Microglial Cell Cultures Research Articles

GET73 modulates lipopolysaccharide- and ethanol-induced increase in cytokine/chemokine levels in primary cultures of microglia of rat cerebral cortex.

- Alcohol-induced pro-inflammatory activation might influence cellular and synaptic pathology, thus contributing to the behavioral phenotypes associated with alcohol use disorders. In the present study, the possible anti-inflammatory properties of N-[(4-trifluoromethyl)-benzyl]4-methoxybutyramide (GET73), a promising therapeutic agent for alcohol use disorder treatment, were evaluated in primary cultures of rat cortical microglia. - Primary cultures of cerebral cortex microglial cells were treated with 100 ng/ml lipopolysaccharide (LPS; 8h, 37°C) or 75 mM ethanol (EtOH; 4 days, 37°C) alone or in the presence of GET73 (1-30 µM). At the end of the incubation period, multiparametric quantification of cytokines/chemokines was performed by using the xMAP technology and Luminex platform. Furthermore, cultured microglial cell viability following the treatment with EtOH and GET73, alone or in combination, has been measured by a colorimetric assay (i.e. MTT assay). - GET73 (10 and 30 µM) partially or fully prevented the LPS-induced increase of IL-6, IL-1β, RANTES/CCL5 protein and MCP-1/CCL2 levels. On the contrary, GET73 failed to attenuate the TNF-α level increase induced by LPS. Furthermore, GET73 treatment (10-30 µM) significantly attenuated or prevented the EtOH-induced increase of TNF-α, IL-6, IL-1β and MCP-1/CCL2 levels. Finally, at all the concentrations tested (1-30 µM), the GET73 treatment did not alter the EtOH-induced reduction of microglial cell viability. - The current results provide the first in vitro evidence of GET73 protective properties against EtOH-induced neuroinflammation. These data add more information on the complex and multifactorial profile of action of the compound, further supporting the significance of developing GET73 as a therapeutic tool for the treatment of individuals with alcohol use disorders.

Identification of human circulating factors following remote ischemic conditioning (RIC): Potential impact on stroke

Remote ischemic conditioning (RIC) is a procedure consisting of short cycles of ischemia applied in a limb that activates endogenous protection in distant organs, such as the brain. Despite the promising outcomes of RIC, the biochemical factors governing inter-organ communication remain largely unexplored, particularly in humans. A pilot study on 20 healthy humans was performed to identify potential circulating biochemical factors involved in RIC signalling. Blood was collected before and immediately, 4 and 22 h after the end of RIC. To characterize the responses triggered by RIC, a combination of biochemical and proteomic analysis, along with functional in vitro tests in human cells, were performed.RIC did not alter the levels of nitric oxide, bilirubin and cell-free mitochondrial DNA. In contrast, carboxyhaemoglobin levels increased following RIC at all time points and young subset, suggesting endogenous production of carbon monoxide that is a cytoprotective gasotransmitter. Additionally, the levels of glutathione and cysteinylglycine bound to proteins also increased after RIC, while glutathione catabolism decreased. Plasma proteomic analysis identified overall 828 proteins. Several steps of statistical analysis (Student's t-test, repeated measures ANOVA, with Holm corrected pairwise p-values <0.05 threshold and fold change higher or lower than 100 %) leaded to the identification of 9 proteins with altered circulating levels in response to RIC at 4h and 22h. All 9 proteins are from extracellular space or exosomes, being involved in inflammation, angiogenesis or metabolism control. In addition, RIC-conditioned plasma from young subjects protected microglial cell culture against inflammatory stimuli, indicating an anti-inflammatory effect of RIC. Nevertheless, other functional tests in neurons or endothelial cells had no effect. Overall, we present some evidence for RIC-induced anti-inflammatory and antioxidant responses in healthy human subjects, in particular in young subjects. This study is a first step towards the disclosure of signalling factors involved in RIC-mediated inter-organ communication.

Sodium alginate hydrogel encapsulating microglia cell lysate subjected to serum starvation for mitigating glioma cells.

