Neuronal-glial Co-cultures Research Articles

Differential expression of miRNA-146a-regulated inflammatory genes in human primary neural, astroglial and microglial cells

Micro RNA-146a (miRNA-146a) is an inducible, 22 nucleotide, small RNA over-expressed in Alzheimer's disease (AD) brain. Up-regulated miRNA-146a targets several inflammation-related and membrane-associated messenger RNAs (mRNAs), including those encoding complement factor-H (CFH) and the interleukin-1 receptor associated kinase-1 (IRAK-1), resulting in significant decreases in their expression (p<0.05, ANOVA). In this study we assayed miRNA-146a, CFH, IRAK-1 and tetraspanin-12 (TSPAN12), abundances in primary human neuronal-glial (HNG) co-cultures, in human astroglial (HAG) and microglial (HMG) cells stressed with Aβ42 peptide and tumor necrosis factor alpha (TNFα). The results indicate a consistent inverse relationship between miRNA-146a and CFH, IRAK-1 and TSPAN12 expression levels, and indicate that HNG, HAG and HMG cell types each respond differently to Aβ42-peptide+TNFα-triggered stress. While the strongest miRNA-146a-IRAK-1 response was found in HAG cells, the largest miRNA-146a-TSPAN12 response was found in HNG cells, and the most significant miRNA-146a-CFH changes were found in HMG cells, the ‘resident scavenging macrophages’ of the brain.

Differentiation of postnatal cerebellar glial progenitors is controlled by Bmi1 through BMP pathway inhibition

Bmi1 is a polycomb group (Pc-G) protein involved in heritable gene repression, maintenance of cell identity, and proliferation. During the development of the central nervous system, Bmi1 is crucial for self-renewal of neural stem cells and for proliferation of neuronal (granule cell) progenitors of the cerebellum. Here, we use loss of function mouse models and in vitro assays--granule cell cultures and glial-neuronal co-cultures--to show that Bmi1 plays a crucial role in specification of glial progenitors during postnatal cerebellar development. Moreover, we demonstrate in in vitro assays that Bmi1 exerts this novel function through repression of BMP pathway and that this is independent of its known role in mediating the cellular response to Shh signaling. Thus modulation of Bmi1 expression in glial progenitors may represent a key event in determining the differentiation potential of these cells.

Xenon and Sevoflurane Protect against Brain Injury in a Neonatal Asphyxia Model

Perinatal hypoxia-ischemia causes significant morbidity and mortality. Xenon and sevoflurane may be used as inhalational analgesics for labor. Therefore, the authors investigated the potential application of these agents independently and in combination to attenuate perinatal injury. Oxygen-glucose deprivation injury was induced in pure neuronal or neuronal-glial cocultures 24 h after preconditioning with xenon and/or sevoflurane. Cell death was assessed by lactate dehydrogenase release or staining with annexin V-propidium iodide. The mediating role of phosphoinositide-3-kinase signaling in putative protection was assessed using wortmannin, its cognate antagonist. In separate in vivo experiments, perinatal asphyxia was induced 4 hours after preconditioning with analgesic doses alone and in combination; infarct size was assessed 7 days later, and neuromotor function was evaluated at 30 days in separate cohorts. The role of phosphorylated cyclic adenosine monophosphate response element binding protein in the preconditioning was assessed by immunoblotting. Both anesthetics preconditioned against oxygen-glucose deprivation in vitro alone and in combination. The combination increased cellular viability via phosphoinositide-3- kinase signaling. In in vivo studies, xenon (75%) and sevoflurane (1.5%) alone as well as in combination (20% xenon and 0.75% sevoflurane) reduced infarct size in a model of neonatal asphyxia. Preconditioning with xenon and the combination of xenon and sevoflurane resulted in long-term functional neuroprotection associated with enhanced phosphorylated cyclic adenosine monophosphate response element binding protein signaling. Preconditioning with xenon and sevoflurane provided long-lasting neuroprotection in a perinatal hypoxic-ischemic model and may represent a viable method to preempt neuronal injury after an unpredictable asphyxial event in the perinatal period.

