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PubMed2006

Relationships between neurons expressing neuronal nitric oxide synthase, degree of microglia activation and animal survival. A study in the rat cortex after transient ischemia.

The focal ischemia obtained in an animal model of middle cerebral artery occlusion (MCAo) causes the "core" of damage in the striatum and the "penumbra" of damage in the fronto-parietal cortex. The latter is mainly functionally affected and shows changes in nNOS and iNOS expression during the acute phase of ischemia. With the aim to study possible relationships between these changes and the affection entity during the animal recovery, we investigated from 24 up to 144 h after reperfusion the expression and content of these two NOS isoforms in the neurons and microglia and the degree of microglia reactivity in the fronto-parietal cortices of rats undertaken to transient MCAo. Evaluation of motor-sensory performances and survival allowed dividing the animals into two groups. Immunohistochemistry, western blot and quantitative analysis demonstrated, both in the ischemic and contralateral cortex of the rats with longer survival, wellness and significantly increased number of the nNOS-IR neurons at 24 h and moderately activated microglia up to 144 h. In the rats not recovering, injured and significantly decreased nNOS-IR neurons, intensely activated microglia and appearance of iNOS-IR were seen at all time points. In conclusion, since the recovery occurs when nNOS-IR neurons are greatly increased, we presume nNOS protect the tissue likely controlling the passage from the state of reactive to that of activated microglia. Moreover, the morphological signs of wellness and the two-fold increase in number of the nNOS-IR neurons appear to be characteristic of the "penumbra" area and could explain why this region is mainly functionally affected.

Animals↗
PubMed2006

Role of microglia in the pathogenesis of osmotic-induced demyelination.

Osmotic demyelination is a serious disease caused by rapid correction of hyponatremia. In humans, demyelinative lesions occur preferentially in the central pons, and thus are termed central pontine myelinolysis. Although accumulation of microglia has been reported in such demyelinative lesions, their role in the pathogenesis of osmotic demyelination remains unclear. We examined the expression of cytokines in microglia that accumulated in the demyelinative lesions in a rat model of osmotic demyelination. Hyponatremia was induced in rats by a combination of dDAVP infusion and liquid diet feeding. After 7 days, serum sodium levels were rapidly corrected by hypertonic saline injection. The rats developed severe motor deficits, and marked demyelinative lesions were found in the midbrain and cerebral cortex. In the area of the demyelinative lesions, massive accumulations of microglia were observed that expressed the proinflammatory cytokines TNF-alpha and IFN-gamma as well as iNOS. In contrast, in hyponatremia corrected rats treated with lovastatin, which is known to inhibit microglial infiltration in various animal models of CNS disease, neurological impairments and the degree of demyelination were significantly ameliorated. Lovastatin also reduced the accumulation of microglia and decreased the expression of TNF-alpha in the demyelinative lesions. These results indicate that microglia play a detrimental role in the pathogenesis of osmotic demyelination by producing proinflammatory cytokines, and further suggest that lovastatin may be useful in repressing the demyelination.

Animals↗
PubMed2003

The isolation and culture of microglia-like cells from the goldfish brain.

We have developed a method for isolating goldfish microglia. Cells were identified as microglia immunohistochemically with NN-2, a monoclonal antibody (MAb) raised against teleost retinal microglial cells, and by their phagocytic abilities. Morphological characterization of the cells identified round, phase-bright cells as well as flattened macrophage-like cells. Ramified cells were also seen but they were rare. Fusion of macrophage-like cells occurred in high density cultures and resulted in the formation of giant cells that disintegrated a few days later. Immunohistochemical studies demonstrated that virtually all of the cells in our cultures were NN-2+ and did not label with either antiGFAP (an astrocyte marker) or MAb 6D2 (an oligodendrocyte marker). Cells identified as microglia were intensely phagocytic and ingested latex microspheres, DiIAcLDL and goldfish myelin in vitro. In addition, we labelled microglial cells in vivo with intracranial injections of fluorescent dextran and found that microglia isolated from these animals contained the dextran and phagocytosed microspheres. We also studied the effect of myelin on microsphere uptake and compared the effect of myelin and opsonized myelin on the phagocytic activity of the cells. Our results showed a clear increase in the phagocytic activity of microglia when incubated with myelin, with an enhanced effect of opsonized myelin.

