Primary Mixed Glial Cultures Research Articles

Differences in Magnetic Particle Uptake by CNS Neuroglial Subclasses: Implications for Neural Tissue Engineering

To analyze magnetic particle uptake and intracellular processing by the four main non-neuronal subclasses of the CNS: oligodendrocyte precursor cells; oligodendrocytes; astrocytes; and microglia. Magnetic particle uptake and processing were studied in rat oligodendrocyte precursor cells and oligodendrocytes using fluorescence and transmission electron microscopy, and the results collated with previous data from rat microglia and astrocyte studies. All cells were derived from primary mixed glial cultures. Significant intercellular differences were observed between glial subtypes: microglia demonstrate the most rapid/extensive particle uptake, followed by astrocytes, with oligodendrocyte precursor cells and oligodendrocytes showing significantly lower uptake. Ultrastructural analyses suggest that magnetic particles are extensively degraded in microglia, but relatively stable in other cells. Intercellular differences in particle uptake and handling exist between the major neuroglial subtypes. This has important implications for the utility of the magnetic particle platform for neurobiological applications including genetic modification, transplant cell labeling and biomolecule delivery to mixed CNS cell populations.

Imipramine affects morphology of microglial cells in the primary mixed rat glial culture

Imipramine affects morphology of microglial cells in the primary mixed rat glial culture

Involvement of early growth response-2 (Egr-2) in lipopolysaccharide-induced neuroinflammation

Early growth response-2 (Egr-2) protein is a transcription factor, which belongs to Egr family which involve in modulating the peripheral immune response, by means of the induction of differentiation of lymphocyte precursors, activation of T and B cells. Egr-2 plays essential roles in peripheral nerve myelination, adipogenesis, tissue repair and fibrosis, immune tolerance; however, its regulation and role in central nervous system (CNS) remain poorly understood. In contrast to Egr-1, which has been extensively investigated, the regulation and function of Egr-2 remains less well characterized. To elaborate whether Egr-2 was involved in CNS injury, we performed a neuroinflammatory model by lipopolysaccharide (LPS) lateral ventral injection in adult rats. Egr-2 expression was strongly induced in active glia cells (astrocytes and microglias) in inflamed brain cortex. In vitro studies indicated that the upregulation of Egr-2 may be involved in the subsequent glia cellular activation following LPS exposure; and knock down of Egr-2 in primary mixed glial cultures (MGC) by siRNA showed that Egr-2 promoted the synthesis of TNF-α. Collectively, these results suggested Egr-2 may be important in host defense in CNS immune response, which might provide a potential target to the treatment of neuroinflammation.

Cobalt Chloride-induced Hypoxia Ameliorates NLRP3-Mediated Caspase-1 Activation in Mixed Glial Cultures

Hypoxia has been shown to promote inflammation, including the release of proinflammatory cytokines, but it is poorly investigated how hypoxia directly affects inflammasome signaling pathways. To explore whether hypoxic stress modulates inflammasome activity, we examined the effect of cobalt chloride (CoCl2)-induced hypoxia on caspase-1 activation in primary mixed glial cultures of the neonatal mouse brain. Unexpectedly, hypoxia induced by oxygen-glucose deprivation or CoCl2 treatment failed to activate caspase-1 in microglial BV-2 cells and primary mixed glial cultures. Of particular interest, CoCl2-induced hypoxic condition considerably inhibited NLRP3-dependent caspase-1 activation in mixed glial cells, but not in bone marrow-derived macrophages. CoCl2-mediated inhibition of NLRP3 inflammasome activity was also observed in the isolated brain microglial cells, but CoCl2 did not affect poly dA:dT-triggered AIM2 inflammasome activity in mixed glial cells. Our results collectively demonstrate that CoCl2-induced hypoxia may negatively regulate NLRP3 inflammasome signaling in brain glial cells, but its physiological significance remains to be determined.

