Depletion of Microglia from Primary Cellular Cultures

Pont-Lezica, Lorena; Colasse, Sabrina; Bessis, Alain

doi:10.1007/978-1-62703-520-0_7

Cited by 9 publications

(9 citation statements)

References 28 publications

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“…Elimination of microglia from astroglia is important for distinguishing the astroglial response to TLR4 stimulation. Ara-C combined with LME eliminates microglia effectively [ 24 , 25 ]. The mechanism by which LME destroys microglia has not been fully established.…”

Section: Discussionmentioning

confidence: 99%

“…Usually, secondary astroglial cultures contain only small amounts (<10 % of total cells) of microglia, as previously described [ 27 , 28 ]. To eliminate all possible metabolic effects of microglial contamination, the treatment with a combination of Ara-C and LME was applied according to the method described by Pont-Lezica et al [ 25 ]. Briefly, 10 μmol/L (final concentration in nutrient medium) of Ara-C was added to the astroglial culture 3 days prior to the assay.…”

Section: Methodsmentioning

confidence: 99%

“…The present study used a conventional secondary astroglial culture prepared from pups of Sprague–Dawley rats. To eliminate microglial cells, the conventional astroglial culture was treated with cytosine β- d -arabinofuranoside hydrochloride (Ara-C), followed by l -leucine methyl ester (LME) [ 24 , 25 ]. This procedure completely eliminates Iba1-positive cells.…”

Section: Introductionmentioning

confidence: 99%

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A possible role of microglia-derived nitric oxide by lipopolysaccharide in activation of astroglial pentose-phosphate pathway via the Keap1/Nrf2 system

Iizumi

Takahashi

Mashima

et al. 2016

J Neuroinflammation

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BackgroundToll-like receptor 4 (TLR4) plays a pivotal role in the pathophysiology of stroke-induced inflammation. Both astroglia and microglia express TLR4, and endogenous ligands produced in the ischemic brain induce inflammatory responses. Reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines produced by TLR4 activation play harmful roles in neuronal damage after stroke. Although astroglia exhibit pro-inflammatory responses upon TLR4 stimulation by lipopolysaccharide (LPS), they may also play cytoprotective roles via the activation of the pentose phosphate pathway (PPP), reducing oxidative stress by glutathione peroxidase. We investigated the mechanisms by which astroglia reduce oxidative stress via the activation of PPP, using TLR4 stimulation and hypoxia in concert with microglia.MethodsIn vitro experiments were performed using cells prepared from Sprague–Dawley rats. Coexisting microglia in the astroglial culture were chemically eliminated using l-leucine methyl ester (LME). Cells were exposed to LPS (0.01 μg/mL) or hypoxia (1 % O2) for 12–15 h. PPP activity was measured using [1-14C]glucose and [6-14C]glucose. ROS and NO production were measured using 2′,7′-dichlorodihydrofluorescein diacetate and diaminofluorescein-FM diacetate, respectively. The involvement of nuclear factor-erythroid-2-related factor 2 (Nrf2), a cardinal transcriptional factor under stress conditions that regulates glucose 6-phosphate dehydrogenase, the rate-limiting enzyme of PPP, was evaluated using immunohistochemistry.ResultsCultured astroglia exposed to LPS elicited 20 % increases in PPP flux, and these actions of astroglia appeared to involve Nrf2. However, the chemical depletion of coexisting microglia eliminated both increases in PPP and astroglial nuclear translocation of Nrf2. LPS induced ROS and NO production in the astroglial culture containing microglia but not in the microglia-depleted astroglial culture. LPS enhanced astroglial ROS production after glutathione depletion. U0126, an upstream inhibitor of mitogen-activated protein kinase, eliminated LPS-induced NO production, whereas ROS production was unaffected. U0126 also eliminated LPS-induced PPP activation in astroglial–microglial culture, indicating that microglia-derived NO mediated astroglial PPP activation. Hypoxia induced astroglial PPP activation independent of the microglia–NO pathway. Elimination of ROS and NO production by sulforaphane, a natural Nrf2 activator, confirmed the astroglial protective mechanism.ConclusionsAstroglia in concert with microglia may play a cytoprotective role for countering oxidative stress in stroke.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0564-0) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A possible role of microglia-derived nitric oxide by lipopolysaccharide in activation of astroglial pentose-phosphate pathway via the Keap1/Nrf2 system