Glioma is the most common malignant tumor in the brain, accounting for over 80% of all primary intracranial tumors. The current clinical treatment has shown certain limitations. Although M1 type microglia can secrete various pro-inflammatory cytokines and are expected to be used for glioma treatment, direct use of microglia may lead to overactivation and trigger immune storms. Therefore, we first found that serum starvation can stimulate the transformation of microglia into M1 type. Subsequently, we found through comparative experiments that the inhibitory effect of microglial cell lysis medium on glioma cells was stronger than that of microglial cell culture medium. Finally, we successfully prepared sodium alginate hydrogel loaded with microglia lysis solution to achieve sustained inhibitory effect on the growth of glioma and avoid its proliferation.

G6PD deficiency mediated impairment of iNOS and lysosomal acidification affecting phagocytotic clearance in microglia in response to SARS-CoV-2

The glucose-6-phosphate dehydrogenase (G6PD) deficiency is X-linked and is the most common enzymatic deficiency disorder globally. It is a crucial enzyme for the pentose phosphate pathway and produces NADPH, which plays a vital role in regulating the oxidative stress of many cell types. The deficiency of G6PD primarily causes hemolytic anemia under oxidative stress triggered by food, drugs, or infection. G6PD-deficient patients infected with SARS-CoV-2 showed an increase in hemolysis and thrombosis. Patients also exhibited prolonged COVID-19 symptoms, ventilation support, neurological impacts, and high mortality. However, the mechanism of COVID-19 severity in G6PD deficient patients and its neurological manifestation is still ambiguous. Here, using a CRISPR-edited G6PD deficient human microglia cell culture model, we observed a significant reduction in NADPH level and an increase in basal reactive oxygen species (ROS) in microglia. Interestingly, the deficiency of the G6PD-NAPDH axis impairs induced nitric oxide synthase (iNOS) mediated nitric oxide (NO) production, which plays a fundamental role in inhibiting viral replication. Surprisingly, we also observed that the deficiency of the G6PD-NADPH axis reduced lysosomal acidification and free radical production, further abrogating the lysosomal clearance of viral particles. Thus, impairment of NO production, lysosomal functions, and redox dysregulation in G6PD deficient microglia altered innate immune response, promoting the severity of SARS-CoV-2 pathogenesis.

Measuring Antibody-Mediated Tau Fibril Uptake in Microglia by Flow Cytometry.

Microglia are brain-resident phagocytic cells, considered to be the innate immune cells of the central nervous system. Microglia respond to both infectious pathogens in the brain and sterile cellular debris, including the proteinaceous aggregates that accumulate in the brains of patients with Alzheimer's disease (AD). Microtubule-associated protein Tau is an intracellular protein that self-aggregates into neurofibrillary tangles in Alzheimer's disease and many other neurodegenerative diseases. Ongoing clinical trials are testing whether therapeutic antibodies specific to Tau protein aggregates can reduce pathological protein deposition and improve the course of disease. Data suggest that Tau-specific antibodies act on extracellular Tau aggregates by promoting uptake into microglia cells and thus preventing its prion-like spread to unaffected neurons. Here we describe a protocol to test the effect of Tau-specific antibodies on Tau uptake into microglia by flow cytometry. Recombinant Tau protein is fibrillized in vitro and tagged with a fluorescent label. Then, fibrillized Tau is incubated with the antibody of interest and applied to microglial cells in culture. Uptake of Tau into microglia is then assessed by measuring fluorescence intensity by flow cytometry. With slight modifications, this assay can be used to test effects of many antibodies, various Tau protein compositions, and different microglial sources in a high-throughput format.

Chemical Analysis of SU-Eohyeol Pharmacopuncture and Its In Vitro Biological Activities.