Animals↗
PubMed2005

Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes.

Cultures of astrocytes can be readily established and are widely used to study the biological functions of these glial cells in isolation. Unfortunately, contamination by microglia can confound results from such studies. Herein, a simple and highly effective modification of a common procedure to remove microglia from astrocyte cultures is described. After becoming confluent, astrocytes were exposed to a mitotic inhibitor for 5-6 days then treated with 50-75 mM l-leucine methyl ester (LME) for 60-90 min. Unlike previous protocols that employed lower LME concentrations on subconfluent cultures or during passage of astrocytes, this protocol effectively depleted microglia from high-density astrocyte monolayers. This was evidenced by the selective depletion of microglial-specific markers. Purified monolayers appeared morphologically normal 24h after LME treatment and expressed nitric oxide synthase-2 (NOS-2) and cyclooxygenase-2 (COX-2) proteins upon stimulation with LPS plus IFNgamma, albeit to a lower level than unpurified monolayers. This difference could be attributed to removal of contaminating microglia from monolayers and not to astrocyte dysfunction, since LME treatment did not alter global protein synthesis and a reactive phenotype could be induced in the purified monolayers. Thus, this modified protocol selectively depletes microglia from high-density primary astrocyte monolayers without compromising their functional integrity.

Animals↗
PubMed2004

P2X7 receptor modulation of beta-amyloid- and LPS-induced cytokine secretion from human macrophages and microglia.

To test whether extracellular ATP can play a role in the neuroimmunopathology of Alzheimer's disease (AD), we evaluated the capacity of the ATP-binding purinoreceptor, P2X7, to modulate cytokine secretion on cultured human macrophages and microglia pre-activated 24 h with the 42 amino acid beta-amyloid peptide (Abeta(1-42)) or lipopolysaccharide. Thirty minutes of exposure to the selective P2X7 agonist 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) resulted in the secretion of IL-1beta after either Abeta(1-42) or LPS stimulation of human macrophages that was dependent on the concentration of the stimulus used to pre-activate the cells. Further tests on human microglia treated with BzATP (300 microM) resulted in a 1.5- and 3.5-fold enhancement of IL-1alpha and IL-1beta secretion, respectively, from cells pre-activated by 10 microM Abeta(1-42) and a 1.6- and 3.9-fold enhancement of IL-1alpha and IL-1beta secretion, respectively, from cells pre-activated by 1 microg/ml LPS. BzATP induction of IL-1alpha and IL-1beta secretion from microglia was completely reversed by pre-incubation of the cells with the P2X7 antagonist, adenosine 5'-triphosphate 2',3'-acyclic dialcohol (oxidized ATP). In contrast to its effects on IL-1alpha and IL-1beta secretion, BzATP induced TNF-alpha after LPS stimulation, but not after stimulation with Abeta(1-42), induced IL-18 secretion regardless of whether microglia were pre-activated and attenuated IL-6 secretion after either LPS or Abeta(1-42) pre-activation. These results demonstrate that extracellular ATP can modulate Abeta-induced cytokine secretion from human macrophages and microglia and thus may play a role in the neuroimmunopathology of AD.

Adenosine Triphosphate↗
PubMed2004

Microglia phagocytose alloreactive CTL-damaged 9L gliosarcoma cells.