Matrix metalloproteinase-9 and urokinase plasminogen activator mediate interleukin-1-induced neurotoxicity

Matrix metalloproteinases (MMPs) are endopeptidases known to mediate acute neuronal injury, but it is unclear whether these proteases are induced by the primary insult or by inflammation associated with injury. We have reported recently that interleukin-1 (IL-1) induces neurotoxicity by an astrocyte-dependent mechanism. The aim of the present study was to test the hypothesis that MMPs mediate IL-1 neurotoxicity in rat, glial–neuronal cocultures. IL-1β induced the release of astrocytic MMP-9 in cocultures, whilst an antagonist of MMP-9 inhibited IL-1β-induced neuronal death. Urokinase plasminogen activator (uPA) was constitutively expressed on neuronal membrane fractions, and amiloride (an antagonist of uPA) or plasminogen activator inhibitor (PAI)-1 significantly reduced IL-1β-induced neurotoxicity. Thus, neuronal uPA contributes to IL-1 neurotoxicity, and may be responsible for activating MMP-9 released from IL-1-primed astrocytes. In summary, IL-1-induced neurotoxicity is dependent on extracellular protease activity, and these mechanisms may contribute to neuronal cell death in CNS diseases.

Neuroprotective actions of endogenous interleukin‐1 receptor antagonist (IL‐1ra) are mediated by glia

The pro-inflammatory cytokine interleukin-1 (IL-1), contributes to neuronal inflammation and cell death induced by ischemia, excitotoxicity, or trauma, while administration of IL-1 receptor antagonist (IL-1ra) reduces neuronal injury. The aim of the present study was to test the hypothesis that endogenous IL-1ra is neuroprotective in vivo and in vitro, and to identify its mechanism of actions. Mice lacking IL-1ra (IL-1ra knock-out (KO]) exhibited a dramatic increase in neuronal injury (3.6-fold increase in infarct size) induced by transient cerebral ischemia compared to wild-type (WT) animals. Basal cell death of cultured cortical neurons from WT and IL-1ra KO was identical, and treatment with NMDA or AMPA (20 microM) increased cell death to the same extent in WT and IL-1ra KO neurons. However, basal and NMDA- or AMPA-induced cells death was significantly higher in glial-neuronal co-cultures from IL-1ra KO than from WT mice. We further showed that pure microglial cultures, but not pure astrocytes cultures, released IL-1ra in response to treatment with conditioned medium from NMDA- or AMPA-treated primary neurons. These results demonstrate that endogenous IL-1ra produced by microglia is neuroprotective in cerebral ischemia or excitotoxicity.

Effects of isoflurane on oxygen‐glucose deprivation‐induced changes of gene expression profiling in glial‐neuronal cocultures

Isoflurane decreases ischemia-induced neuronal injury. The mechanisms of this effect are largely unknown. We hypothesize that isoflurane induces expression of protective stress genes and decreases expression of apoptosis-related genes. The mRNA expression of about 1300 genes related to neurobiology in rat glial-neuronal cocultures was evaluated by microarray technology. Four experimental conditions were examined: control; 2% isoflurane; oxygen-glucose deprivation (OGD, to simulate ischemia in vitro); or isoflurane (2%) plus OGD. There were four immediate early genes/transcription factors (early growth response 1, c-fos, nerve growth factor-induced factor A and Knox-24) whose mRNA expression was increased to more than 1.4-fold of control levels under the conditions of isoflurane, OGD or isoflurane plus OGD. Isoflurane increased the mRNA expression of heme oxygenase, a 32-kDa heat-shock protein, and decreased the mRNA expression of caspase-2, calpain 1 and the Bcl-2-associated death agonist. These isoflurane effects were still apparent under the condition of isoflurane plus OGD. The mRNA expression of Gbeta1, early growth response 1 and the death effector domain-containing protein DEFT in the samples used for microarray assay was determined by reverse transcriptase-polymerase chain reaction, and the results were consistent with the patterns of changes across the experimental conditions as revealed by microarray technology. Our data suggest that the effects of isoflurane on the mRNA expression of multiple genes in glial-neuronal cocultures are consistent with its neuroprotection against ischemia. A coordinated change in expression of many genes (increased expression of potentially protective gene and decreased expression of potentially damaging genes) after the exposure of isoflurane was revealed by this study.

Dexmedetomidine produces its neuroprotective effect via the α 2A-adrenoceptor subtype

