, especially in aged patients. Surgery-induced neuroinflammation and blood-brain barrier (BBB) dysfunction play a crucial role in the pathogenesis of PND. Interleukin-17A (IL-17A) increases after surgical stress and will be involved in BBB dysfunction. However, the effect of IL-17A on BBB function during PND remains poorly understood. METHODS: Male wild-type C57BL/6J mice (15 months old) received tibial fracture surgery and fixation to establish the PND model. All the mice were injected intraperitoneally with an IL-17A-neutralizing antibody (Abs) or isotype-control Abs 30 min before tibial fracture surgery. Animal behaviour tests conducted 24 h after surgery included the contextual fear conditioning and Y maze tests. Serum and hippocampus IL-17A levels and hippocampus IL-6 and IL-1ß levels were detected by ELISA. BBB function was detected by Evans blue (EB) test. Hippocampus matrix metalloproteinase-2 (MMP-2)- and MMP-9-positive cells were detected by immunohistochemistry. Hippocampus albumin, occludin, claudin-5 and IL-17A receptors were detected by Western blot. For the in vitro experiment, bEnd.3 cells were incubated with IL-17A. Cell IL-17A receptors were detected by immunofluorescence. Cellular MMP-2, MMP-9, occludin, and claudin-5 were detected by Western blot. RESULTS: Tibial fracture surgery promoted memory impairment, increased levels of IL-17A and IL-17A receptors, inflammatory factor production and BBB dysfunction. IL-17A Abs inhibited this effect, including improving memory function, decreasing inflammatory factor production and alleviating BBB disruption, indicated by decreased tight junctions (TJs) and increased MMPs after surgery. The in vitro study suggested that recombinant IL-17A could upregulate the expression of IL-17A receptors, decrease TJs and increase the level of MMPs in bEnd.3 cells. CONCLUSIONS: Our results suggested that IL-17A-promoted BBB disruption might play an important role in the pathogenesis of PND.

Effect of tryptase on mouse brain microvascular endothelial cells via protease-activated receptor 2.

BACKGROUND: Mast cells (MCs), the 'first responders' in brain injury, are able to disrupt the blood-brain barrier (BBB), but the underlying mechanism is not well understood. Tryptase is the most abundant MC secretory product. Protease-activated receptor 2 (PAR-2) has been identified as a specific receptor for tryptase, which is abundantly expressed in brain microvascular endothelial cells. The BBB comprises brain microvascular endothelial cells that display specialised molecular properties essential for BBB function and integrity. Therefore, the purpose of the present study was to investigate the effects of tryptase on mouse brain microvascular endothelial cell line bEnd3 and its potential mechanisms of action. METHODS: Induction of mouse brain microvascular endothelial cell activation by tryptase was examined. Then, mouse brain microvascular endothelial cells were pretreated with a PAR-2 antagonist and stimulated with tryptase. Cellular activation, proinflammatory cytokine production, expression of PAR-2, Toll-like receptors (TLRs) and mitogen-activated protein kinases (MAPK), nuclear factor kappa B (NF-kappa B) phosphorylation were assessed. RESULTS: Tryptase upregulated the production of VCAM-1, MMPs (MMP9 and MMP2), TLR4 and TNF-α and downregulated the expression of the tight junction proteins occludin and claudin-5 in mouse brain microvascular endothelial cell. Among the MAPK and NF-kappa B pathway, ERK and NF-kappa B were activated by tryptase. All of these effects could be eliminated by the PAR-2 inhibitor. CONCLUSION: Based on our findings, we conclude that tryptase can trigger brain microvascular endothelial cell activation and proinflammatory mediator release. These findings may further clarify the involvement and mechanism of tryptase in BBB disruption.

Histamine upregulates the expression of histamine receptors and increases the neuroprotective effect of astrocytes.