Intracranial adoptive transfers of alloreactive cytotoxic T lymphocytes (aCTL) for brain tumor treatment were safe and showed promise in preclinical and early clinical trials. To better understand the endogenous immune responses that may ensue following cellular therapy with aCTL, we examined the ability of microglia to phagocytose aCTL-damaged and undamaged rat 9L gliosarcoma cells in vitro and in vivo. In vitro, 5.5+/-0.9% of microglial cells isolated from adult tumor-bearing rat brains phagocytosed aCTL-damaged 9L cells, whereas microglia did not bind to or ingest undamaged 9L cells. Addition of supernates from either 9L cell cultures or from aCTL+9L co-incubate cell cultures to microglia did not significantly alter their ability to bind to or phagocytose damaged glioma cells even though the latter contained T helper 1 and 2 cytokines. At 3 days following intracranial 9L cell infusion, 17.5+/-0.1% of the microglia phagocytosed CFSE-labeled aCTL-damaged 9L tumor cells within the adult rat brain, confirming the in vitro data. The results suggest that microglia within the tumor microenvironment of the adult rat glioma model selectively remove damaged, but not undamaged, glioma cells.

Animals↗
PubMed2005

Fractalkine-upregulated milk-fat globule EGF factor-8 protein in cultured rat microglia.

Fractalkine is the only known member of the CX(3)C-chemokine family, and so is its receptor CX(3)CR1. Fractalkine, typically is expressed by neurons where it is inserted in the plasma membrane ("chemokine on a stalk"). It can, however, be clipped off by a specific enzyme and diffuse into the extracellular space. CX(3)CR1 is primarily expressed by microglia, the phagocytes of the brain. This study was aimed at studying gene expression changes in cultured rat microglia upon fractalkine stimulation using gene chip technology. Six genes turned out to be upregulated, amongst which milk-fat globule EGF factor-8 protein (MFG-E8) was the most surprising, but also the most revealing one. We hypothesize that it serves as a bridging molecule between apoptotic cells (neurons) and microglia. Since the docking to microglia is, in part, mediated by members of the integrin family, six of these molecules have been-post hoc-included in real-time PCR confirmations of chip results. Two of them-integrin alpha(2) and integrin beta(5)-were upregulated as well. These data provide a much closer look into molecular mechanisms involved in apoptosis of neurons and their removal by microglia.

Animals↗
PubMed2005

Leptomeningeal cells activate microglia and astrocytes to induce IL-10 production by releasing pro-inflammatory cytokines during systemic inflammation.

The leptomeninges covering the surface of the brain parenchyma play the physical role at the cerebrospinal fluid-blood barrier. We report here that leptomeningeal cells may transduce peripheral proinflammatory signals to the central anti-inflammatory response through the activation of glial cells in the brain parenchyma. After adjuvant injection, both microglia and astrocytes in the cerebral cortex localized in the proximity of the leptomeninges were activated. The protein levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) in the cortical extracts were significantly increased at different time after adjuvant injection. The TNF-alpha immunoreactivity was most prominent in the leptomeninges covering astrocytes. On the other hand, the IL-10 immunoreactivity was observed in both activated microglia and astrocytes localized along the leptomeninges. Cultured leptomeningeal cells covering the cerebral cortex released TNF-alpha which was significantly increased by lipopolysaccharide (LPS). Upon stimulation with LPS, cultured leptomeningeal cells also secreted interleukin-1beta and interleukin-6 with differential time-courses. When primary cultured rat astrocytes and microglia were treated with the conditioned medium of LPS-activated cultured leptomeningeal cells, the immunoreactivity of IL-10 was markedly increased. These observations strongly suggest that leptomeningeal cells release pro-inflammatory cytokines to activate both microglia and astrocytes during systemic inflammation. The activated astrocytes and microglia may in turn regulate anti-inflammatory response in the brain by providing IL-10.

Animals↗
PubMed2005

Recognition of Staphylococcus aureus-derived peptidoglycan (PGN) but not intact bacteria is mediated by CD14 in microglia.

