Migration Of Microglial Cells Research Articles

Netrin-1 Is an Important Mediator in Microglia Migration

Microglia migrate to the cerebral cortex during early embryonic stages. However, the precise mechanisms underlying microglia migration remain incompletely understood. As an extracellular matrix protein, Netrin-1 is involved in modulating the motility of diverse cells. In this paper, we found that Netrin-1 promoted microglial BV2 cell migration in vitro. Mechanism studies indicated that the activation of GSK3β activity contributed to Netrin-1–mediated microglia migration. Furthermore, Integrin α6/β1 might be the relevant receptor. Single-cell data analysis revealed the higher expression of Integrin α6 subunit and β1 subunit in microglia in comparison with classical receptors, including Dcc, Neo1, Unc5a, Unc5b, Unc5c, Unc5d, and Dscam. Microscale thermophoresis (MST) measurement confirmed the high binding affinity between Integrin α6/β1 and Netrin-1. Importantly, activation of Integrin α6/β1 with IKVAV peptides mirrored the microglia migration and GSK3 activation induced by Netrin-1. Finally, conditional knockout (CKO) of Netrin-1 in radial glial cells and their progeny led to a reduction in microglia population in the cerebral cortex at early developmental stages. Together, our findings highlight the role of Netrin-1 in microglia migration and underscore its therapeutic potential in microglia-related brain diseases.

Emodin relieves morphine-stimulated BV2 microglial activation and inflammation through the TLR4/NF-κB/NLRP3 pathway.

The objective of this study is to disclose the role of emodin, a natural anthraquinone derivative that has been proposed to suppress microglial activation and inflammation, in morphine tolerance. Here, cell counting kit-8 method assayed the viability of BV2 microglial cells treated by ascending concentrations of emodin. In emodin-pretreated BV2 microglial cells challenged with morphine with or without transfection of toll-like receptor 4 (TLR4) overexpression plasmids, transwell assay measured cell migration. Immunofluorescence staining and western blot detected the expression of microglial markers. Inflammatory levels were subjected to ELISA and western blot. BODIPY 581/591 C11 assay estimated lipid reactive oxygen species activity. Iron assay kit examined total iron content. Western blot tested the expression of ferroptosis- and TLR4/nuclear factor-kappaB (NF-κB)/NOD-like receptor 3 (NLRP3) pathway-associated proteins. Molecular docking predicted the binding affinity of emodin to TLR4. Emodin was noted to obstruct the migration, activation, inflammatory response, and ferroptosis of BV2 microglial cells induced by morphine. In addition, emodin had a high binding affinity with TLR4 and inactivated TLR4/NF-κB/NLRP3 pathway in morphine-challenged BV2 microglial cells. Upregulation of TLR4 partially countervailed the protective role of emodin against morphine-elicited BV2 microglial cell migration, activation, inflammation, and ferroptosis. Accordingly, emodin might target TLR4 and act as an inactivator of TLR4/NF-κB/NLRP3 pathway, thus inhibiting BV2 microglial activation and inflammation to mitigate morphine tolerance.

Lead inhibits microglial cell migration via suppression of store-operated calcium entry

Lead (Pb) is a non-biodegradable environmental pollutant that can lead to neurotoxicity by inducing neuroinflammation. Microglial activation plays a key role in neuroinflammation, and microglial migration is one of its main features. However, whether Pb affects microglial migration has not yet been elucidated. Herein, the effect of Pb on microglial migration was investigated using BV-2 microglial cells and primary microglial cells. The results showed that cell activation markers (TNF-α and CD206) in BV-2 cells were increased after Pb treatment. The migration ability of microglia was inhibited by Pb. Both store-operated calcium entry (SOCE) and the Ca2+ release-activated Ca2+ (CRAC) current were downregulated by microglia treatment with Pb in a dose-dependent manner. However, there was no statistical difference in the protein levels of stromal interaction molecule (STIM) 1, STIM2, or Ca2+ release-activated Ca2+ channel protein (Orai) 1 in microglia. The external Ca2+ influx and cell migration ability were restored to a certain extent after overexpression of either STIM1 or its CRAC activation domain in microglia. These results indicated that Pb inhibits microglial migration by downregulation of SOCE and impairment of the function of STIM1.