BACKGROUND: Astrocytes have attracted increasing attention over recent decades for their role in neuroinflammation. Histamine, a major aminergic brain neurotransmitter, has an important influence on the main activities of astrocytes, such as ion homeostasis, energy metabolism, and neurotransmitter clearance. However, little is known about the impact of histamine on astrocyte immunomodulatory function. METHODS: The expression of all known histamine receptor subtypes was examined in primary astrocytes. Then, primary astrocytes were pretreated with selective histamine receptor antagonists and stimulated with histamine. Cellular activation, proinflammatory cytokine production, and expression of neurotrophic factors were assessed. RESULTS: Astrocytes could constitutively express three histamine receptors (H1R, H2R, and H3R), and these three histamine receptors could be selectively upregulated to varying degrees upon histamine treatment. Histamine also dose-dependently stimulated astrocyte activation and subsequent production of glial cell-derived neurotrophic factor (GDNF), whereas it suppressed the secretion of the proinflammatory factors tumor necrosis factor-alpha (TNF-α) and interleukin-1ß (IL-1ß). The effects of histamine were completely abolished by either an H1R or H3R antagonist, while an H2R antagonist attenuated the effects partly. CONCLUSIONS: The present study identified the expression of H1R, H2R, and H3R on astrocytes. We also demonstrated that negative regulation of astrocytic TNF-α and IL-1ß production and the enhancement of astrocytic GDNF stimulated by histamine were receptor-mediated processes in which all three of the expressed histamine receptors (H1R, H2R, and H3R) were involved. These findings may further clarify the involvement and mechanism of astrocyte activation in neuroinflammation.

Induction of Microglial Activation by Mediators Released from Mast Cells.

BACKGROUND/AIMS: Microglia are the resident immune cells in the brain and play a pivotal role in immune surveillance in the central nervous system (CNS). Brain mast cells are activated in CNS disorders and induce the release of several mediators. Thus, brain mast cells, rather than microglia, are the "first responders" due to injury. However, the functional aspects of mast cell-microglia interactions remain uninvestigated. METHODS: Conditioned medium from activated HMC-1 cells induces microglial activation similar to co-culture of microglia with HMC-1 cells. Primary cultured microglia were examined by flow cytometry analysis and confocal microscopy. TNF- alpha and IL-6 were measured with commercial ELISA kits. Cell signalling was analysed by Western blotting. RESULTS: In the present study, we found that the conditioned medium from activated HMC-1 cells stimulated microglial activation and the subsequent production of the pro-inflammatory factors TNF-α and IL-6. Co-culture of microglia and HMC-1 cells with corticotropin-releasing hormone (CRH) for 24, 48 and 72 hours increased TNF-α and IL-6 production. Antagonists of histamine receptor 1 (H1R), H4R, proteinase-activated receptor 2 (PAR2) or Toll-like receptor 4 (TLR4) reduced HMC-1-induced pro-inflammatory factor production and MAPK and PI3K/AKT pathway activation. CONCLUSIONS: These results imply that activated mast cells trigger microglial activation. Interactions between mast cells and microglia could constitute a new and unique therapeutic target for CNS inflammation-related diseases.

Lithium Ameliorates LPS-Induced Astrocytes Activation Partly via Inhibition of Toll-Like Receptor 4 Expression.

BACKGROUND/AIMS: Astrocytes are critical for the development of postoperative cognitive dysfunction (POCD). In addition, astrocytes express toll-like receptors 4 (TLR4) and build up responses to innate immune triggers by releasing pro-inflammatory molecules. The pathogenesis of neurological disorders often involves the activation of astrocytes and associated inflammatory processes. Lithium, a primary drug for the treatment of bipolar disorder, has recently been suggested to have a role in neuroprotection during neurodegenerative diseases. In this study, we aimed to investigate whether lithium can ameliorate LPS-induced astrocytes activation via inhibition of TLR4 expression. METHODS: Primary astrocytes cells were pretreated with lithium and stimulated with lipopolysaccharide (LPS). Cellular activation, cytokine production, and TLR4 expression, were assessed. RESULTS: Lithium significantly inhibited LPS-induced astrocytes activation and pro-inflammatory cytokine production, as well as LPS-induced TLR4 expression. CONCLUSIONS: Lithium can inhibit LPS-induced TLR4 expression and astrocytes activation. These results indicate that lithium plays an important role in astrocytes activation and neuroinflammation-related diseases, which may open new avenues for neuroscience and biomedical research, and also offers new insight into the treatment of POCD.

Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells.

BACKGROUND/AIMS: Microglia are an essential player in central nervous system inflammation. Recent studies have demonstrated that the astrocytic chemokine, CCL2, is associated with microglial activation in vivo. However, CCL2-induced microglial activation has not yet been studied in vitro. The purpose of the current study was to understand the role of astrocyte-derived CCL2 in microglial activation and to elucidate the underlying mechanism(s). METHODS: Primary astrocytes were pre-treated with CCL2 siRNA and stimulated with TNF-α. The culture medium (CM) was collected and added to cultures of microglia, which were incubated with and without CCR2 inhibitor. Microglial cells were analyzed by quantitative RT-PCR to determine whether they polarized to the M1 or M2 state. Microglial migratory ability was assessed by transwell migration assay. RESULTS: TNF-α stimulated the release of CCL2 from astrocytes, even if the culture media containing TNF-α was replaced with fresh media after 3 h. CM from TNF-α-stimulated astrocytes successfully induced microglial activation, which was ascertained by increased activation of M1 and enhanced migration ability. In contrast, CM from astrocytes pretreated with CCL2 siRNA showed no effect on microglial activation, compared to controls. Additionally, microglia pre-treated with RS102895, a CCR2 inhibitor, were resistant to activation by CM from TNF-α-stimulated astrocytes. CONCLUSION: This study demonstrates that the CCL2/CCR2 pathway of astrocyte-induced microglial activation is associated with M1 polarization and enhanced migration ability, indicating that this pathway could be a useful target to ameliorate inflammation in the central nervous system.

Activated brain mast cells contribute to postoperative cognitive dysfunction by evoking microglia activation and neuronal apoptosis.

BACKGROUND: Neuroinflammation plays a key role in the occurrence and development of postoperative cognitive dysfunction (POCD). Microglia, the resident immune cells in the brain, has been increasingly recognized to contribute to neuroinflammation. Although brain mast cells (MCs) are the "first responder" in the brain injury rather than microglia, little is known about the functional aspects of MCs-microglia interactions. METHODS: Male Sprague-Dawley (SD) rats were injected intracerebroventricular with MC stabilizer Cromolyn (100 µg/µl), MC stimulator C48/80 (1 µg/µl), or sterile saline 30 min before open tibial fracture surgery, and the levels of neuroinflammation and memory dysfunction were tested 1 and 3 days after surgery. In addition, the effect of activated MCs on microglia and neurons was determined in vitro. RESULTS: Tibial fracture surgery induced MCs degranulation, microglia activation, and inflammatory factors production, which initiated the acute brain inflammatory response and neuronal death and exhibited cognitive deficit. Site-directed preinjection of the "MCs stabilizer" disodium cromoglycate (Cromolyn) inhibited this effect, including decrease of inflammatory cytokines, reduced MCs degranulation, microglia activation, neuronal death, and improved cognitive function 24 h after the surgery. In vitro study, we found that the conditioned medium from lipopolysaccharide (LPS)-stimulated mast cells line (P815) could induce primary microglia activation through mitogen-activated protein kinase (MAPK) pathway signaling and subsequent production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). In addition, the activated P815 could directly induce neuronal apoptosis and synapse injury with microglia independently. Cromolyn could inhibit P815 activation following improved microglia activation and neuronal loss. CONCLUSIONS: These results implicate that activated MCs could trigger microglia activation and neuronal damage, resulting in central nervous system (CNS) inflammation, and communications of MCs with microglia and neuron could constitute a new and unique therapeutic target for CNS immune inflammation-related diseases.

α-Lipoic acid inhibits sevoflurane-induced neuronal apoptosis through PI3K/Akt signalling pathway.