Iizumi

Takahashi

Mashima

et al. 2016

J Neuroinflammation

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“…Primary hippocampal neuron cultures were obtained from Sprague-Dawley rat pups (< 24 h), which were purchased from the Chinese Academy of Military Science (Beijing, China), and cultured according to the procedure described previously [48][49]. Briefly, the brain was dissected and the hippocampus was removed.…”

Section: Hippocampal Neurons Culturementioning

confidence: 99%

Autophagy activation aggravates hippocampal neuronal apoptosis caused by intermittent hypoxia

Tan¹,

Li²,

Meng³

et al. 2018

Oncotarget

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Unlike the role of autophagy in continuous hypoxia, the role of autophagy in intermittent hypoxia is unclear. To investigate whether autophagy is involved in intermittent hypoxia-caused hippocampal neuronal apoptosis, we studied the activity and action of autophagy in hippocampal neurons after exposure to intermittent hypoxia. We found the expression of autophagy-related proteins and the autolysosome formation were upregulated, and exposure to intermittent hypoxia led to a significant increase in light chain 3 (LC3) turnover and a decrease in SQSTM1/ p62 level in hippocampal neurons. Furthermore, accompanying autophagy activation, the level of hypoxia inducible factor-1α (HIF-1α) was increased. Conversely, inhibition of HIF-1α by chemical inhibitor or small interfering RNA (siRNA) downregulated the expression of autophagy-related proteins and blocked autophagy activation. We also found reactive oxygen species (ROS) increased after exposure to intermittent hypoxia, and inhibition of ROS by antioxidants decreased LC3-II expression. What's more, activation of autophagy by rapamycin exacerbated the apoptosis induced by intermittent hypoxia, whereas inhibition of autophagy by chloroquine or autophagyrelated genes siRNA (Beclin1, Atg5 and Atg7) ameliorated intermittent hypoxiainduced apoptosis. Additionally, X-linked inhibitor of apoptosis protein (XIAP), an apoptosis inhibitor protein, was decreased significantly after autophagy activation. Taken together, HIF-1α and ROS-mediated autophagy activation aggravated hippocampal neuronal apoptosis caused by intermittent hypoxia, and the decrease of XIAP might be one of the mechanisms of hippocampal neuronal apoptosis caused by autophagy activation.

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“…The primary neuronal cell cultures were performed as previously described, with minor modifications (16). Briefly, the neonatal brains were removed and dissected in D-Hank's solution.…”

Section: Introductionmentioning

confidence: 99%

Angiotensin II protects cortical neurons against oxygen-glucose deprivation-induced injury in vitro

et al. 2013

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Abstract. Ischemic cerebrovascular disease is a common type of cerebrovascular disease and the leading cause of disability and mortality worldwide. Therefore, it is crucial to elucidate its pathogenesis and develop novel therapeutic strategies. This study was performed to investigate whether angiotensin (Ang) II exerts a protective effect against cerebral ischemia̸reperfusion (I/R) injury in vitro. The primary cultured neurons were prepared and an I/R model was established by incubation of cortical neurons with Na 2 S 2 O 4 , followed by culture in fresh medium. The protective effect of Ang II and its underlying mechanisms were investigated by morphology observation, MTT assay, flow cytometry analysis and reverse transcription-polymerase chain reaction (RT-PCR). The data demonstrated that Ang II significantly ameliorated the neuronal injury caused by oxygen-glucose deprivation. Furthermore, Ang II increased cell viability through inhibiting cell apoptosis. The RT-PCR results revealed that Ang II was able to reverse the increased bax mRNA and the decreased bcl2 mRNA expression. Of note, the protective activity of Ang II may be attenuated by co-treatment with Ang II type 2 (AT2) receptor blockade (PD123319), but not Ang II type 1 (AT1) receptor blockade (valsartan). These findings suggested that Ang II exerted a protective effect against neuronal injury induced by oxygen-glucose deprivation through decreasing cell apoptosis. Therefore, Ang II may be used as a potential therapeutic target in the future.

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Depletion of Microglia from Primary Cellular Cultures

Cited by 9 publications

References 28 publications

A possible role of microglia-derived nitric oxide by lipopolysaccharide in activation of astroglial pentose-phosphate pathway via the Keap1/Nrf2 system

A possible role of microglia-derived nitric oxide by lipopolysaccharide in activation of astroglial pentose-phosphate pathway via the Keap1/Nrf2 system

Autophagy activation aggravates hippocampal neuronal apoptosis caused by intermittent hypoxia

Angiotensin II protects cortical neurons against oxygen-glucose deprivation-induced injury in vitro

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