Which of the three α 2-adrenoceptor subtypes of α 2A, α 2B, or α 2C mediates the neuroprotective effect of dexmedetomidine was examined in cell culture as well as in an in vivo model of neonatal asphyxia. Dexmedetomidine dose-dependently attenuated neuronal injury (IC 50=83±1 nM) in neuronal-glial co-cultures derived from wild-type mice; contrastingly, dexmedetomidine did not exert neuroprotection in injured cells from transgenic mice (D79N) expressing dysfunctional α 2A-adrenoceptors. An α 2A-adrenoceptor subtype-preferring antagonist 2-[(4,5-Dihydro-1 H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1 H-isoindole maleate (BRL44408) completely reversed dexmedetomidine-induced neuroprotection, while other subtype-preferring antagonists 2-[2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione dihydrochloride (ARC239) (α 2B) and rauwolscine (α 2C) had no significant effect on the neuroprotective effect of dexmedetomidine in neuronal-glial co-cultures. Dexmedetomidine also protected against exogenous glutamate induced cell death in pure cortical neuron cultures assessed by flow cytometry and reduced both apoptotic and necrotic types of cell death. Likewise this neuroprotective effect was antagonised by BRL44408 but not ARC239 or rauwolscine. Dexmedetomidine exhibited dose-dependent protection against brain matter loss in vivo (IC 50=40.3±6.1 μg/kg) and improved the neurologic functional deficit induced by the hypoxic-ischemic insult. Protection by dexmedetomidine against hypoxic-ischemic-induced brain matter loss was reversed by the α 2A-adrenoceptor subtype-preferring antagonist BRL44408; neither ARC239 nor rauwolscine reversed the neuroprotective effect of dexmedetomidine in vivo. Our data suggest that the neuroprotective effect of dexmedetomidine is mediated by activation of the α 2A adrenergic receptor subtype.

Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis.

Ischemic brain injury resulting from stroke arises from primary neuronal losses and by inflammatory responses. Previous studies suggest that erythropoietin (EPO) attenuates both processes. Although EPO is clearly antiapoptotic for neurons after experimental stroke, it is unknown whether EPO also directly modulates EPO receptor (EPO-R)–expressing glia, microglia, and other inflammatory cells. In these experiments, we show that recombinant human EPO (rhEPO; 5,000 U/kg body weight, i.p.) markedly reduces astrocyte activation and the recruitment of leukocytes and microglia into an infarction produced by middle cerebral artery occlusion in rats. In addition, ischemia-induced production of the proinflammatory cytokines tumor necrosis factor, interleukin 6, and monocyte chemoattractant protein 1 concentration is reduced by >50% after rhEPO administration. Similar results were also observed in mixed neuronal-glial cocultures exposed to the neuronal-selective toxin trimethyl tin. In contrast, rhEPO did not inhibit cytokine production by astrocyte cultures exposed to neuronal homogenates or modulate the response of human peripheral blood mononuclear cells, rat glial cells, or the brain to lipopolysaccharide. These findings suggest that rhEPO attenuates ischemia-induced inflammation by reducing neuronal death rather than by direct effects upon EPO-R–expressing inflammatory cells.

Combination of xenon and isoflurane produces a synergistic protective effect against oxygen-glucose deprivation injury in a neuronal-glial co-culture model.

Combination of xenon and isoflurane produces a synergistic protective effect against oxygen-glucose deprivation injury in a neuronal-glial co-culture model.

Novel diketopiperazine enhances motor and cognitive recovery after traumatic brain injury in rats and shows neuroprotection in vitro and in vivo.

The authors developed a novel diketopiperazine that shows neuroprotective activity in a variety of in vitro models, as well as in a clinically relevant experimental model of traumatic brain injury (TBI) in rats. Treatment with 1-ARA-35b (35b), a cyclized dipeptide derived from a modified thyrotropin-releasing hormone (TRH) analog, significantly reduced cell death associated with necrosis (maitotoxin), apoptosis (staurosporine), or mechanical injury in neuronal-glial cocultures. Rats subjected to lateral fluid percussion-induced TBI and then treated with 1 mg/kg intravenous 35b thirty minutes after trauma showed significantly improved motor recovery and spatial learning compared with vehicle-treated controls. Treatment also significantly reduced lesion volumes as shown by magnetic resonance imaging, and decreased the number of TUNEL-positive neurons observed in ipsilateral hippocampus. Unlike TRH or traditional TRH analogs, 35b treatment did not change mean arterial pressure, body temperature, or thyroid-stimulating hormone release, and did not have analeptic activity. Moreover, in contrast to TRH or typical TRH analogs, 35b administration after TBI did not alter free-magnesium concentration or cellular bioenergetic state. Receptor-binding studies showed that 35b did not act with high affinity at 50 classical receptors, channels, or transporters. Thus, 35b shows none of the typical physiologic actions associated with TRH, but possesses neuroprotective actions in vivo and in vitro, and appears to attenuate both necrotic and apoptotic cell death.

Traumatic injury in vitro induces IEG mRNAs in cultured glial cells, suppressed by co-culture with neurons.