Sevoflurane is a widely used anaesthetic agent, including in anaesthesia of children and infants. Recent studies indicated that the general anaesthesia might cause the cell apoptosis in the brain. This issue raises the concerns about the neuronal toxicity induced by the application of anaesthetic agents, especially in the infants and young children. In this study, we used Morris water maze, western blotting and immunohistochemistry to elucidate the role of α-lipoic acid in the inhibition of neuronal apoptosis. We found that sevoflurane led to the long-term cognitive impairment in the young rats. This adverse effect may be caused by the neuronal death in the hippocampal region, mediated through PI3K/Akt signalling pathway. We also showed that α-lipoic acid offset the effect of sevoflurane on the neuronal apoptosis and cognitive dysfunction. This study elucidated the potential clinical role of α-lipoic acid, providing a promising way in the prevention and treatment of long-term cognitive impairment induced by sevoflurane general anesthesia.

Enhancement of LPS-induced microglial inflammation response via TLR4 under high glucose conditions.

BACKGROUND: Microglia activation mediated by toll-like receptor 4 (TLR4) plays an important role in neuroinflammation and postoperative cognitive dysfunction (POCD). Diabetes mellitus (DM) has been recently suggested as an independent risk factor for POCD. In this study, we investigate the potential exacerbation of the inflammatory response in primary microglia due to high glucose conditions. METHODS: Primary microglial cells were exposed to normal glucose (25 mmol/L) and high glucose (35 mmol/L) levels alone or with lipopolyscaccharide (LPS 0, 2, 5, 10 ng/mL). The pro-inflammatory response of the cells was assessed by measuring changes in cytokine levels and the evaluation of associated signaling pathways. RESULTS: Neither high glucose nor low LPS (≤5 ng/ml) alone had an effect on TNF-a and IL-6 levels, but the combination of low LPS and high glucose stimulated the inflammatory response. Analyses of the associated signaling pathways demonstrated that high glucose enhanced the LPS-induced microglial activation via the TLR4/JAK2/STAT3 pathway. CONCLUSION: This study demonstrates that high glucose, one of the key abnormalities characteristic of DM, can augment LPS-induced microglial activation and inflammatory cytokine levels through the TLR4/JAK2/STAT3 pathway, offering new insight into the pathophysiological relationship between DM and POCD.

IL-17A is implicated in lipopolysaccharide-induced neuroinflammation and cognitive impairment in aged rats via microglial activation.

BACKGROUND: Neuroinflammation is considered a risk factor for impairments in neuronal function and cognition that arise with trauma, infection, and/or disease. IL-17A has been determined to be involved in neurodegenerative diseases such as multiple sclerosis. Recently, IL-17A has been shown to be upregulated in lipopolysaccharide(LPS)-induced systemic inflammation. This study aims to explore the role of IL-17A in LPS-induced neuroinflammation and cognitive impairment. METHODS: Male Sprague-Dawley (SD) rats were injected intraperitoneally with LPS (500 µg/kg), and IL-17A expression in serum and in the hippocampus was examined 6, 12, 24, and 48 h later. Then, we investigated whether IL-17A-neutralizing antibodies (IL-17A Abs, 1 mg/kg) prevented neuroinflammation and memory dysfunction in aged rats that received LPS (500 µg/kg) injection. In addition, the effect of IL-17A on microglial activation in vitro was determined using ELISA and immunofluorescence. RESULTS: LPS injection increased the expression of IL-17A in serum and in the hippocampus. IL-17A Abs improved LPS-induced memory impairment. In addition, IL-17A Abs prevented the LPS-induced expression of TNF-α, IL-6 and inflammatory proteins, and of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) as well as the activation of microglia in the brain. IL-17A Abs also inhibited the expression of amyloid precursor protein (APP) and BACE1 and increased the expression of the synaptic marker PSD95 in the aged rats treated with LPS. In an in vitro study, we found that recombinant IL-17A could simulate microglial activation and increase production of pro-inflammatory cytokines. CONCLUSION: Taken together, our results suggest that IL-17A was involved in LPS-induced neuroinflammation and cognitive impairment in aged rats via microglial activation. Anti-IL-17A may represent a new therapeutic strategy for the treatment of endotoxemia-induced neuroinflammation and cognitive dysfunction.