Recognition of Staphylococcus aureus and its cell-wall component peptidoglycan (PGN) by microglia is mediated, in part, by Toll-like receptor 2 (TLR2). However, the pattern recognition receptor (PRR) CD14 can also bind PGN and enhance TLR2-mediated signaling in macrophages, suggesting a similar phenomenon might occur in microglia. To assess the functional significance of CD14 on microglial activation, we evaluated the responses of primary microglia isolated from CD14 knockout (KO) and wild type (WT) mice. PGN-dependent microglial activation was partially CD14-dependent as demonstrated by the attenuated expression of TNF-alpha, macrophage inflammatory protein-2 (MIP-2/CXCL2), and the soluble PRR pentraxin-3 in CD14 KO microglia compared to WT cells. In contrast, microglial responses to intact S. aureus occurred primarily via a CD14-independent manner. Collectively, these findings reveal the complex nature of gram-positive bacterial recognition by microglia, which occurs, in part, via CD14.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Involvement of COX-1 and up-regulated prostaglandin E synthases in phosphatidylserine liposome-induced prostaglandin E2 production by microglia.

After engulfment of apoptotic cells through phosphatidylserine (PS)-mediated recognition, microglia secrete prostaglandin E2 (PGE2), a potent anti-inflammatory molecule in the central nervous system. Despite the clinical significance, the mechanism underlying PGE2 production by phagocytosis of apoptotic cells is poorly understood. In the present study, we used PS liposomes to elucidate the phagocytic pathway for PGE2 production in microglia, because PS liposomes mimic the effects of apoptotic cells on microglia/macrophages. The level of PGE2 in the culture medium of primary cultured rat microglia was significantly increased by PS liposomes treatment but not by phosphatidylcholine liposomes treatment. The specific ligand for class B scavenger receptor (SR-B), high density lipoprotein, significantly suppressed PS liposome-induced PGE2 production. PS liposomes were immediately phagocytosed by microglia and sorted to endosomes/lysosomes. Cyclooxygenase (COX)-2 and membrane-bound prostaglandin E synthase-1 (mPGES-1) were induced by treatment with lipopolysaccharide (LPS) but not with PS liposomes. On the other hand, mPGES-2 and cytosolic PGES (cPGES) that are functionally coupled with COX-1 were upregulated after treatment with PS liposomes or LPS. Furthermore, PS liposome-induced PGE2 production was significantly suppressed by indomethacin, a preferential COX-1 inhibitor, but not by NS-398, a selective COX-2 inhibitor. PS liposomes induced activation of p44/p42 extracellular signal-regulated kinase (ERK) but not p38 mitogen-activated protein kinase in SR-BI independent manner. These observations strongly suggest that the up-regulation of terminal PGESs that are preferentially coupled with COX-1, especially mPGES-2, plays the pivotal role in PS liposome-induced PGE2 production by microglia. Although SR-BI plays an essential role in PS liposome-induced PGE2 production, other PS-recognizing receptors, possibly PS-specific receptor, could also promote PGE2 production by transducing intracellular signals including p44/p42 ERK after PS liposomes treatment.

Animals↗
PubMed2004

Suramin inhibits spinal cord microglia activation and long-term hyperalgesia induced by formalin injection.

UNLABELLED: Previous studies in our laboratory have shown that long-term (a period of weeks) increases in pain-related behavior were correlated with the activation of spinal microglia after subcutaneous injection of formalin into the dorsal surface of 1 hind paw. The present study examined whether intrathecal delivery of suramin (a P2 receptor antagonist) blocks microglia activation and long-term hyperalgesia induced by formalin injection. Suramin was administered by using an osmotic pump attached to an intrathecal catheter. Suramin delivery (1.25 microg/kg/h) began 1 day before the formalin injection and lasted for 4 days. Rats were observed by using a modified hot plate test before and at different times after formalin injection. The spinal cord was surveyed for changes in microglia labeling as shown by OX-42 staining at different times after formalin injection. Suramin decreased both the hyperalgesic sensitivity to the thermal stimuli and microglial activation induced by formalin injection as compared to the saline-treated group. This suggests that adenosine triphosphate is one potential mediator that activates spinal cord microglia and enhances pain-related behavior in the formalin model. PERSPECTIVE: This report suggests that blocking specific spinal P2 receptors might decrease the central enhancement of pain caused by peripheral injury and inflammation. One mechanism might be by blocking the activation of spinal microglia. Thus, P2 antagonists might have therapeutic usefulness in certain pain conditions.