Introduction: Pharmacopuncture (PA) is widely used in traditional Korean medicine to treat various diseases, including abdominal obesity, nervous system diseases, and musculoskeletal disorders. In the present study, we attempted to identify the chemical components of SU-Eohyeol PA (SUEHP), comprising extracts of eight medicinal herbs and Cervi Parvum Cornu, using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) and evaluated the in vitro anti-inflammatory and antioxidant activities of SUEHP. Methods: Volatile components of SUEHP were identified by GC-MS analysis of the n-hexane, dichloromethane (DCM), and distilled water-acetonitrile (DW-CAN) solvent fractions. LC-MS was performed to identify small metabolites of SUEHP using a water-methanol solvent fraction. In vitro anti-inflammatory and antioxidant potential of SUEHP was evaluated using cell-free biochemical assays and a BV2 microglial cell culture system. Results: GC-MS of SUEHP using the n-hexane, DCM, and DW-ACN solvent fractions detected 32 components. The major components (>3%) were 9-octadecenamide (25.9%) and eicosane (5.4%) in n-hexane fraction, 9-octadecenamide (29.1%) and cis-11-octadecenoic acid (3.1%) in DCM fraction, and 9-octadecenamide (31.1%) and 9-octadecenoic acid (17.9%) in DW-CAN fraction. LC-MS of SUEHP using a water-methanol solvent fraction detected 36 primary metabolites, including (a)symmetric dimethylarginine, L-pyroglutamic acid, n,n-dimethylglycine, n-acetyl-L-aspartic acid, and amino acids. Enrichment analysis and subsequent network analysis of the primary metabolites suggested their association with neurodegenerative diseases, including Alzheimer's disease and schizophrenia. Cell-free biochemical assays and molecular signaling studies of lipopolysaccharide-stimulated BV2 murine microglial cells demonstrated the anti-inflammatory and antioxidant activities of SUEHP. Conclusion: The present study identified the biochemically active components of SUEHP and suggested their therapeutic potential against diseases related to inflammatory and oxidative stress.

Investigating the Role of APOE Genotype in Human Microglia

AbstractBackgroundAlthough the pivotal role of apolipoprotein E () in AD has been recognized for three decades, its function in different brain cells is far from understood. Knockout studies of the APOE gene may shed light of the function of ApoE. The aim of the present study was to unravel its function in induced pluripotent stem cell (iPSC) derived microglia.MethodWe studied two human iPSC‐derived microglia cell cultures, one derived from a healthy control (Bioni010‐C – endogenous genotype apolipoprotein E34 (APOE34)) and one from an AD patient (UKBi011‐A – endogenous genotype APOE44) and their edited isogenic cell‐lines (for UKBi011‐A, APOE22 and APOE33 and for Bioni010‐C, hemizygous due to gene editing, APOE2‐ and APOE3‐ (32971461, 29753274)). Additionally, APOE KO lines were generated from both backgrounds. We performed whole genome transcriptomic (IlluminaTruSequencing) and proteomic (TMT‐labelled Nano‐LC peptidomic MS) analysis (n = six per genotype). For transcriptomic data, log2‐normalized intensities were analysed with (in R); there were 13115 unique transcripts. For proteomics analysis, after global normalization, the data were locally normalized (by protein), the limma package was used to calculate FDR‐corrected p‐values (1% cut off); there were 3975 (Bioni010 samples) and 4349 (UKBi011 samples) proteins. In the quality control analysis for the proteomics, we excluded one outlier of the six per line based on principle component/Euclidean distance analysis. We extracted pairwise comparisons to APOE KO in transcriptomic (|logFC|&gt;0.5, FDR &lt;0.05) and proteomic (|logFC|&gt;0 and FDR &lt;0.05) data.ResultTwenty transcripts (including APOE) were concordantly differentially expressed across all comparisons between APOE‐expressing and KO. These are broadly involved in processes related to mitochondrial function/organization, apoptosis, transcriptional regulation, immune functions, extracellular matrix, and energy metabolism among others. Seven proteins were concordantly differentially expressed across all pairwise comparisons with APOE KO, involved in extracellular matrix, immune signalling, energy metabolism, and cytoskeletal structure.ConclusionOur study finds APOE knockout leads to a number of changes related to immune, mitochondrial, and extracellular matrix genes, suggesting that APOE in human iPSC‐derived microglia cells influences immune and mitochondrial functions.

The two synthetic cannabinoid compounds 4'-F-CBD and HU-910 efficiently restrain inflammatory responses of brain microglia and astrocytes.