Asymmetric Production of Nitric Oxide in Mouse Primary Cortical Mixed Glial Cell Cultures Treated With Lipopolysaccharide

Activated glial cells produce many toxic molecules, including cytokines and nitric oxide (NO). There is evidence that excess NO production plays a key role in neuronal cell death. Previous research has demonstrated that cortical glial cells from the left and right cortices of the brain secrete cytokines asymmetrically. However, no evidence to date exists about whether glial cell-produced NO is produced asymmetrically as well. The results of this study show that NO production and inducible NO synthase gene expression are both significantly higher in the right hemisphere-derived mixed glial cell compared with cultures derived from the left.

Glial proteome changes in response to moderate hypothermia

Reactive glia plays a central role in neuroinflammation associated with secondary damage after brain injury. In order to understand the global effects of therapeutic hypothermia on glial activation and neuroinflammation, we performed proteomic profiling of glial cultures following inflammatory stimulation and hypothermic exposure. Primary mixed glial cultures prepared from mouse brains were stimulated with lipopolysaccharide and interferon-γ under normothermic (37°C) or moderate hypothermic (29°C) conditions, and their proteome profiles were compared by LC-ESI-MS/MS. Differentially expressed proteins were determined by high-throughput label-free quantification. Under hypothermic conditions, 64 and 16 proteins were upregulated (≥1.5-fold) and downregulated (≤ 0.7-fold), respectively, compared to normothermic conditions. More importantly, hypothermia altered the abundance of 143 proteins that were either increased or decreased by inflammatory stimulation. The results were validated for several proteins (ICAM-1, STAT-1, YWHAB, and IFIT-3) by Western blot analysis. Pathway and network analysis indicate that hypothermia influences various biological functions of glia such as molecular transport, cell movement, immune response, cell death, and stress response. In conclusion, moderate hypothermia seems to have a significant effect on the protein expression profiles of brain glia and possibly ensuing neuroinflammation. These proteins may be involved in the protective mechanism of hypothermia against brain injuries.

An efficient and economical culture approach for the enrichment of purified oligodendrocyte progenitor cells

Oligodendrocyte progenitor cell (OPC) culture has provided a powerful approach to mechanistically investigate the proliferation and differentiation of oligodendroglia. However, existing culture methods (including the traditional shake-off method) have limitations, particularly their low productivities. Therefore, we developed a simplified and highly efficient method to produce a large yield of OPCs with low expense by using specialised modified media, in which B104-conditioned medium (B104-CM) instead of growth factors was used as a mitogenic source for OPC propagation, while a modified OPC isolation-medium was applied to improve the isolation of OPCs. First, we withdrew foetal bovine serum when primary mixed glial cultures were 65–75% confluent and substituted with modified oligodendrocyte growth medium to enrich OPCs. Second, we employed a chemical-based method to isolate and purify OPCs from mixed glial cultures using a modified oligodendrocyte isolation medium. As a result, our approach produced a high yield of purified OPCs, approximately 90-fold higher than that produced via the traditional shake-off method. Importantly, the purified OPCs produced via our modified approach maintained typical capacities of proliferation and differentiation observed in oligodendrocyte lineage cells. Together, our modified method provides a highly efficient approach to OPC culture for oligodendrocyte research.

Acetaminophen induces oligodendrocyte precursor cell death and reactive astroglia in a primary mixed glial cell culture

Brain damage in acetaminophen (AP) overdose is associated with liver failure but its direct effects has not been evaluated.AimTo study the effect of toxic and non‐toxic doses of AP on astrocyte and oligodendrocyte precursor cell (OPC) viability in a primary mixed glial cell culture.MYMPrimary cultures of mixed glial cells were carried out according to McCarthy and de Vellis (1980). Cells were treated with vehicle (DMSO) or AP (1 and 20 mM). After 48 hs cell viability was assessed; astrocyte marker (GFAP) and OPC marker (PDGFR‐alpha) expression was evaluated by western‐blot (WB) and immunocytochemistry.ResultsThe 1 mM dose of AP did not produce significant differences in cell viability, while 20 mM exposure decreased in 40% cell viability (P<0.001). The 20 mM dose produced the death of the 90% of the OPC in the culture and a decrease of 89% of PDGFR‐alpha expression (P<0.001) with an increase in GFAP optical density/cell and WB expression (47% and 97% respectively) (P<0.05). The 1mM dose did not produce significant changes in GFAP but produce a 25% significant decreased in PDGFR‐alpha expression (p<0.05).ConclusionAP produces direct toxic effects on glial cells characterized by oligodendrocyte death and the induction of reactive astroglia.