Glial changes following traumatic injury to glial monolayers as well as to neuronal-glial co-culture systems in vitro were examined with a focus on the expression of mRNAs coding for the immediate early genes (IEG) c-fos, c-jun and zif/268, demonstrated using in situ hybridization. Glial cells along scratch wound lines extended cytoplasmic processes as early as 10 min post-injury and the whole wound was covered with gliosis by 24 h. For complete restoration in the case of glial cells co-cultured with neurons, this required 48 h. Induction of the three IEG mRNAs was eminent along the edges of scratch wound, peaking at 30-60 min post-injury and subsiding by 3 h. The peak expression of IEG mRNAs was delayed to 1-3 h post-injury and became undetectable at 6 h in neuronal-glial co-cultures. The data suggest that mechanical injury to glial cells causes gliosis and the expression of IEG mRNAs, which are suppressed by co-culture with neurons, indicating some influence of neuronal-glial interactions.

Glia modulate the response of murine cortical neurons to excitotoxicity: glia exacerbate AMPA neurotoxicity

We have developed "pure" neuronal cultures (< 1% astrocytes) from mouse neocortex to study the effect of glial cells on the response of neurons to injury. Cortical neurons were found to require glial-conditioned medium to survive. Immature neurons, 2-4 d in vitro, deprived of glial-conditioned medium, underwent apoptosis over 48 hr, as suggested by condensed nuclear morphology, DNA fragmentation, and protection by inhibition of macromolecular synthesis. Apoptosis induced by trophic factor deprivation has been described for other neuronal populations, such as superior cervical ganglion and dorsal root ganglion cells. Cortical neurons in pure culture provide another neuronal population for the study of apoptosis induced by trophic factor deprivation. We then studied the interaction of neurons and glia under excitotoxic conditions. Experiments on mature cultures showed that pure neuronal cultures were at least 10-fold more sensitive to acute glutamate exposure than were neuronal-glial ("mixed") cocultures. The difference in sensitivity between pure neurons and mixed cultures was reduced when mixed cultures were treated with the glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (trans-PDC). In 24 hr exposure to N-methyl-D-aspartate (NMDA), or oxygen, glucose deprivation, pure neurons were more sensitive than mixed cultures; trans-PDC again increased the sensitivity of mixed cultures to nearly that of pure neuronal cultures. In contrast, mixed and pure neuronal cultures exposed to NMDA for 10 min, or to kainate for 24 hr, had similar injury dose-response curves, suggesting that glial glutamate uptake is a less important protective mechanism in these excitotoxic injuries. Surprisingly, pure neurons were less sensitive than mixed cultures to (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) toxicity at concentrations up to 100 microM. This does not reflect astrocyte toxicity, as AMPA at concentrations to 1 mM did not injure astrocyte cultures. Glial cultures showed increased levels of glutamate in the extracellular medium in response to exposure to AMPA, but not NMDA or kainate. However, pure neuronal and mixed cultures exposed to the same concentration of AMPA did not have elevated levels of glutamate in the media. We found that glia were generally neuroprotective under excitotoxic conditions, likely through their ability to clear extracellular glutamate. However, the presence of glia exacerbated AMPA neurotoxicity.

Maintenance of Neuronal Glutathione by Glial Cells

Glutathione levels in neurons and glial cells were investigated in a neuronal-glial coculture and in separate cultures. Brain cell suspensions obtained from cerebral hemispheres of fetal rats were cultured, and after 5 days the glutathione content of this cell population, consisting mainly of neurons and astroglial cells, was 23.0 nmol/mg of cell protein, with a significantly high content in glial cells (28.0 nmol/mg of protein) in comparison with neurons (18.8 nmol/mg of protein). When the neurons and glial cells were separated and recultured in fresh medium, neuronal glutathione rapidly decreased, whereas glial glutathione remained unchanged. Cysteine is a rate-limiting precursor for glutathione synthesis, and its level was also decreased in neurons, but not in glial cells. Cysteine was taken up rapidly by both neurons and glial cells, but cystine was taken up only by glial cells. This accounts for the rapid decrease of glutathione in the cultured neurons, because the culture medium contains cystine, but not cysteine. It was also found that the cultured glial cells released cysteine into the medium. These results suggest that neurons maintain their glutathione level by taking up cysteine provided by glial cells.