To study the anti-inflammatory potential of the two synthetic cannabinoids 4'-F-CBD and HU-910, we used post-natal brain cultures of mouse microglial cells and astrocytes activated by reference inflammogens. We found that 4'-F-CBD and HU-910 efficiently curtailed the release of TNF-α, IL-6, and IL-1β in microglia and astrocytes activated by the bacterial Toll-Like Receptor (TLR)4 ligand LPS. Upon LPS challenge, 4'-F-CBD and HU-910 also prevented the activation of phenotypic activation markers specific to microglia and astrocytes, that is, Iba-1 and GFAP, respectively. In microglial cells, the two test compounds also efficiently restrained LPS-stimulated release of glutamate, a non-cytokine inflammation marker for these cells. The immunosuppressive effects of the two cannabinoid compounds were concentration-dependent and observable between 1 and 10 μM. These effects were not dependent on cannabinoid or cannabinoid-like receptors. Both 4'-F-CBD and HU-910 were also capable of restraining the inflammogenic activity of Pam3CSK4, a lipopeptide that activates TLR2, and of BzATP, a prototypic agonist of P2X7 purinergic receptors, suggesting that these two cannabinoids could exert immunosuppressive effects against a variety of inflammatory stimuli. Using LPS-stimulated microglia and astrocytes, we established that the immunosuppressive action of 4'-F-CBD and HU-910 resulted from the inhibition of ROS produced by NADPH oxidase and subsequent repression of NF-κB-dependent signaling events. Our results suggest that 4'-F-CBD and HU-910 may have therapeutic utility in pathological conditions where neuroinflammatory processes are prominent.

P12.08.A CELL-EXTRINSIC D-2-HYDROXYGLUTARATE PROMOTES HYPERMETHYLATION AT LINEAGE-SPECIFIC REGULATORY REGIONS TO PREVENT MICROGLIAL ACTIVATION IN IDH-MUTANT GLIOMAS

Abstract BACKGROUND Tumor-associated macrophages and microglia (TAMs) are highly abundant myeloid cells in diffuse gliomas. Their composition and transcriptional programs differ according to the IDH mutation status of glioma cells. The underlying mechanisms remain until now little known. MATERIAL AND METHODS We compared bulk DNA methylome (Methylation EPIC array) and transcriptome of TAMs (CD11b+ purified by magnetic-activated cell sorting) from 25 IDH-mutant and 11 IDH-wildtype gliomas. The contamination of the CD11b+ fraction by tumor cells was determined by the detection of IDH and/or TERT mutations using droplet digital PCR. Only samples with a purity of at least 95% were used for analyses. To determine experimentally direct effects of D-2-hydroxyglutarate (D-2-HG), which is the oncometabolite produced and realeased into the microenvironment by IDH-mutated glioma cells, we used primary cultures of human microglial cells obtained from glioma surgery or epilepsy surgery. We exposed microglia or not to (D-2-HG) during 14 days, and analyzed their DNA methylome and transcriptome. RESULTS TAMs from IDH-mutant gliomas compared with TAMs from IDH-wildtype gliomas exhibit decreased expression of pro-inflammatory pathways (mainly IFN-γ, IFN-α, and TNF-α signaling) and genes related to G2-M checkpoint, glycolysis and Epithelial-Mesenchymal Transition (EMT). We uncovered that TAMs from IDH-mutant gliomas exhibit significant hypermethylation at promoters and enhancers linked to decreased expression of genes involved in inflammatory responses. Notably, these hypermethylated regions were enriched for binding motifs of transcription factors controlling an environment-dependent transcriptional network in microglial cells (such as PU.1, AP-1, C/EBP and RUNX). These findings were confirmed by comparing methylome data of TAMs from IDH-mutant gliomas and methylome of microglia from normal brains available in the literature. A 14 days exposition to D-2-HG of human microglial cells in vitro partially recapitulates the observed methylation changes. Transcriptome analyses of these cells show that this oncometabolite dampens the IFN-α/γ pathways and attenuates the response to LPS while inducing a metabolic shift toward oxidative phosphorylation. CONCLUSION Our findings establish a link between an extrinsic cell-effect of D-2-HG and the hypermethylation of DNA of microglial cells at regulatory regions in IDH-mutant gliomas. Our observations support the concept that oncometabolites may disrupt directly the function of immune cells of the tumor microenvironment.