On the potential therapeutic role of taurine in retinal degenerative diseases

Taurine, considered a non‐essential amino acid that differs from other amino acids in that it contains a sulfonic acid instead of a carboxyl group, is the most abundant amino acid in the retina. Taurine plays a critical role in maintaining retinal health and the survival of retinal neurons, although its exact function in the retina is not yet fully understood. Interestingly, taurine shares structural similarities with β‐alanine, another non‐proteinogenic amino acid that can act as a competitive inhibitor of taurine receptors, allowing β‐alanine to be used to induce taurine depletion. β‐alanine induced taurine depletion results in the activation and migration of microglial cells in the retina, oxidative stress in the inner and outer nuclear and ganglion cell layers, loss of retinal synaptic connectivity, and impaired phagocytic capacity of retinal pigment epithelial cells. In addition, taurine depletion leads to photoreceptor and retinal ganglion cell loss. Most of these detrimental events are exacerbated by light exposure. These findings suggest that taurine levels are associated with retinal sensitivity to light.Given the important role of taurine in the retina, it has been proposed as a promising therapeutic candidate for retinal degenerative diseases. These diseases, which can be inherited or acquired, are a leading cause of irreversible blindness worldwide. Age‐related macular degeneration is the most common acquired cause of photoreceptor degeneration and a major public health concern due to the aging population. Retinitis pigmentosa is the most common inherited retinal degenerative disease, which is a heterogeneous group of sight‐threatening disorders. These diseases share photoreceptor degeneration as a common feature, typically due to genetic defects affecting the photoreceptors or the retinal pigment epithelium but can also be influenced by environmental factors. Additionally, inflammation and oxidative stress contribute to their development and progression. These diseases represent a major economic burden to healthcare systems and society. Therefore, it is imperative to develop preventive and/or therapeutic strategies that can impede or halt their progression.Taurine supplementation is a promising therapeutic approach for these diseases due to its antioxidant, anti‐apoptotic, and anti‐inflammatory properties. Our group has investigated the effects of taurine supplementation in the Royal College of Surgeons (RCS) rat, an established model of inherited retinal degeneration that manifests an impaired phagocytic capacity of the retinal pigment epithelium due to a MERKT gene mutation. Our findings demonstrate that taurine supplementation improves functional photoreceptor survival, decreases glial activation (including decreased GFAP expression in Müller cells and inhibition of microglial activation and migration), reduces oxidative stress in the outer and inner nuclear layers, suppresses apoptosis, and preserves synaptic connections in the RCS rat retina. Therefore, taurine is essential for retinal health and may provide neuroprotection against retinal degeneration thanks to its multifaceted properties.

Effect of Proinflammatory S100A9 Protein on Migration and Proliferation of Microglial Cells.

Alzheimer's disease (AD) is a multifactorial disease affecting aging population worldwide. Neuroinflammation became a focus of research as one of the major pathologic processes relating to the disease onset and progression. Proinflammatory S100A9 is the central culprit in the amyloid-neuroinflammatory cascade implicated in AD and other neurodegenerative diseases. We studied the effect of S100A9 on microglial BV-2 cell proliferation and migration. The responses of BV-2 cells to S100A9 stimulation were monitored in real-time using live cell microscopy, transcriptome sequencing, immunofluorescence staining, western blot analysis, and ELISA. We observed that a low dose of S100A9 promotes migration and proliferation of BV-2 cells. However, acute inflammatory condition (i.e., high S100A9 doses) causes diminished cell viability; it is uncovered that S100A9 activates TLR-4 and TLR-7 signaling pathways, leading to TNF-α and IL-6 expression, which affect BV-2 cell migration and proliferation in a concentration-dependent manner. Interestingly, the effects of S100A9 are not only inhibited by TNF-α and IL-6 antibodies. The addition of amyloid-β (Aβ) 1-40 peptide resumes the capacities of BV-2 cells to the level of low S100A9 concentrations. Based on these results, we conclude that in contrast to the beneficial effects of low S100A9 dose, high S100A9 concentration leads to impaired mobility and proliferation of immune cells, reflecting neurotoxicity at acute inflammatory conditions. However, the formation of Aβ plaques may be a natural mechanism that rescues cells from the proinflammatory and cytotoxic effects of S100A9, especially considering that inflammation is one of the primary causes of AD.

Treponema pallidum membrane protein Tp47 induced autophagy and inhibited cell migration in HMC3 cells via the PI3K/AKT/FOXO1 pathway.