Cannabinoid receptor agonists modulate oligodendrocyte differentiation by activating PI3K/Akt and the mammalian target of rapamycin (mTOR) pathways

The endogenous cannabinoid system participates in oligodendrocyte progenitor differentiation in vitro. To determine the effect of synthetic cannabinoids on oligodendrocyte differentiation, we exposed differentiating cultures of oligodendrocytes with cannabinoid CB(1), CB(2) and CB(1)/CB(2) receptor agonists and antagonists. The response of the PI3K/Akt and the mammalian target of rapamycin (mTOR) signalling pathways were studied as effectors of cannabinoid activity. Purified oligodendrocyte progenitor cells (OPC) obtained from primary mixed glial cell cultures were treated for 48 h with CB(1), CB(2) and CB(1) /CB(2) receptor agonists (ACEA, JWH133 and HU210, respectively) in the presence or absence of the antagonists AM281 (CB(1) receptor) and AM630 (CB(2) receptor). Moreover, inhibitors of the phosphatidylinositol 3-kinase (PI3K)/Akt and mTOR pathways (LY294002 and rapamycin, respectively) were used to study the involvement of these pathways on cannabinoid-induced OPC maturation. ACEA, JWH133 and HU-210 enhanced OPC differentiation as assessed by the expression of stage specific antigens and myelin basic protein (MBP). Moreover, this effect was blocked by the CB receptor antagonists. ACEA, JWH133 and HU210 induced a time-dependent phosphorylation of Akt and mTOR, whereas the inhibitors of PI3K/Akt (LY294002) or of mTOR (rapamycin) reversed the effects of HU-210 on oligodendrocyte differentiation and kinase activation. Activation of cannabinoid CB(1) or CB(2) receptors with selective agonists accelerated oligodendrocyte differentiation through the mTOR and Akt signalling pathways.

Imipramine and fluoxetine influence phenotype of microglial cells in the rat primary mixed glial cell cultures

Imipramine and fluoxetine influence phenotype of microglial cells in the rat primary mixed glial cell cultures

Moclobemide exerts anti-inflammatory effect in lipopolysaccharide-activated primary mixed glial cell culture

An increasing body of evidence indicates that glial activation and neuroinflammation play an important role in the pathogenesis of psychiatric and neurodegenerative diseases. Activated glial cells secrete various cytokines that influence neurotransmission, hypothalamus-pituitary-adrenal axis activity, neuronal plasticity and neurogenesis. It has been suggested that alterations in cytokine networks are involved in the mechanism of action of antidepressant drugs. Until now, only a few studies demonstrated that some tricyclic antidepressants and selective serotonin reuptake inhibitors reduced production of pro-inflammatory cytokines in brain glia cells. We have investigated for the first time whether the antidepressant, moclobemide (a reversible selective inhibitor of monoamine oxidase-A) has an influence on pro-inflammatory cytokines [interleukin (IL)-1β and tumor necrosis factor (TNF)-α] and anti-inflammatory cytokine (IL-10) in primary rat mixed glial cell cultures stimulated by lipopolysaccharide (LPS). Our results showed that moclobemide used in a wide range of concentrations diminished LPS-stimulated IL-1β and TNF-α mRNAs expression in cellular extracts and remarkably reduced the levels of both pro-inflammatory cytokines in culture medium. In opposite to this, the drug had no influence on IL-10 mRNA and slightly reduced IL-10 concentration. Moreover, moclobemide decreased LPS-stimulated translocation of NFκB p65 subunit into cellular nuclei. These results suggest that moclobemide exerts anti-inflammatory effect in the central nervous system because it affects the balance between pro- and anti-inflammatory cytokines (IL-1β, TNF-α/IL-10) in primary mixed glial cell cultures.