Adenosine Triphosphate↗
PubMed2005

Microglia activated by IL-4 or IFN-gamma differentially induce neurogenesis and oligodendrogenesis from adult stem/progenitor cells.

Cell renewal in the adult central nervous system (CNS) is limited, and is blocked in inflammatory brain conditions. We show that both neurogenesis and oligodendrogenesis of adult neural progenitor cells in mice are blocked by inflammation-associated (endotoxin-activated) microglia, but induced by microglia activated by cytokines (IL-4 or low level of IFN-gamma) associated with T-helper cells. Blockage was correlated with up-regulation of microglial production of tumor necrosis factor-alpha. The effect induced by IL-4-activated microglia was mediated, at least in part, by insulin-like growth factor-I. The IL-4-activated microglia showed a bias towards oligodendrogenesis whereas the IFN-gamma-activated microglia showed a bias towards neurogenesis. It thus appears that microglial phenotype critically affects their ability to support or impair cell renewal from adult stem cell.

Animals↗
PubMed2004

Microglia-derived glial cell line-derived neurotrophic factor could protect Sprague-Dawley rat astrocyte from in vitro ischemia-induced damage.

The interrelationship between microglia and astrocytes in cerebral ischemia was determined in vitro by adding in vitro ischemia-induced supernatant from microglia into astrocytes under the same conditions (glucose-, oxygen- and serum-free). The involvement of glial cell line-derived neurotrophic factor (GDNF) was further investigated by immunoblocking assay and Western blot analysis. Results showed that microglia-derived supernatant protected against in vitro ischemia-induced damage of astrocytes and this protection was pre-blocked by anti-GDNF but not normal rabbit serum. In addition, in vitro ischemia appeared to induce the expression of GDNF in microglia. These results indicate that microglia-derived protection on astrocytes during in vitro ischemia is GDNF-dependent.

Animals↗
PubMed2005

Molecular mechanisms responsible for microglia-derived protection of Sprague-Dawley rat brain cells during in vitro ischemia.

Microglia-derived protection of brain cells (microglia, astrocytes, and neurons) during in vitro ischemic stress (deprivation of glucose, oxygen, and serum) was determined. Trypan blue exclusion assay, immunoblocking assay, Western blot analysis, and ELISA assay were used to determine the molecular mechanisms responsible for the microglia-derived protection. Results demonstrated that supernatants from the ischemic microglia protected all three cell-types from ischemia-induced damage by releasing the transforming growth factor-beta1 (TGF-beta1) and glial cell line-derived neurotrophic factor (GDNF). The protection of microglia was TGF-beta1 related, whereas astrocytes protection was GDNF-dependent. The protection of neurons was TGF-beta1 and GDNF independent, and the molecular nature responsible for their protection remains to be determined. These results indicate contribution from the surrounding cells and the types of receptors expressed on different brain cells probably also play an important role in determining their fate against ischemia.

Animals↗
PubMed2004

Enhancement of urokinase-type plasminogen activator (uPA) secretion, but not that of substrate plasminogen (PGn), by rat microglia stimulated with neuronal conditioned medium.

Assuming the presence of intercellular interactions between injured motoneurons and microglia in the axotomized facial nucleus, we investigated the effects of neuronal conditioned medium (NCM) on the release of urokinase-type plasminogen activator (uPA) from microglia. Zymography revealed that NCM markedly enhanced the release of uPA from microglia, although NCM itself did not contain a significant amount of uPA. In contrast, the secretion of plasminogen (PGn), a substrate of uPA, was not promoted by the NCM treatment. The specific effect of NCM was found to be quite distinct from that of microglial activators, interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS), which reduce uPA release from microglia. In summary, Neuron-derived soluble molecules appear to stimulate microglia to enhance the production/release of uPA, but not that of PGn, by a mechanism independent of the activation reaction by IFN-gamma or LPS.