The migratory ability of microglia facilitates their rapid transport to a site of injury to kill and remove pathogens. However, the effect of Treponema pallidum membrane proteins on microglia migration remains unclear. The effect of Tp47 on the migration ability and autophagy and related mechanisms were investigated using the human microglial clone 3 cell line. Tp47 inhibited microglia migration, the expression of autophagy-associated protein P62 decreased, the expression of Beclin-1 and LC3-II/LC3-I increased, and the autophagic flux increased in this process. Furthermore, autophagy was significantly inhibited, and microglial cell migration was significantly increased after neutralisation with an anti-Tp47 antibody. In addition, Tp47 significantly inhibited the expression of p-PI3K, p-AKT, and p-mTOR proteins, and the sequential activation of steps in the PI3K/AKT/mTOR pathways effectively prevented Tp47-induced autophagy. Moreover, Tp47 significantly inhibited the expression of p-FOXO1 protein and promoted FOXO1 nuclear translocation. Inhibition of FOXO1 effectively suppressed Tp47-induced activation of autophagy and inhibition of migration. Treponema pallidum membrane protein Tp47-induced autophagy and inhibited cell migration in HMC3 Cells via the PI3K/AKT/FOXO1 pathway. These data will contribute to understanding the mechanism by which T. pallidum escapes immune killing and clearance after invasion into the central nervous system.

Mitochonic acid 5 promotes the migration of mouse microglial BV‑2 cells in the presence of LPS‑induced inflammation via Mfn2‑associated mitophagy.

Effective strategies are needed to prevent the development of neuroinflammation, which is associated with nervous system disease,\\r\\nin patients. A previous study indicated that mitochonic acid 5 (MA‑5) may promote the survival of microglial cells via mitofusin 2 (Mfn2)‑associated mitophagy in response to lipopolysaccharide (LPS)‑induced inflammation. The current study investigated the role and underlying mechanisms of MA‑5 in the migration of BV‑2 cells following LPS‑mediated inflammation. The results of the present study revealed that MA‑5 promoted migration and upregulated the expression of F‑actin, C‑X‑C motif chemokine receptor (CXCR) 4 and CXCR7 in BV‑2 cells in response to LPS‑induced inflammation. The results also indicate that MA‑5 did not promote migration or upregulate the expression of F‑actin, CXCR4 or CXCR7 following the inhibition of Mfn2. Overall, the results of the present study suggest that MA‑5 may promote the migration of microglial cells via Mfn2‑associated mitophagy following LPS‑induced inflammation.

Microfluidic Wound-Healing Assay for ECM and Microenvironment Properties on Microglia BV2 Cells Migration

Microglia cells, as the resident immune cells of the central nervous system (CNS), are highly motile and migratory in development and pathophysiological conditions. During their migration, microglia cells interact with their surroundings based on the various physical and chemical properties in the brain. Herein, a microfluidic wound-healing chip is developed to investigate microglial BV2 cell migration on the substrates coated with extracellular matrixes (ECMs) and substrates usually used for bio-applications on cell migration. In order to generate the cell-free space (wound), gravity was utilized as a driving force to flow the trypsin with the device. It was shown that, despite the scratch assay, the cell-free area was created without removing the extracellular matrix coating (fibronectin) using the microfluidic assay. It was found that the substrates coated with Poly-L-Lysine (PLL) and gelatin stimulated microglial BV2 migration, while collagen and fibronectin coatings had an inhibitory effect compared to the control conditions (uncoated glass substrate). In addition, the results showed that the polystyrene substrate induced higher cell migration than the PDMS and glass substrates. The microfluidic migration assay provides an in vitro microenvironment closer to in vivo conditions for further understanding the microglia migration mechanism in the brain, where the environment properties change under homeostatic and pathological conditions.