Expression of Platelet-Derived Growth Factor and Its Receptor in Rat Neuronal and Astroglial Cultures

Platelet-derived growth factor (PDGF) is a potent mitogen and differentiation signal for glial cells in the central nervous system. Several lines of evidence have indicated that neurons are one source of PDGF. We present data suggesting that they may respond to a PDGF signal as well. Immunofluorescence techniques have been used to demonstrate the presence of PDGF and its receptor in both neurons and glial cells isolated from developing rat brain and maintained in vitro. Similar findings hold either in medium supplemented with serum or under serum-free conditions. PDGF receptor β subunit (PDGF-Rβ)-like immunoreactivity was found encrusting the surface of cultured neurons. Virtually all neurons in this study contained PDGF-Rβ on the surface of the perikaryon and on neuronal processes down to their fine distal tips. PDGF receptor was found on neurons cultured in serum-containing or serum-free medium, and in cultures with few glial cells as well as neuronal-glial co-cultures. Neurons and glial cells also contain PDGF subunits (designated PDGF-A and PDGF-B), revealed by immunofluorescence staining. Isoform-specific antibodies stain cultured neurons and glia in either serum-containing or serum-free medium. The identity of stained cells was confirmed using double- or triple-labeling immunofluorescence procedures for simultaneous localization of PDGF isoforms, PDGF receptor, phosphorylated neurofilament protein, and/or glial fibrillary acidic protein. These studies indicate that both neurons and glia contain platelet-derived growth factor and carry the PDGF receptor on their cell surface in vitro. While the localization of PDGF and its receptor have previously been considered separately, the present study demonstrates that both the growth factor and the receptor are expressed by the same population of neurons, indicating that PDGF may act us an autocrine factor regulating neuronal differentiation.

Serotonin promotes region-specific glial influences on cultured serotonin and dopamine neurons.

To test the hypothesis that glia mediate interactions between embryonic serotonergic (5-HT) neurons and dopamine neurons, we studied the effects of 5-HT in co-cultures of E14 raphe neurons of mesencephalic dopamine neurons and radial glia/astrocytes derived from the same (homotypic) or opposite (heterotypic) brain region using a dose (10(-5) M) that would produce 5-HT uptake into glial cells as well as activate 5-HT receptors. Morphometric analysis of 5-HT and tyrosine hydroxylase (TH) immunoreactive neurons revealed regional differences in the effects of 5-HT (and nialamide) on survival, cell soma size, and dendrite-like neurite outgrowth in neuronal-glial co-cultures. In general, 5-HT had more significant effects on both types of monoamine neuron when they were cultured with mesencephalic glia (GSN). Stimulatory effects of 5-HT on growth of TH neurons in GSN cultures suggest that developing raphe axons, which reach the mesencephalon during the early differentiation of these neurons, may enhance the influence of local glial-derived trophic factors. Likewise, the promotion of 5-HT neuronal survival in these cultures suggests that glial factors in the mesencephalon may contribute to the support of 5-HT neurons in addition to the influences of raphe glia. The inhibitory effects of 5-HT on neurite outgrowth by raphe neurons in GSN co-cultures indicates enhanced sensitivity of these neurons to the inhibitory effects of 5-HT in the presence of mesencephalic glia. The region-specific effects of 5-HT and nialamide in glial co-cultures suggest that raphe and mesencephalic glia may express different capacities for 5-HT uptake, receptors, and/or monoamine oxidase (MAO) activities. These characteristics could be important for the specificity of growth-regulatory influences of glial cells on the development of brain monoamine neurons.

Glutamate Neurotoxicity in Culture Depends on the Presence of Glutamine: Implications for the Role of Glial Cells in Normal and Pathological Brain Development

Glutamate toxicity was studied in neuronal (SC9), glial (WC5), and neuroblastoma-glioma hybrid cell lines. In all three cell types, glutamate had a dual effect, depending on the concentration of glutamine in the culture medium. An expected dose-dependent cytotoxicity of the amino acid was observed when cells were cultured in medium containing the standard glutamine concentration (1-4 mM), but when the culture's glutamine content was decreased to 0.15-0.5 mM, glutamate had an apparent opposite, growth-promoting effect. The specificity of glutamate effect was indicated by the following: (a) it was stereospecific, with the L and not the D isomer being active; (b) monosodium aspartate was inactive in the presence of either high or low glutamine; and (c) monosodium glutamate and monopotassium glutamate had a similar dual effect. Furthermore, the glutamate receptor antagonist gamma-glutamylglycine blocked the amino acid cytotoxicity in a dose-dependent fashion. As glial cells are a major source of glutamine in the brain, neuronal-glial co-cultures were used to analyze the possible role of glial cells in glutamate neurotoxicity. It was found that SC9 cells were more sensitive to glutamate when co-cultured with WC5 cells. Continuous depolarization of the SC9 cells with KCl decreased cell number, but glutamate had no additive neurotoxic effect when added with KCl. We suggest that glutamine, glial cells, and neuronal activation play roles in modulating glutamate neurotoxicity, in developing as well as aged brains. It is tempting to speculate also that alterations in the glutamate/glutamine ratio under pathological conditions may take part in the etiology of some neurodegenerative diseases.