Unprecedented Neoverrucosane and Cyathane Diterpenoids with Anti-Neuroinflammatory Activity from Cultures of the Culinary-Medicinal Mushroom Hericium erinaceus.

The culinary medicinal mushroom Hericium erinaceus holds significant global esteem and has garnered heightened interest within increasingly ageing societies due to its pronounced neuroprotective and anti-neuroinflammatory properties. Within this study, two novel diterpenes, 16-carboxy-13-epi-neoverrucosane (1) and Erinacine L (2); three known xylosyl cyathane diterpenoids, Erinacine A (3), Erinacine C (4), and Erinacine F (5); and four lanostane-type triterpenoids, and three cyclic dipeptides (10-12), in addition to orcinol (13), were isolated from the rice-based cultivation medium of H. erinaceus. Their structures were determined by NMR, HR-ESI-MS, ECD, and calculated NMR. Compound 1 marks a pioneering discovery as the first verrucosane diterpene originating from basidiomycetes, amplifying the scope of fungal natural product chemistry, and the intricate stereochemistry of Compound 5 has been comprehensively assessed for the first time. Compounds 2-5 not only showed encouraging neurotrophic activity in rat adrenal pheochromocytoma PC-12 cells, but also significantly inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV2 microglia cell cultures with IC50 values as low as 5.82 ± 0.18 μM. To elucidate the mechanistic underpinnings of these bioactivities, molecular docking simulation was used to analyze and support the interaction of 1 and 2 with inducible NO synthase (iNOS), respectively. In particular, compound 2, a cyathane-xyloside containing an unconventional hemiacetal moiety, is a compelling candidate for the prevention of neurodegenerative diseases. In summation, this investigation contributes substantively to the panorama of fungal diterpene structural diversity, concurrently furnishing additional empirical substantiation for the role of cyathane diterpenes in the amelioration of neurodegenerative afflictions.

Lipopolysaccharide augments microglial GABA uptake by increasing GABA transporter-1 trafficking and bestrophin-1 expression.

Gamma-aminobutyric acid (GABA), the principal inhibitory neurotransmitter in the brain, affects numerous immune cell functions. Microglia, the brain's resident innate immune cells, regulate GABA signaling through GABA receptors and express the complete GABAergic machinery for GABA synthesis, uptake, and release. Here, the use of primary microglial cell cultures and ex vivo brain tissue sections allowed for demonstrating that treatment with lipopolysaccharide (LPS) increased microglial GABA uptake as well as GABA transporter (GAT)-1 trafficking. This effect was not entirely abolished by treatment with GAT inhibitors (GAT-Is). Notably, LPS also induced microglial upregulation of bestrophin-1 (BEST-1), a Ca2+ -activated Cl- channel permeable to GABA. Combined administration of GAT-Is and a BEST-1 inhibitor completely abolished LPS-induced microglial GABA uptake. Interestingly, increased microglial GAT-1 membrane turnover via syntaxin 1A was detected in LPS-treated cultures after BEST-1 blockade. Altogether, these findings provided evidence for a novel mechanism through which LPS may trigger the inflammatory response by directly altering microglial GABA clearance and identified the GAT-1/BEST-1 interplay as a potential novel mechanism involved in brain inflammation.

Role of omentin-1 in susceptibility to anxiety and depression like behaviors

Neuro-inflammation and blood-brain barrier (BBB) dysfunction are associated with depression. Evidence shows that adipokines enter the brain from the circulation, which regulates depressive behaviors. Omentin-1 is a newly identified adipocytokine that has anti-inflammatory effects, but little is known about its role in neuro-inflammation and mood-relevant behavior. Our results showed omentin-1 knockout mice (Omentin-1−/−) increased susceptibility to anxiety and depressive-like behaviors, which are associated with abnormalities of cerebral blood flow (CBF) and impaired BBB permeability. Moreover, omentin-1 depletion significantly increased hippocampal pro-inflammatory cytokines (IL-1β, TNFα, IL-6), caused microglial activation, inhibited hippocampus neurogenesis, and resulted in autophagy impairment by dysregulating ATG genes. Omentin-1 deficiency also sensitized mice to the behavioral changes induced by lipopolysaccharide (LPS), suggesting that omentin-1 could rescue neuro-inflammation by acting as an anti-depressant. Our in vitro microglia cell culture data confirmed that recombinant omentin-1 suppresses microglial activation and pro-inflammatory cytokine expression induced by LPS. Our study suggests that omentin-1 can be used as a promising therapeutic agent for the prevention or treatment of depression by providing a barrier-promoting effect and an endogenous anti-inflammatory balance to downregulate the proinflammatory cytokines.

