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Effects of ischemic post-conditioning on neuronal VEGF regulation and microglial polarization in a rat model of focal cerebral ischemia.

ABSTRACT: Ischemic postconditioning is increasingly being investigated as a therapeutic approach for cerebral ischemia. However, the majority of studies are focused on the acute protection of neurons per se. Whether and how postconditioning affects multiple cells in the recovering neurovascular unit remains to be fully elucidated. Here, we asked whether postconditioning may modulate help-me signaling between injured neurons and reactive microglia. Rats were subjected to 100 min of focal cerebral ischemia, then randomized into a control versus postconditioning group. After 3 days of reperfusion, infarct volumes were significantly reduced in animals treated with postconditioning, along with better neurologic outcomes. Immunostaining revealed that ischemic postconditioning increased expression of vascular endothelial growth factor (VEGF) in neurons within peri-infarct regions. Correspondingly, we confirmed that VEGFR2 was expressed on Iba1-positive microglia/macrophages, and confocal microscopy showed that in postconditioned rats, these cells were polarized to a ramified morphology with higher expression of M2-like markers. Treating rats with a VEGF receptor 2 kinase inhibitor negated these effects of postconditioning on microglia/macrophage polarization. In vitro, postconditoning after oxygen-glucose deprivation up-regulated VEGF release in primary neuron cultures, and adding VEGF to microglial cultures partly shifted their M2-like markers. Altogether, our findings support the idea that after postconditioning, injured neurons may release VEGF as a 'help-me' signal that promotes microglia/macrophage polarization into potentially beneficial phenotypes.

SUBMITTER: Esposito E

PROVIDER: S-EPMC6105375 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Project description:BackgroundActivated microglia are polarized into the M1 or M2 phenotype. We previously reported that electroacupuncture (EA) effectively prevented nuclear factor-κB (NF-κB) nuclear translocation and improved neuronal C-X-C motif 3 chemokine ligand 1 (CX3CL1) expression, repressing microglial activation by upregulating neuronal cylindromatosis (CYLD) expression in the periischemic cortex. However, the potential mechanisms are unclear. Therefore, we explored whether EA improved CYLD protein expression to regulate microglial polarization-mediated neuroinflammation and the potential mechanisms in an ischemic stroke model.MethodsA middle cerebral artery occlusion/reperfusion (MCAO/R) model was established in male Sprague-Dawley (SD) rats. The rats were treated with EA at the Baihui, Hegu and Taichong acupoints once daily beginning 2 h after focal cerebral ischemia. CYLD gene interference was used to investigate the role of CYLD in microglial polarization. We used neurobehavioral evaluations and TTC staining to examine the neuroprotective effect of EA via CYLD upregulation. Immunofluorescence and RT-qPCR were used to measure NLRP3 activation, M1/M2 microglial activation, pro-/anti-inflammatory gene mRNA expression and crosstalk (CX3CL1/CX3CR1 axis) between neurons and microglia. Western blotting was used to assess the underlying molecular mechanism.ResultsCYLD inhibited M1 microglial activation and improved M2 microglial activation after 72 h of reperfusion. CYLD overexpression decreased the NLRP3 mRNA level. CYLD suppressed microglial overactivation by inhibiting NLRP3 activation. CYLD gene silencing partially weakened EA improvement of neurological function deficits and reduction of infarct volumes after 72 h reperfusion. In addition, EA inhibited M1-like phenotypic microglial activation and promoted M2-like phenotypic microglia through upregulating CYLD expression. Finally, EA-mediated modulation of the CX3CL1/CX3CR1 axis and NLRP3 inflammasome was reversed by CYLD gene silencing in the periischemic cortex.ConclusionEA-induced upregulation of neuronal CYLD expression plays anti-inflammatory and neuroprotective roles and regulates the interaction between neurons and microglia, thereby suppressing M1 and improving M2 microglial activation in the periischemic cortex.

