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The pyrin domain-containing protein 3 (NLRP3) inflammasome drives the profound cerebral ischemia and reperfusion injury (I/R) and mediates the secretion of IL-1β (interleukin-1β), which exerts a subsequent cascade of inflammatory injury. The NLRP3-activated-microglial manipulation in adjacent neuronal and endothelial NLRP3 activation has been confirmed in our previous studies. In the present study, we extended the cognition of how microglia mediated neuronal and endothelial NLRP3-IL-1β signaling during cerebral ischemia and reperfusion injury. In vitro, Neuro-2a and bEND3 cells were cultured alone or co-cultured with BV2 cells and oxygen-glucose deprivation/reoxygenation (OGD/R) was performed. In vivo, transient middle cerebral artery occlusion (tMCAO) rat models and lentiviral silencing targeting IL-1R1 were performed. The NLRP3 inflammasome activation was evaluated by enzyme-linked immunosorbent assay, western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence. In the co-culture system after OGD/R treatment, NLRP3 inflammasomes in neurons and endothelial cells were activated by microglial IL-1β via IL-1β/IL-1R1/TRAF6 signaling pathway, with the basal protein level of NLRP3. In addition, ruptured lysosomes engulfing ASC specks which were possibly secreted from microglia triggered the enhanced NLRP3 expression. In cortices of tMCAO rats at 24 h of reperfusion, silencing IL-1R1, mainly presented in neurons and endothelial cells, was efficient to block the subsequent inflammatory damage and leukocyte brain infiltration, leading to better neurological outcome. Neuronal and endothelial NLRP3 inflammasomes were activated by microglia in cerebral ischemia and reperfusion injury mainly via IL-1β/IL-1R1/TRAF6 signaling, which might be therapeutically targetable., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Idebenone is a well-appreciated mitochondrial protectant while the mechanisms underlying the neuroprotection in cerebral ischemia and reperfusion (I/R) remain elusive. It has been manifested NLRP3 inflammasom activation contributed to I/R induced damage. It raises questions how exactly NLRP3 inflammasom was activated in microglia and neuron and whether idebenone reverses the process in I/R., Methods: I/R rat model was utilized and BV2, primary microglia and PC12 cells were subjected to oxygen-glucose deprivation (OGD). Then, western-blotting, q-PCR, immunofluorescence staining, ELISA, flow cytometry and immunoprecipitation analysis were performed., Results: We found ROS-NLRP3 singaling was activated in BV2 cells at OGD/R 24 h. Importantly, microglial NLRP3 activation was essential for NLRP3 activation in PC12 cells under microglial-neuronal co-culture circumstance, which has been confirmed to induced neuronal apoptosis. Further, we found mitochondrial dysfunction in OGD/R led to mt-DNA translocation as well as generation of mt-ROS, resulting cytosolic accumulation of oxidized mt-DNA. Ultimately, oxidized mt-DNA binding to NLRP3 contributed to further activation of NLRP3 and dramatically augmented inflammation in BV2 and PC12 cells. Furthermore, idebenone treatment inhibited the process, thus suppressing the NLRP3-mediated inflammatory injury after OGD/R. In vivo, NLRP3 was activated in microglia of I/R rats and inhibition of NLRP3 was observed in idebenone treatment group, which had less neurological deficit and less infarct volume., Interpretation: Our data revealed the anti-inflammatory effects of idebenone via suppressing activation of NLRP3 and ameliorating NLRP3-mediating damage in I/R, which may provide new insight in therapeutic strategy for ischemic stroke., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Although post-ischemic inflammation induced by the innate immune response is considered an essential step in the progression of cerebral ischemia injury, the role of triggering receptor expressed on myeloid cells 2 (TREM2) in the pathogenesis of ischemic stroke remains to be elucidated. Here, we found that the transcriptional and post-transcriptional levels of TREM2 were increased in cultured primary microglia after oxygen-glucose deprivation and reoxygenation and in the ischemic penumbra of the cerebral cortex after middle cerebral artery occlusion (MCAO) and reperfusion in mice. TREM2 was mainly expressed in microglia, but not in astrocytes, neurons, or oligodendrocytes in mice subjected to MCAO. Manipulating TREM2 expression levels in vitro and in vivo significantly regulated the production of pro- and anti-inflammatory mediators after ischemic stroke. TREM2 overexpression markedly suppressed the inflammatory response and neuronal apoptosis. By contrast, TREM2 gene silencing intensified the inflammatory response, increased neuronal apoptosis and infarct volume, and further exacerbated neurological dysfunction. Our study demonstrated that TREM2 protects against cerebral ischemia/reperfusion injury through the aspect of post-ischemic inflammatory response and neuronal apoptosis. Pharmacological targeting of TREM2 to suppress the inflammatory response may provide a new approach for developing therapeutic strategies in the treatment of ischemic stroke and other cerebrovascular diseases.

Purpose: The family of Toll-like receptors (TLRs) has recently been reported to play a role in ischemic injury, but the time course and cell types of the post-stroke TLR9 upregulation remain unclear. In this study, we investigated the dynamic changes of TLR9 expression and the expression of TLR9 in neurons and glial cells after cerebral ischemia reperfusion in mice., Methods: Focal cerebral ischemia was induced by middle cerebral artery occlusion for 90 min in male C57BL/6 mice. The TLR9 expression levels in the tissue surrounding the infarct were detected by Western Blot at 6 h, 3 d, 7 d, 14 d, 21 d, and 28 d after reperfusion. The expression of TLR9 in neurons and glial cells was observed by immunofluorescence staining., Results: The expression of TLR9 protein first increased and then decreased, with the peak observed at 14 d-21 d. Only small punctate intracellular TLR9 was occasionally observed in the neurons at each time point, and the TLR9-positive rate showed no difference at different time points. By contrast, the activated microglia gathered at the margin of the infarct, and the intracellular TLR9 changed from scattered small punctate to coarse and lumpy. The TLR9-positive rate of microglia was first increased and then decreased with time, with the peak observed at 3 d. No positive TLR9 staining was found in the astrocytes and oligodendrocytes., Conclusions: TLR9 expression showed dynamic changes for a long period of time and microglias were the main brain cells to express TLR9 after cerebral ischemia and reperfusion.

Following cerebral infarction, hypoxic tissues remains in the ischemic cortex for long periods of time. Glial fibrillary acidic protein (GFAP) is a specific marker of astrocytes, which is thought to be essential for neuronal survival. We aimed to clarify the relationship between hypoxic tissue and astrocytes following cerebral infarction. Rats with middle cerebral artery occlusion were randomly divided into a 1.5-hour ischemia-reperfusion(1.5-hour IR) group and a permanent ischemia (PI) group. Hypoxic tissue and GFAP fluorescence intensity in the ischemic cortex were observed postoperatively on days 1, 3, 7, and 14. Results showed that hypoxic tissue was present from day 1 to 14 in the 1.5-hour IR group and on days 1 and 3 in the PI group. The GFAP fluorescence intensity in the 1.5-hour IR group was stronger than that in the PI group at the same time point of observation. Over time, GFAP expression increased and peaked at 7 days in each group, followed by a decrease in signal. In hypoxic tissue, the GFAP fluorescence intensity was stronger than that in the surrounding tissue at all observation time points. These data indicate that astrocytes were strongly activated in hypoxic tissue induced by temporary ischemia followed by reperfusion. The activation of astrocytes may partially contribute to the survival and repair of hypoxic tissue following brain ischemia.