Propofol inhibits neuroinflammation and metabolic reprogramming in microglia in vitro and in vivo.

Microglial activation-induced neuroinflammation is closely related to the development of sepsis-associated encephalopathy. Accumulating evidence suggests that changes in the metabolic profile of microglia is crucial for their response to inflammation. Propofol is widely used for sedation in mechanically ventilated patients with sepsis. Here, we investigate the effect of propofol on lipopolysaccharide-induced neuroinflammation, neuronal injuries, microglia metabolic reprogramming as well as the underlying molecular mechanisms. The neuroprotective effects of propofol (80mg/kg) in vivo were measured in the lipopolysaccharide (2mg/kg)-induced sepsis in mice through behavioral tests, Western blot analysis and immunofluorescent staining. The anti-inflammatory effects of propofol (50μM) in microglial cell cultures under lipopolysaccharide (10ng/ml) challenge were examined with Seahorse XF Glycolysis Stress test, ROS assay, Western blot, and immunofluorescent staining. We showed that propofol treatment reduced microglia activation and neuroinflammation, inhibited neuronal apoptosis and improved lipopolysaccharide-induced cognitive dysfunction. Propofol also attenuated lipopolysaccharide-stimulated increases of inducible nitric oxide synthase, nitric oxide, tumor necrosis factor-α, interlukin-1β and COX-2 in cultured BV-2 cells. Propofol-treated microglia showed a remarkable suppression of lipopolysaccharide-induced HIF-1α, PFKFB3, HK2 expression and along with downregulation of the ROS/PI3K/Akt/mTOR signaling pathway. Moreover, propofol attenuated the enhancement of mitochondrial respiration and glycolysis induced by lipopolysaccharide. Together, our data suggest that propofol attenuated inflammatory response by inhibiting metabolic reprogramming, at least in part, through downregulation of the ROS/PI3K/Akt/mTOR/HIF-1α signaling pathway.

Hydrogen peroxide signaling modulates neuronal differentiation via microglial polarization and Wnt/β-catenin pathway.

Reactive oxygen species (ROS) are generated within the cell and serve as second messengers in fundamental cellular processes under physiologic conditions. Although the deleterious effects of high-level ROS associated with oxidative stress are well established, it is unclear how the developing brain reacts to redox changes. Our aim is to investigate how redox alteration affects neurogenesis and the mechanism that underlies it.We investigated in vivo microglial polarization and neurogenesis in zebrafish after hydrogen peroxide (H2O2) incubation. To quantify intracellular H2O2 levels in vivo, a transgenic zebrafish line that expresses Hyper and termed Tg(actb2:hyper3)ka8 was used. Then, in vitro studies with N9 microglial cells, 3-dimensional neural stem cell (NSC)-microglia coculture, and conditioned medium experiments are carried out to comprehend the mechanism underlying the changes in neurogenesis upon redox modulation.In zebrafish, exposure to H2O2 altered embryonic neurogenesis, induced M1 polarization in microglia, and triggered the Wnt/β-catenin pathway. N9 microglial cell culture experiments revealed that exposure to H2O2 resulted in M1 polarization in microglial cells, and this polarization was mediated by the Wnt/β-catenin pathway. Redox modulation of microglia interfered with NSC differentiation in coculture experiments. Neuronal differentiation was significantly higher in NSCs cocultured with H2O2-treated microglia when compared to control microglia. Wnt inhibition prevented the effects of H2O2-treated microglia on NSCs. No significant alterations were observed in conditioned medium experiments.Our findings point to a robust interplay between microglia and neural progenitors influenced by the redox state. Intracellular H2O2 levels can interfere with neurogenesis by altering the phenotypic state of the microglia via the Wnt/β-catenin system.