In vitro differentiation of lineage‐negative bone marrow cells into microglia‐like cells

Microglia are believed to be the only resident immune cells in the CNS, originating from hematopoietic-derived myeloid cells and invading the CNS during development. However, the detailed mechanisms of differentiation and transformation of microglial cells are not fully understood. Here, we demonstrate that murine microglial cells show two morphological forms in vitro, namely, small round cells expressing CD11b, Iba1, triggering receptor expressing on myeloid cells-2 (TREM2), and weakly expressing major histocompatibility complex class II and large flat cells expressing only CD11b and Iba1. Moreover, lineage-negative bone marrow (LN) cells cultured with primary mixed glial culture cells could differentiate into only the small round microglia-like cells, despite the absence of CCR2 and Gr-1 expression. Addition of macrophage colony stimulating factor (M-CSF) to LN cell culture allowed the proliferation and expression of TREM2 in LN cells, and the addition of neutralizing anti-M-CSF antibodies suppressed the proliferation of LN cells despite the expression of TREM2. When LN cells were cultured with M-CSF, the number of small round cells in the culture was considerably low, indicating that the small round morphology of the immature cells is not maintained in the presence of only M-CSF. On the other hand, when LN cells were grown in the presence of astrocytes, the small round cells were maintained at a concentration of approximately 30% of the total population. Therefore, cell-cell contact with glial cells, especially astrocytes, may be necessary to maintain the small round shape of the immature cells expressing TREM2.

Ambivalent effects of compound C (dorsomorphin) on inflammatory response in LPS-stimulated rat primary microglial cultures

It was proven that compound C displays beneficial effects in models of inflammatory-induced anemia, ischemic stroke, and fibrodysplasia ossificans progressiva. Compound C influence on microglia, playing a major role in neuroinflammation, has not been evaluated yet. The aim of the present study was to determine the effect of compound C on cytokine release, NO, and reactive oxygen species (ROS) production. The rat microglial cultures were obtained by shaking the primary mixed glial cultures. Cytokine and nitrite concentrations were assayed using ELISA kits. ROS were assayed with nitroblue tetrazolium chloride. AMPK activity was assayed using the SAMS peptide. The expression of arginase I, NF-kappaB p65, and hypoxia-inducible factor-1 alpha (HIF-1 alpha) was evaluated using Western blot. Compound C displayed ambivalent effect depending on microglia basal activity. It up-regulated the release of TNF alpha and NO production and increased the expression of arginase I in non-stimulated microglia. However, compound C down-regulated IL-1 beta, IL-6 and TNF alpha release, NO, ROS production, and AMPK activity, diminished NF-kappaB and HIF-1 alpha expression, as well as increased arginase I expression in lipopolysaccharide (LPS)-stimulated microglia. Compound C did not affect iNOS expression and IL-10 and TGF-beta release in non-stimulated and LPS-stimulated microglia. The observed alterations in the release or production of inflammatory mediators may be explained by the changes in NF-kappaB, HIF-1 alpha, and arginase I expression and 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolinum bromide values in response to LPS, whereas the basis for the compound C effect on non-stimulated microglia remains to be investigated.

Microglia Activated with the Toll-Like Receptor 9 Ligand CpG Attenuate Oligomeric Amyloid β Neurotoxicity in in Vitro and in Vivo Models of Alzheimer’s Disease