Understanding the role of microglia in the onset of photoreceptor degeneration

AbstractPhotoreceptor degenerative diseases are currently the leading cause of irreversible blindness in developed countries and a major cause of irreversible blindness in the world. The most common forms of these diseases are age‐related macular degeneration and retinitis pigmentosa, both diseases cause a profound visual impairment due to photoreceptor loss. In the early stages of photoreceptor degeneration, glial cells have a major role to play, presumably leading to extensive retinal remodelling. The mammalian retina contains three types of glial cells. Two types of macroglial cells: astrocytes and Müller cells, and microglial cells. Microglial cells are the major resident immune cells in the retina and, although these cells and neuroinflammation have been implicated in the pathology of many neurodegenerative diseases, their role and function in photoreceptor degenerations are not yet fully understood. During retinal degeneration, microglial cells change their morphology, become activated and travel from the inner to the outer layers of the retina to phagocytose the dying photoreceptors, while astrocytes and Müller cells become hypertrophic, and overexpress glial fibrillary acidic protein (GFAP) filling the space left by dead photoreceptors to form a glial seal. It is known that activated microglial cells can be either neuroprotective or neurodestructive during photoreceptor degenerations, and therefore it is not yet clear whether activated microglial cells may increase or decrease photoreceptor loss. Microglial cell activation, migration and proliferation occur simultaneously with the onset of photoreceptor degeneration, prior to the loss of the vast majority of photoreceptors. During photoreceptor loss, microglial cells reach the outer nuclear and outer segment layers. These events suggest that photoreceptor death is the trigger for the activation and migration of microglia to the outer retinal layers, where they phagocytose the dying photoreceptors and remove cellular debris. By carrying out these functions, microglial cells in the retina may affect photoreceptor survival or death, eventually phagocytizing stressed but still viable photoreceptors. Interestingly, inhibition of microglial cell activation and migration during photoreceptor degeneration results in improved cone outer segment morphology and increased photoreceptor survival, which suggest that the role of activated microglial cells may be more neurodestructive than neuroprotective and that treatments with anti‐inflammatory drugs that modulate microglial reactivity may be considered in the early stages of photoreceptor degenerations.

Neuroprotective effects of intraocular bone marrow‐derived mononuclear cell transplants in inherited retinal degenerations

AbstractInherited retinal degenerations are diseases characterized by photoreceptor loss secondary to genetic defects. At present, only a minority are susceptible of genetic treatment and therefore are a frequent cause of blindness in infancy and adulthood.Retinitis Pigmentosa (RP) is the most frequent inherited retinal degeneration. This disease has a prevalence of around 1 in 4000 individuals and is caused by mutations in more than 300 genes. RP is characterized by progressive loss of photoreceptors, rods and then cones that may die secondarily to rod loss.The rats P23H and Royal college of Surgeon (RCS) are two well characterized animal models of RP because they bear genetic defects observed in RP patients. The P23H rat suffers an autosomal dominant mutation of the rhodopsin gene that causes protein misfolding and endoplasmic reticulum stress in rods, which die first followed by cones. The RCS rat has a mutation of MERTK gene, that impedes the normal photoreceptor outer segment phagocytosis by RPE cells thus causing simultaneous rod and cone degeneration.Therapies for inherited photoreceptor degeneration aim to prevent or delay death of the photoreceptors, or in specific circumstances try to replace them.The Bone marrow derived‐mononuclear cells (BM‐MNCs) are a mononuclear CD34+ subpopulation of bone marrow‐derived cells that can be separated from the other cellular fractions of the bone marrow aspirate using a Ficoll density gradient‐based protocol. The BM‐MNCs is a mixture of specific bone marrow cell subtypes in which is possible to find cells: Lin+, Lin−, CD34+, c‐Kit+, CD133+ or Sca‐1+ HSCs, endothelial precursor cells, and Mesenchymal Stem Cells.BM‐MNCs transplantation has shown promising results in different animal models of neuronal degeneration, including inherited photoreceptor degenerations.In our studies we have performed a syngeneic intravitreal or subretinal transplantation of BM‐MNCs in a P23H‐1 and RCS rats, and we have documented that both increase photoreceptor survival. They also improve the morphology of the photoreceptors outer segment, decrease synaptic degeneration in the outer plexiform layer and reduce glial fibrillary acidic protein (GFAP) expression in Müller cells. However, the transplanted cells did not penetrate the retina, only survived for up to 15 days, and did not modify microglial cell activation and migration or the electroretinographic responses. Their beneficial effects are maybe due to a paracrine trophic effect achieved through release of neurotrophic, anti‐angiogenic and/or immunomodulatory factors.In conclusion, the intra ocular transplantation BM‐MNCs shows photoreceptor neuroprotective actions in inherited retinal degenerations. These cells may have the potential to slow down photoreceptor loss, and therefore could be indicated for treatment of the early stages of photoreceptor degenerations.