Resistance to Degradation of Silk Fibroin Hydrogels Exposed to Neuroinflammatory Environments.

Central nervous system (CNS) diseases represent an extreme burden with significant social and economic costs. A common link in most brain pathologies is the appearance of inflammatory components that can jeopardize the stability of the implanted biomaterials and the effectiveness of therapies. Different silk fibroin scaffolds have been used in applications related to CNS disorders. Although some studies have analyzed the degradability of silk fibroin in non-cerebral tissues (almost exclusively upon non-inflammatory conditions), the stability of silk hydrogel scaffolds in the inflammatory nervous system has not been studied in depth. In this study, the stability of silk fibroin hydrogels exposed to different neuroinflammatory contexts has been explored using an in vitro microglial cell culture and two in vivo pathological models of cerebral stroke and Alzheimer's disease. This biomaterial was relatively stable and did not show signs of extensive degradation across time after implantation and during two weeks of in vivo analysis. This finding contrasted with the rapid degradation observed under the same in vivo conditions for other natural materials such as collagen. Our results support the suitability of silk fibroin hydrogels for intracerebral applications and highlight the potentiality of this vehicle for the release of molecules and cells for acute and chronic treatments in cerebral pathologies.

Extracellular vesicles released by hair follicle and adipose mesenchymal stromal cells induce comparable neuroprotective and anti-inflammatory effects in primary neuronal and microglial cultures

Background aimsDespite intensive research, to date, there is no effective treatment for neurodegenerative diseases. Among the different therapeutic approaches, recently, the use of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) has gained attention. MethodsIn the present work, we focused on medium/large extracellular vesicles (m/lEVs) derived from hair follicle–-derived (HF) MSCs, comparing their potential neuroprotective and anti-inflammatory effect against adipose tissue (AT)-MSC–derived m/lEVs. ResultsThe obtained m/lEVs were similar in size with comparable expression of surface protein markers. The neuroprotective effect of both HF-m/lEVs and AT-m/lEVs was statistically significant in dopaminergic primary cell cultures, increasing cell viability after the incubation with 6-hidroxydopamine neurotoxin. Moreover, the administration of HF-m/lEVs and AT-m/lEVs counteracted the lipopolysaccharide-induced inflammation in primary microglial cell cultures, decreasing the levels of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-1β. ConclusionsTaken together, HF-m/lEVs demonstrated comparable potential with that of AT-m/lEVs as multifaceted biopharmaceuticals for neurodegenerative disease treatment.

Resveratrol alleviated neuroinflammation induced by pseudorabies virus infection through regulating microglial M1/M2 polarization

BackgroundPseudorabies virus (PRV) infections in susceptible non-porcine species trigger uncontrolled inflammations and eventually fatal encephalitis. Resveratrol (Res) has broad pharmacological functions including anti-virus, anti-inflammation, and neuroprotective. PurposeWe attempted to investigate the potential of Res in ameliorating PRV infection pathology in mice and decipher the mechanism of Res in treating PRV. MethodsThe mice were infected by PRV to investigate the protective effect of Res. Blood-brain barrier (BBB) permeability, H&E/Nissl/TUNEL staining, Real-time PCR and ELISA analyses were performed. Primary microglia and neuron were isolated from mice and cultured. The co-culture model of microglia and neuron was established by transwell. Immunofluorescence assay and flow cytometry were used. ResultsIn this study, we showed that Res ameliorated brain damage by reducing BBB permeability in PRV-infected mice, and diminished the expressions of MMP-2, MMP-9 and ZO-1 in the cortex. Pathological changes of neurons by H&E/Nissl/TUNEL staining suggested that Res could alleviate neuronal lesions. Moreover, Res inhibited the expressions of pro-inflammatory factors (IL-6, TNF-α) and chemokines (CCL3, CXCL10, MCP-1), but increased the expressions of anti-inflammatory factors (IL-4, IL-10) and neurotrophic factor (TGF-β, NGF and GDNF) in brain. In vitro cultured microglia cells, Res could suppress M1 microglia polarization and activate M2 microglia polarization. Co-culture of PRV-infected microglia with neuron cells by transwell system indicated that Res alleviated inflammatory response and neuronal apoptosis. ConclusionThis study provided evidence that Res could protect mice from PRV-induced encephalitis through regulation of microglia polarization and neuronal apoptosis suggesting the potential for treatment of viral encephalitis.