Project description:Cerebral ischemia is a severe, acute condition, normally caused by cerebrovascular disease, and results in high rates of disability, and death. Phagoptosis is a newly recognized form of cell death caused by phagocytosis of viable cells, and has been reported to contribute to neuronal loss in brain tissue after ischemic stroke. Previous data indicated that exposure of phosphatidylserine to viable neurons could induce microglial phagocytosis of such neurons. Phosphatidylserine can be reversibly exposed to viable cells as a result of a calcium-activated phospholipid scramblase named TMEM16F. TMEM16F-mediated phospholipid scrambling on platelet membranes is critical for hemostasis and thrombosis, which plays an important role in Scott syndrome and has been confirmed by much research. However, few studies have investigated the association between TMEM16F and phagocytosis in ischemic stroke. In this study, a middle-cerebral-artery occlusion/reperfusion (MCAO/R) model was used in adult male Sprague-Dawley rats in vivo, and cultured neurons were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate cerebral ischemia-reperfusion (I/R) injury in vitro. We found that the protein level of TMEM16F was significantly increased at 12 h after I-R injury both in vivo and in vitro, and reversible phosphatidylserine exposure was confirmed in neurons undergoing I/R injury in vitro. Additionally, we constructed a LV-TMEM16F-RNAi transfection system to suppress the expression of TMEM16F during and after cerebral ischemia. As a result, TMEM16F knockdown alleviated motor function injury and decreased the microglial phagocytosis of viable neurons in the penumbra through inhibiting the "eat-me" signal phosphatidylserine. Our data indicate that reducing neuronal phosphatidylserine-exposure via deficiency of TMEM16F blocks phagocytosis of neurons and rescues stressed-but-still-viable neurons in the penumbra, which may contribute to reducing infarct volume and improving functional recovering.

Project description:BackgroundThe pathogenic roles of receptor-mediated sphingosine 1-phosphate (S1P) signaling in cerebral ischemia have been evidenced mainly through the efficacy of FTY720 that binds non-selectively to four of the five S1P receptors (S1P1,3,4,5). Recently, S1P1 and S1P2 were identified as specific receptor subtypes that contribute to brain injury in cerebral ischemia; however, the possible involvement of other S1P receptors remains unknown. S1P3 can be the candidate because of its upregulation in the ischemic brain, which was addressed in this study, along with underlying pathogenic mechanisms.MethodsWe used transient middle cerebral artery occlusion/reperfusion (tMCAO), a mouse model of transient focal cerebral ischemia. To identify S1P3 as a pathogenic factor in cerebral ischemia, we employed a specific S1P3 antagonist, CAY10444. Brain damages were assessed by brain infarction, neurological score, and neurodegeneration. Histological assessment was carried out to determine microglial activation, morphological transformation, and proliferation. M1/M2 polarization and relevant signaling pathways were determined by biochemical and immunohistochemical analysis.ResultsInhibiting S1P3 immediately after reperfusion with CAY10444 significantly reduced tMCAO-induced brain infarction, neurological deficit, and neurodegeneration. When S1P3 activity was inhibited, the number of activated microglia was markedly decreased in both the periischemic and ischemic core regions in the ischemic brain 1 and 3 days following tMCAO. Moreover, inhibiting S1P3 significantly restored the microglial shape from amoeboid to ramified microglia in the ischemic core region 3 days after tMCAO, and it attenuated microglial proliferation in the ischemic brain. In addition to these changes, S1P3 signaling influenced the proinflammatory M1 polarization, but not M2. The S1P3-dependent regulation of M1 polarization was clearly shown in activated microglia, which was affirmed by determining the in vivo activation of microglial NF-?B signaling that is responsible for M1 and in vitro expression levels of proinflammatory cytokines in activated microglia. As downstream effector pathways in an ischemic brain, S1P3 influenced phosphorylation of ERK1/2, p38 MAPK, and Akt.ConclusionsThis study identified S1P3 as a pathogenic mediator in an ischemic brain along with underlying mechanisms, involving its modulation of microglial activation and M1 polarization, further suggesting that S1P3 can be a therapeutic target for cerebral ischemia.

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Effects of ischemic post-conditioning on neuronal VEGF regulation and microglial polarization in a rat model of focal cerebral ischemia.

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