Roles of Ca2+ activity in injury-induced migration of microglia in zebrafish in vivo

Microglia are the resident immune cells in the brain. It is well known that brain injury can activate the microglia and induce its directional migration towards the injury sites for exerting immune functions. While extracellular ATP released from the injury site mediates the directionality of activated microglia's migration, what endows activated microglia with migration capability remains largely unexplored. In the present study, we used the larval zebrafish as an in vivo model to visualize the dynamics of both morphology and Ca2+ activity of microglia during its migration evoked by local brain injury. We found that, in response to local injury, activated microglia exhibited an immediate Ca2+ transient and later sustained Ca2+ bursts during its migration towards the local injury site. Furthermore, suppression of Ca2+ activities significantly retarded microglial cell migration. Thus, our study suggests that intracellular Ca2+ activity is required for activated microglia's migration.

Activin A is a novel chemoattractant for migration of microglial BV2 cells

BackgroundMicroglia are involved in many neurodegenerative diseases and repairment of traumatic injury to the CNS. Activin A is a neurotrophic and neuroprotective factor that can regulate the activities of macrophages/microglia. However, the effects of activin A on the migration of microglia are still unclear. In this study, the role of activin A in regulation of the microglia migration was investigated with the murine microglial BV2 cell. MethodsThe levels of cytokines were detected by enzyme-linked immunosorbent assay (ELISA). The protein expression was examined by Western blotting. The adhesion of BV2 cells was assayed by real-time cell analysis (RTCA). The migration of BV2 cells was determined by transwell chamber and microfluidics device. Smad3 was overexpressed or knocked down in BV2 cells by transfection of Smad3 or Smad3 shRNA-expressing plasmids. ResultsActivin A inhibited the release of nitric oxide (NO) and inflammatory cytokines of TNF-α and IL-6 and the expression of TNF-α and IL-6 mRNA by BV2 cells. In contrast, activin A promoted the production of TGF-β1. Activin A inhibited adhesion, promoted wound healing and migration which is related to the expression of N-cadherin and E-cadherin expression. Additionally, Smad3 overexpression in BV2 cells decreased the levels of TNF-α and IL-6, and promoted the wound healing, whereas Smad3 knockdown showed the opposite effects. ConclusionsThese findings revealed that activin A regulated the biological behavior of BV2 cells via Smad3 signaling, suggesting that activin A may serve as a potential treatment target for neuroinflammation and glia scar formation in nervous system.

Intravitreal and subretinal syngeneic bone marrow mononuclear stem cell transplantation improves photoreceptor survival but does not ameliorate retinal function in two rat models of retinal degeneration.

To study and compare effects of syngeneic bone marrow mononuclear stem cells (BM-MNCs) transplants on inherited retinal degeneration in two animal models with different etiologies: the RCS and the P23H-1 rats. To compare the safety and efficacy of two methods of intraocular delivery: subretinal and/or intravitreal. A suspension of BM-MNCs was injected subretinally or intravitreally in the left eyes of P23H-1 and RCS rats at post-natal day (P) 21. At different survival intervals after the injection: 7, 15, 30 or 60 days, the retinas were cross-sectioned, and photoreceptor survival and glial cell responses were investigated using immunodetection of cones (anti-cone arrestin), synaptic connections (anti-bassoon), microglia (anti-Iba-1), astrocytes and Müller cells (anti-GFAP). Electroretinographic function was also assessed longitudinally. Intravitreal injections (IVIs) or subretinal injections (SRIs) of BM-MNCs did not produce adverse effects. The transplanted cells survived for up to 15 days but did not penetrate the retina. Both IVIs and SRIs increased photoreceptor survival, decreased synaptic degeneration and glial fibrillary acidic protein (GFAP) expression in Müller cells but did not modify microglial cell activation and migration or the electroretinographic responses. Intravitreal and subretinal syngeneic BM-MNCs transplantation decreases photoreceptor degeneration and shows anti-gliotic effects on Müller cells but does not ameliorate retinal function. Moreover, syngeneic BM-MNCs transplants are more effective than the xenotransplants of these cells. BM-MNC transplantation has potential therapeutic effects that merit further investigation.

Overexpression of Foxc1 ameliorates sepsis‑associated encephalopathy by inhibiting microglial migration and neuroinflammation through the IκBα/NF‑κB pathway.