Exploring neuroprotective effect of trazodone in a microglia cell culture model of neuroinflammation

Exploring neuroprotective effect of trazodone in a microglia cell culture model of neuroinflammation

A Novel Anti-Inflammatory Formulation Comprising Celecoxib and Cannabidiol Exerts Antidepressant and Anxiolytic Effects.

Background: Ample research shows that anti-inflammatory drugs, particularly celecoxib, exert antidepressant effects, especially in patients with microglia activation. However, substantial cardiovascular adverse effects limit celecoxib's usefulness. Given that cannabidiol (CBD) exerts anti-inflammatory, microglia-suppressive, and antidepressant effects, we hypothesized that it may potentiate the therapeutic effects of celecoxib. Methods: The effects of celecoxib, CBD, and their combination were examined in murine models of antidepressant- and anxiolytic-like behavioral responsiveness, including the forced swim test (FST), elevated plus maze (EPM), lipopolysaccharide (LPS)-induced neuroinflammation, and chronic social defeat stress (CSDS), as well as in microglia cell cultures. Results: Acute administration of a combination of celecoxib plus CBD, at doses that had no effects by themselves (10 and 5 mg/kg, respectively), produced significant antidepressant- and anxiolytic-like effects in the FST and EPM, in male and female mice. In the LPS model, combinations of celecoxib (10 or 20 mg/kg) plus CBD (30 mg/kg) reversed the anxiety-like behavior in the open-field test (OFT) and anhedonia in the sucrose preference test (SPT), with minimal effects of celecoxib or CBD by themselves. In the CSDS paradigm, a combination of celecoxib plus CBD (each at 30 mg/kg) reversed the deficits in the OFT, EPM, social exploration, and SPT, whereas celecoxib or CBD by themselves had partial effects. In BV2 microglia cultures stimulated with LPS or α-synuclein, CBD markedly potentiated the suppressive effects of celecoxib over TNFα (tumor necrosis factor-α) and IL (interleukin)-1β secretion. Conclusions: Combinations of celecoxib plus CBD produce efficacious antidepressant- and anxiolytic-like effects, which may depend on their synergistic microglia-suppressive effects.

Neuroinflammation and microglial activation as target for therapeutic strategy: in vitro and in vivo models of study

AbstractBackgroundAlzheimer's disease (AD) is characterized by the extracellular accumulation of senile plaques composed of beta‐amyloid (Aβ) and the intracellular deposition of neurofibrillary tangles composed of hyperphosphorylated tau (Tp). Aβ oligomers (AβO) induce damage beginning with loss of synapses, followed by neurite network disorganization and neuronal death. Neuroinflammation plays an essential role in the disease. In AD patient brains, reactive microglia are closely colocalized with amyloid plaques, indicating their close link. Aβ‐activated microglia produce neurotoxic cytokines/chemokines which subsequently cause neuronal dysfunction and death.MethodUsing rat primary cultures of cortical neurons and microglial cells, generating a solution of AβO/protofibrils, playing with the concentration and time of exposure, we were able to mimic the early effects (acute neuronal loss) and the neuroinflammatory response.ResultAt the beginning of the exposure, we observed a Aβ phagocytosis, followed by a strong increase of TREM2/OX41 marker associated with cytokines release and CD206 (M2 marker) decrease over the time.In addition, microglial activation was deeply studied in the hippocampus of aged mice infused with the AβO/protofibril preparation. We observed progressive Tp accumulation associated with neurodegeneration and progressive activation of microglial cells (transition from a phagocytosis stage to a pro‐inflammatory stage with over‐expression of IBA1, CD68 immunoreactivity and production of pro‐inflammatory cytokines).ConclusionIn this work, we proposed vitro/vivo models showing differential and consecutive microglial responses to Aβ. These models are useful for targeting microglial in the pre‐disease period (in vitro) and for modulating the microglial response in the brain once the disease process is underway, and hold promise as a disease‐modifying treatment strategy.