Soluble oligomeric amyloid beta (oAbeta) 1-42 causes synaptic dysfunction and neuronal injury in Alzheimer's disease (AD). Although accumulation of microglia around senile plaques is a hallmark of AD pathology, the role of microglia in oAbeta1-42 neurotoxicity is not fully understood. Here, we showed that oAbeta but not fibrillar Abeta was neurotoxic, and microglia activated with unmethylated DNA CpG motif (CpG), a ligand for Toll-like receptor 9, attenuated oAbeta1-42 neurotoxicity in primary neuron-microglia co-cultures. CpG enhanced microglial clearance of oAbeta1-42 and induced higher levels of the antioxidant enzyme heme oxygenase-1 in microglia without producing neurotoxic molecules such as nitric oxide and glutamate. Among subclasses of CpGs, class B and class C activated microglia to promote neuroprotection. Moreover, intracerebroventricular administration of CpG ameliorated both the cognitive impairments induced by oAbeta1-42 and the impairment of associative learning in Tg2576 mouse model of AD. We propose that CpG may be an effective therapeutic strategy for limiting oAbeta1-42 neurotoxicity in AD.

Influence of imipramine, moclobemide and fluoxetine on pro-inflammatory (TNF-α, IL-1β) and anti-inflammatory (IL-10) cytokines in lipopolysaccharide-stimulated primary rat mixed glial cell cultures

Influence of imipramine, moclobemide and fluoxetine on pro-inflammatory (TNF-α, IL-1β) and anti-inflammatory (IL-10) cytokines in lipopolysaccharide-stimulated primary rat mixed glial cell cultures

P3-368: Misfolded truncated tau protein in the extraneuronal space: Inducer of neuronal death and inflammatory response

A pronounced increase of tau protein levels in the cerebrospinal fluid was found in most patients with Alzheimer's disease. This may reflect the increase of tau protein release from intracellular to the extracellular space. Previously it has been shown that different tau variants may induce neuronal death in the neuroblastoma cell lines. However it is not clear which tau modification is responsible for neuronal death and whether tau may also induce inflammatory response. Different recombinant truncated tau species and human tau isoforms were isolated and purified to homogeneity. The purified proteins were fractionated by gel electrophoresis and stained with Coomassie blue and silver stain or immunolabeled with anti-tau antibody DC25. The neurotoxicity was analyzed in the primary neuronal cultures using ToxiLight Non-Destructive Cytotoxicity Assay and CellTiter-Glo Luminescent Cell Viability Assay. In vitro primary rat mixed glial cultures were used for detection of tau mediated inflammatory response. NO release was evaluated measuring nitrite accumulation in the conditioned cell culture medium. Morphological changes of activated microglia were detected by immunohistochemical staining of immunomarkers CD11b/CD18 and CD68. Human misfolded truncated tau species but not full length tau isoforms displayed in vitro neurotoxic properties when incubated with primary neurons for 24h. Moreover, when rat primary mixed cultures were exposed to the misfolded tau proteins, dose dependent release of nitric oxide and adenylate kinase occurred. In parallel, microglia changed its morphology from ramified to the amoeboid, thus suggesting its activated state. Our results demonstrate that different misfolded tau species induce neuronal death and simultaneously promote inflammatory response mediated by microglia. Moreover, activated microglia underwent morphological transformation, presented full inflammatory repertoire and induced signaling pathways leading to NO release. The presented data suggest that not only intraneuronal misfolded tau trigger neurodegenerative cascade, but also extracellularly located misfolded tau is efficient promoter of neurodegeneration, neuroinflammation and neuronal death.

Pro-inflammatory Cytokines Modulate Glial Apolipoprotein E Secretion

Alzheimer's disease (AD) is a neurological disorder characterized by plaques and an elevated immune response. Specifically, increased expression of interleukin (IL)-1 and tumour necrosis factor (TNF)-alpha, has been observed in AD cerebrospinal fluid and temporal brain tissue. Both of these immunomodulators were shown to carry genetic variants that increase the risk of developing AD. Studies have also established the apolipoprotein E (apoE) gene to be a risk factor for AD with epsilon4 carriers having been found to show lower levels of brain apoE. In the present study, treatment of primary rat mixed glial cell cultures with IL-1beta induced a significant increase in extracellular apoE protein. In contrast, treatment primary rat astrocyte and mixed glial cell cultures with TNF-alpha significantly reduced extracellular apoE protein levels. These results are consistent with the notion that elevated cytokine expression directly modulates immunosuppression and indirectly apoE-mediated neuronal remodeling.