Sepsis-associated encephalopathy (SAE) is a common and severe complication of sepsis. The cognitive dysfunction that ensues during SAE has been reported to be caused by impairments of the hippocampus. Microglia serves a key role in neuroinflammation during SAE through migration. Forkhead box C1 (Foxc1) is a member of the forkhead transcription factor family that has been found to regulate in cell migration. However, the role of Foxc1 in neuroinflammation during SAE remains unknown. In the present study, the mechanistic role of Foxc1 on microglial migration, neuroinflammation and neuronal apoptosis during the occurrence of cognitive dysfunction in SAE was investigated. A microglia-mediated inflammation model was induced by LPS in BV-2 microglial cells in vitro, whilst a SAE-related cognitive impairment model was established in mice using cecal ligation and perforation (CLP) surgery. Cognitive function in mice was evaluated using the Morris Water Maze (MWM) trial. Lipopolysaccharide (LPS) treatment was found to trigger BV-2 cell migration, inflammation and neuronal apoptosis. In addition, CLP surgery induced cognitive injury, which was indicated by longer latencies and shorter dwell times in the goal quadrant compared with those in the Sham group in the MWM trial. LPS treatment or CLP induction decreased the expression of Foxc1 and inhibitor of NF-κB (IκΒα) whilst increasing that of p65, IL-1β and TNF-α. After Foxc1 was overexpressed, the cognitive dysfunction of mice that underwent CLP surgery was improved, with the expression of IκBα also increased, microglial cell migration, the expression of p65, IL-1β and TNF-α and neuronal apoptosis were all decreased in vivo and in vitro, which were in turn reversed by the inhibition of IκBα in vitro. Overall, these results suggest that the overexpression of Foxc1 inhibited microglial migration whilst suppressing the inflammatory response and neuronal apoptosis by regulating the IκBα/NF-κB pathway, thereby improving cognitive dysfunction during SAE.

JQ1 inhibits high glucose-induced migration of retinal microglial cells by regulating the PI3K/AKT signaling pathway.

Diabetic retinopathy (DR) is one of the main leading causes of visual impairment worldwide. The current study elucidates the role of JQ1 in DR. A diabetic model was constructed by STZ injection and a high-fat diet. After establishment of the diabetic model, rats were assigned to treatment groups: 1) control, 2) diabetic model, and 3) diabetic+JQ1 model. In vitro Transwell and wound-healing assays were used to measure BV2 cell viability by stimulation with low glucose and high glucose with or without JQ1 and 740Y-P. Pathological methods were used to analyze DR, and Western blotting was used to analyze protein expression. Identification of enriched pathways in DR was performed by bioinformatics. Histopathological examination demonstrated that JQ1 rescued the loss of retinal cells and increased the thickness of retinal layers in diabetic rats. JQ1 attenuated high glucose-stimulated BV2 microglial motility and migration. The bioinformatics analysis implied that the Pl3K-Akt signaling pathway was enriched in DR. JQ1 decreased the phosphorylation of PI3K and AKT as well as the immunostaining of PI3K in BV2 cells. 740Y-P (a PI3K agonist) significantly reversed the decrease in p-PI3K and p-AK in BV2 cells. Additionally, JQ1 decreased the protein expression of p-PI3K, p-AKT, and MMP2/9 and immunostaining of PI3K in retinal tissues of rats. JQ1 suppresses the PI3K/Akt cascade by targeting MMP expression, thus decreasing the viability and invasion capacity of retinal microglia, suggesting an interesting treatment target for DR.

Tailored Therapeutic Doses of Dexmedetomidine in Evolving Neuroinflammation after Traumatic Brain Injury.