Animals↗
PubMed2006

Amyloid-beta peptides induce several chemokine mRNA expressions in the primary microglia and Ra2 cell line via the PI3K/Akt and/or ERK pathway.

Alzheimer's disease (AD) is characterized by the presence of senile plaques composed primarily of amyloid-beta peptide (Abeta) in the brain. Microglia have been reported to surround these Abeta plaques, which have opposite roles, provoking a microglia-mediated inflammatory response that contributes to neuronal cell loss or the removal of Abeta and damaged neurons. To perform these tasks microglia migrate to the sites of Abeta secretion. We herein analyzed the process of chemokine expression induced by Abeta stimulation in primary murine microglia and Ra2 microglial cell line. We found that Abeta1-42 induced the expressions of CCL7, CCL2, CCL3, CCL4 and CXCL2 in the microglia. The signal transduction pathway for the expression of CCL2 and CCL7 mRNA induced by Abeta1-42 was found to depend on phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK), whereas the pathway for CCL4 depended only on PI3K/Akt. These inflammatory chemokine expressions by Abeta stimulation emphasize the contribution of neuroinflammatory mechanisms to the pathogenesis of AD.

Amyloid beta-Peptides↗
PubMed2004

Neuroprotective role of phosphodiesterase inhibitor ibudilast on neuronal cell death induced by activated microglia.

The phosphodiesterase inhibitor, ibudilast, has many effects on lymphocytes, endothelial cells, and glial cells. We examined the neuroprotective role of ibudilast in neuron and microglia co-cultures. Ibudilast significantly suppressed neuronal cell death induced by the activation of microglia with lipopolysaccharide (LPS) and interferon (IFN)-gamma. To examine the mechanisms by which ibudilast exerts a neuroprotective role against the activation of microglia, we examined the production of inflammatory and anti-inflammatory mediators and trophic factors following ibudilast treatment. In a dose-dependent manner, ibudilast suppressed the production of nitric oxide (NO), reactive oxygen species, interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha and enhanced the production of the inhibitory cytokine, IL-10, and additional neurotrophic factors, including nerve growth factor (NGF), glia-derived neurotrophic factor (GDNF), and neurotrophin (NT)-4 in activated microglia. Thus, ibudilast-mediated neuroprotection was primarily due to the inhibition of inflammatory mediators and the upregulation of neurotrophic factor. In the CA1 region of hippocampal slices, long-term potentiation (LTP) induced by high frequency stimulation (HFS) could be inhibited with LPS and interferon-gamma stimulation. Ibudilast returned this LTP inhibition to the levels observed in controls. These results suggest that ibudilast may be a useful neuroprotective and anti-dementia agent counteracting neurotoxicity in activated microglia.

Animals↗
PubMed2004

The radical scavenger edaravone prevents oxidative neurotoxicity induced by peroxynitrite and activated microglia.

The free radical scavenger edaravone has been used as an anti-oxidative agent in acute ischemic brain disorders. We examined the effect of edaravone on the production of nitric oxide (NO), reactive oxygen species (ROS) and proinflammatory cytokines by activated microglia, and we also examined its neuroprotective role in cortical neuronal cultures oxidatively stressed by the peroxynitrite donor N-morpholinosydnonimine (SIN-1) or activated microglia. Edaravone significantly suppressed the production of NO and ROS by activated microglia, though it did not suppress production of inflammatory cytokines. In addition, edaravone significantly suppressed neuronal cell death and dendrotoxicity induced by either SIN-1 or activated microglia in a dose-dependent manner. These results suggest that edaravone may function as a neuroprotective agent counteracting oxidative neurotoxicity arising from activated microglia, as occurs in either inflammatory or neurodegenerative disorders of the central nervous system.

Animals↗