The actions of leptin on survival and hydrogen peroxide toxicity in primary mixed glial cells of rat

Studies indicate that leptin is involved in not only energy expenditure and food intake, but also in protection against apoptosis, in inflammation and in stimulation of proliferation in many cell types. However, leptin treatment increases the oxidative stress in many cell culture studies. This contradiction evoked a question of whether leptin acts as an oxidant or antioxidant on glial cells. We investigated the effect of leptin on glial cell survival and hydrogen peroxide (H2O2)-induced toxicity in vitro. The survival rate of the cells was determined by using 3-(4,5-D-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, thyazolyl blue (MTT) method. The cells obtained from the whole brain of 1–3 day-old rat were treated with 1, 10, 100 and 1000 ng/mL leptin for 24 or 72 h. Either the pretreatment of leptin alone for 5 h or leptin combined simultaneously with H2O2 or well known antioxidant glutathione (GSH) were applied to the cells. Malondialdehyde (MDA) levels were measured in cell lysates to which leptin was added for 24 h. The 100 and 1000 ng/mL leptin treatment for 72 h increased the glial viability by 19% and 36%, respectively. The dose of H2O2 that killed 75% of the cells was determined as 100 µM. GSH at different doses was applied as a positive control to the cells and the dose of 500 µM completely eliminated toxic effect of 100 µM H2O2. Either the pretreatment of leptin alone for 5 h or leptin combined simultaneously with H2O2 could not eliminate H2O2-caused toxicity. Furthermore, respective leptin doses did not change the glia MDA level. We suggest that leptin can increase glia survival dose dependently, but can not eliminate H2O2-induced oxidation in primary mixed glial cell culture.

NGF promotes microglial migration through the activation of its high affinity receptor: Modulation by TGF-β

Activation and mobilization of microglia are early events in the majority of brain pathologies. Among the signalling molecules that can affect microglial behaviour, we investigated whether nerve growth factor (NGF) was able to influence microglial motility. We found that NGF induced chemotaxis of microglial cells through the activation of TrkA receptor. In addition, NGF chemotactic activity was increased in the presence of low concentrations (≤ 0.2 ng/ml) of transforming growth factor-β (TGF-β), which at this concentration showed chemotactic activity per se. On the contrary, NGF-induced microglial migration was reduced in the presence of chemokinetic concentration of TGF-β (≥ 2 ng/ml). Finally, both basal and NGF-induced migratory activity of microglial cells was increased after a long-term exposure of primary mixed glial cultures to 2 ng/ml of TGF-β. Our observations suggest that both NGF and TGF-β contribute to microglial recruitment. The chemotactic activities of these two pleiotropic factors could be particularly relevant during chronic diseases in which recruited microglia remove apoptotic neurons in the absence of a typical inflammatory reaction.

Attenuation of neuroinflammation and Alzheimer's disease pathology by liver x receptors

Alzheimer's disease (AD) is an age-dependent neurodegenerative disease that causes progressive cognitive impairment. The initiation and progression of AD has been linked to cholesterol metabolism and inflammation, processes that can be modulated by liver x receptors (LXRs). We show here that endogenous LXR signaling impacts the development of AD-related pathology. Genetic loss of either Lxralpha or Lxrbeta in APP/PS1 transgenic mice results in increased amyloid plaque load. LXRs regulate basal and inducible expression of key cholesterol homeostatic genes in the brain and act as potent inhibitors of inflammatory gene expression. Ligand activation of LXRs attenuates the inflammatory response of primary mixed glial cultures to fibrillar amyloid beta peptide (fAbeta) in a receptor-dependent manner. Furthermore, LXRs promote the capacity of microglia to maintain fAbeta-stimulated phagocytosis in the setting of inflammation. These results identify endogenous LXR signaling as an important determinant of AD pathogenesis in mice. We propose that LXRs may be tractable targets for the treatment of AD due to their ability to modulate both lipid metabolic and inflammatory gene expression in the brain.