Understanding the secondary damage mechanisms of traumatic brain injury (TBI) is essential for developing new therapeutic approaches. Neuroinflammation has a pivotal role in secondary brain injury after TBI. Activation of NLRP3 inflammasome complexes results in the secretion of proinflammatory mediators and, in addition, later in the response, microglial activation and migration of the peripheral immune cells into the injured brain are observed. Therefore, these components involved in the inflammatory process are becoming a new treatment target in TBI. Dexmedetomidine (Dex) is an effective drug, widely used over the past few years in neurocritical care units and during surgical operations for sedation and analgesia, and has anti-inflammatory effects, which are shown in in vivo studies. The aim of this original research is to discuss the anti-inflammatory effects of different Dex doses over time in TBI. Brain injury was performed by using a weight-drop model. Half an hour after the trauma, intraperitoneal saline was injected into the control groups and 40 and 200μg/kg of Dex were given to the drug groups. Neurological evaluations were performed with the modified Neurological Severity Score before being killed. Then, the mice were killed on the first or the third day after TBI and histopathologic (hematoxylin-eosin) and immunofluorescent (Iba1, NLRP3, interleukin-1β, and CD3) findings of the brain tissues were examined. Nonparametric data were analyzed by using the Kruskal-Wallis test for multiple comparisons, and the Mann-Whitney U-test was done for comparing two groups. The results are presented as mean ± standard error of mean. The results showed that low doses of Dex suppress NLRP3 and interleukin-1β in both terms. Additionally, high doses of Dex cause a remarkable decrease in the migration and motility of microglial cells and T cells in the late phase following TBI. Interestingly, the immune cells were influenced by only high-dose Dex in the late phase of TBI and it also improves neurologic outcome in the same period. In the mice head trauma model, different doses of Dex attenuate neuroinflammation by suppressing distinct components of the neuroinflammatory process in a different timecourse that contributes to neurologic recovery. These results suggest that Dex may be an appropriate choice for sedation and analgesia in patients with TBI.

BAY61‑3606 attenuates neuroinflammation and neurofunctional damage by inhibiting microglial Mincle/Syk signaling response after traumatic brain injury.

Neuroinflammatory processes mediated by microglial activation and subsequent neuronal damage are the hallmarks of traumatic brain injury (TBI). As an inhibitor of the macrophage-inducible C-type lectin (Mincle)/spleen tyrosine kinase (Syk) signaling pathway, BAY61-3606 (BAY) has previously demonstrated anti-inflammatory effects on some pathological processes, such as acute kidney injury, by suppressing the inflammatory macrophage response. In the present study, the potential effects of BAY on microglial phenotype and neuroinflammation after TBI were investigated. BAY (3 mg/kg) was first administered into mice by intraperitoneal injection after TBI induction in vivo and microglia were also treated with BAY (2 µM) in vitro. The levels of inflammatory factors in microglia were assessed using reverse transcription-quantitative PCR and ELISA. Cortical neuron, myelin sheath, astrocyte and cerebrovascular endothelial cell markers were detected using immunofluorescence. The levels of components of the Mincle/Syk/NF-κB signaling pathway [Mincle, phosphorylated (p)-Syk and NF-κB], in addition to proteins associated with inflammation (ASC, caspase-1, TNF-α, IL-1β and IL-6), apoptosis (Bax and Bim) and tight junctions (Claudin-5), were measured via western blotting and ELISA. Migration and chemotaxis of microglial cells were evaluated using Transwell and agarose spot assays. Neurological functions of the mice were determined in vivo using the modified neurological severity scoring system and a Morris water maze. The results of the present study revealed that the expression levels of proteins in the Mincle/Syk/NF-κB signaling pathway (including Mincle, p-Syk and p-NF-κB), inflammatory cytokines (TNF-α, IL-1β and IL-6), proteins involved in inflammation (ASC and caspase-1), apoptotic markers (Bax and Bim) and the tight junction protein Claudin-5 were significantly altered post-TBI. BAY treatment reversed these effects in both the cerebral cortex extract-induced cell model and the controlled cortical impact mouse model. BAY was also revealed to suppress activation of the microglial proinflammatory phenotype and microglial migration. In addition, BAY effectively attenuated TBI-induced neurovascular unit damage and neurological function deficits. Taken together, these findings provided evidence that BAY may inhibit the Mincle/Syk/NF-κB signaling pathway in microglia; this in turn could attenuate microglia-mediated neuroinflammation and improve neurological deficits following TBI.

Pharmacological modulation of cytokines correlating neuroinflammatory cascades in epileptogenesis.

Epileptic seizure-induced brain injuries include activation of neuroimmune response with activation of microglia, astrocytes cells releasing neurotoxic inflammatory mediators underlies the pathophysiology of epilepsy. A wide spectrum of neuroinflammatory pathways is involved in neurodegeneration along with elevated levels of inflammatory mediators indicating the neuroinflammation in the epileptic brain. Therefore, the neuroimmune response is commonly observed in the epileptic brain, indicating elevated cytokine levels, providing an understanding of the neuroinflammatory mechanism contributing to seizures recurrence. Clinical and experimental-based evidence suggested the elevated levels of cytokines responsible for neuronal excitation and blood-brain barrier (BBB) dysfunctioning causing the drug resistance in epilepsy. Therefore, the understanding of the pathogenesis of neuroinflammation in epilepsy, including migration of microglial cells releasing the inflammatory cytokines indicating the correlation of elevated levels of inflammatory mediators (interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) triggering the generation or recurrence of seizures. The current review summarized the knowledge regarding elevated inflammatory mediators as immunomodulatory response correlating multiple neuroinflammatory NF-kB, RIPK, MAPK, ERK, JNK, JAK-STAT signaling cascades in epileptogenesis. Further selective targeting of inflammatory mediators provides beneficial therapeutic strategies for epilepsy.

PACAP and VIP Modulate LPS-Induced Microglial Activation and Trigger Distinct Phenotypic Changes in Murine BV2 Microglial Cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two structurally related immunosuppressive peptides. However, the underlying mechanisms through which these peptides regulate microglial activity are not fully understood. Using lipopolysaccharide (LPS) to induce an inflammatory challenge, we tested whether PACAP or VIP differentially affected microglial activation, morphology and cell migration. We found that both peptides attenuated LPS-induced expression of the microglial activation markers Iba1 and iNOS (### p < 0.001), as well as the pro-inflammatory mediators IL-1β, IL-6, Itgam and CD68 (### p < 0.001). In contrast, treatment with PACAP or VIP exerted distinct effects on microglial morphology and migration. PACAP reversed LPS-induced soma enlargement and increased the percentage of small-sized, rounded cells (54.09% vs. 12.05% in LPS-treated cells), whereas VIP promoted a phenotypic shift towards cell subpopulations with mid-sized, spindle-shaped somata (48.41% vs. 31.36% in LPS-treated cells). Additionally, PACAP was more efficient than VIP in restoring LPS-induced impairment of cell migration and the expression of urokinase plasminogen activator (uPA) in BV2 cells compared with VIP. These results suggest that whilst both PACAP and VIP exert similar immunosuppressive effects in activated BV2 microglia, each peptide triggers distinctive shifts towards phenotypes of differing morphologies and with differing migration capacities.

NF-κB activation in retinal microglia is involved in the inflammatory and neovascularization signaling in laser-induced choroidal neovascularization in mice

PurposeTo evaluate Nuclear Factor NF-κB (NF-κB) signaling on microglia activation, migration, and angiogenesis in laser-induced choroidal neovascularization (CNV). MethodsNine-week-old C57BL/6 male mice were randomly assigned to IMD-0354 treated or untreated groups (5 mice, 10 eyes per group). CNV was induced with a 532-nm laser. Laser spots (power 250 mW, spot size 100 μm, time of exposure 50 ms) were created in each eye using a slit-lamp delivery system. Selective inhibitor of nuclear factor kappa-B kinase subunit beta (IKK2) inhibitor IMD-0354 (10 μg) was delivered subconjunctivally; vehicle-treated mice were the control. The treatment effect on CNV development was assessed at five days post-CNV induction in vivo in C57BL/6 and Cx3cr1gfp/wt mice by fluorescent angiography, fundus imaging, and ex vivo by retinal flatmounts immunostaining and Western blot analysis of RPE/Choroidal/Scleral complexes (RCSC) lysates. In vitro evaluations of IMD-0354 effects were performed in the BV-2 microglial cell line using lipopolysaccharide (LPS) stimulation. ResultsIMD-0354 caused a significant reduction in the fluorescein leakage and size of the laser spot, as well as a reduction in microglial cell migration and suppression of phospho-IκBα, Vascular endothelial growth factor (VEGF-A), and Prostaglandin-endoperoxide synthase 2 (COX-2). In vivo and ex vivo observations demonstrated reduced lesion size in mice, CD68, and Allograft inflammatory factor 1 (IBA-1) positive microglia cells migration to the laser injury site in IMD-0354 treated eyes. The data further corroborate with GFP-positive cells infiltration of the CNV site in Cx3cr1wt/gfp mice. In vitro IMD-0354 (10–25 ng/ml) treatment reduced NF-κB activation, expression of COX-2, caused decreased Actin-F presence and organization, resulting in reduced BV-2 cells migration capacity. ConclusionThe present data indicate that NF-κB activation in microglia and it's migration capacity is involved in the development of laser CNV in mice. Its suppression by NF-κB inhibition might be a promising therapeutic strategy for wet AMD.