Maximilian Franke1, Michael Bieber2, Peter Kraft3, Alexander N R Weber4, Guido Stoll5, Michael K Schuhmann6. 1. Department of Neurology, University Hospital Würzburg, Josef-Schneider-Straße 11, 97080 Würzburg, Germany. Electronic address: franke_m1@ukw.de. 2. Department of Neurology, University Hospital Würzburg, Josef-Schneider-Straße 11, 97080 Würzburg, Germany. Electronic address: bieber_m@ukw.de. 3. Department of Neurology, Klinikum Main-Spessart, Grafen-von-Rieneck-Str. 5, 97816 Lohr, Germany. Electronic address: peter.kraft@klinikum-msp.de. 4. Interfaculty Institute of Cell Biology, Department of Immunology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany; iFIT - Cluster of Excellence (EXC 2180) "Image-Guided and Functionally Instructed Tumor Therapies", University of Tübingen, Germany. Electronic address: alexander.weber@uni-tuebingen.de. 5. Department of Neurology, University Hospital Würzburg, Josef-Schneider-Straße 11, 97080 Würzburg, Germany. Electronic address: stoll_g@ukw.de. 6. Department of Neurology, University Hospital Würzburg, Josef-Schneider-Straße 11, 97080 Würzburg, Germany. Electronic address: schuhmann_m@ukw.de.
Abstract
PURPOSE: Cerebral ischemia induces a profound neuro-inflammatory response, but the underlying molecular mechanisms are poorly understood. Inflammasomes (NLRP1, NLRP3, NLRC4, AIM2) are intracellular multi-protein complexes which can induce sets of pro-inflammatory cyto- and chemokines, and thereby guide inflammation. We, here, assessed the functional role of NLRP3 in ischemia/reperfusion (I/R) injury in a mouse model of transient cerebral ischemia. METHODS: Ischemic stroke was induced in C57Bl/6 mice by 60 min transient middle cerebral artery occlusion (tMCAO) and 3, 7 or 23 h of reperfusion, a paradigm of I/R injury. The expression patterns of inflammasomes in the ischemic hemispheres were evaluated by semiquantitative real-time PCR and Western Blot analysis accompanied by protein localization using immunocytochemistry. Finally, animals were treated with the inflammasome inhibitors Sulforaphane, Genipin, MCC950 or vehicle, directly before or upon recanalization after tMCAO. Stroke outcome was assessed, including infarct size and functional deficits, local inflammatory response, neuronal survival as well as blood-brain barrier function on day 1 after tMCAO. RESULTS: After tMCAO the relative gene expression levels of NLRP3 increased 20-30x within 1 day in the ischemic hemisphere which translated into an increased expression of NLRP3 in neurons. Accordingly, the gene expression levels of the NLRP3-modulator, Bruton's Tyrosine Kinase (BTK), and the NLRP3-inducible cytokine IL-1β significantly rose. Lesser or non-significant changes were seen for the other inflammasomes. Application of inflammasome inhibitors covering all inflammasomes or specifically NLRP3 significantly reduced infarct volumes when given before or after tMCAO and was accompanied by clear evidence for reduced activation of caspase 1. This stroke attenuating effect coincided with less immune cell infiltration in the ischemic hemisphere and preservation of the blood-brain barrier integrity. CONCLUSIONS: Our data show that induction of the NLRP3 inflammasome in neurons drives neuroinflammation in acute ischemic stroke. Early blockade of NLRP3 protects from I/R injury by mitigating inflammation and stabilizing the blood-brain barrier.
PURPOSE: Cerebral ischemia induces a profound neuro-inflammatory response, but the underlying molecular mechanisms are poorly understood. Inflammasomes (NLRP1, NLRP3, NLRC4, AIM2) are intracellular multi-protein complexes which can induce sets of pro-inflammatory cyto- and chemokines, and thereby guide inflammation. We, here, assessed the functional role of NLRP3 in ischemia/reperfusion (I/R) injury in a mouse model of transient cerebral ischemia. METHODS: Ischemic stroke was induced in C57Bl/6 mice by 60 min transient middle cerebral artery occlusion (tMCAO) and 3, 7 or 23 h of reperfusion, a paradigm of I/R injury. The expression patterns of inflammasomes in the ischemic hemispheres were evaluated by semiquantitative real-time PCR and Western Blot analysis accompanied by protein localization using immunocytochemistry. Finally, animals were treated with the inflammasome inhibitors Sulforaphane, Genipin, MCC950 or vehicle, directly before or upon recanalization after tMCAO. Stroke outcome was assessed, including infarct size and functional deficits, local inflammatory response, neuronal survival as well as blood-brain barrier function on day 1 after tMCAO. RESULTS: After tMCAO the relative gene expression levels of NLRP3 increased 20-30x within 1 day in the ischemic hemisphere which translated into an increased expression of NLRP3 in neurons. Accordingly, the gene expression levels of the NLRP3-modulator, Bruton's Tyrosine Kinase (BTK), and the NLRP3-inducible cytokine IL-1β significantly rose. Lesser or non-significant changes were seen for the other inflammasomes. Application of inflammasome inhibitors covering all inflammasomes or specifically NLRP3 significantly reduced infarct volumes when given before or after tMCAO and was accompanied by clear evidence for reduced activation of caspase 1. This stroke attenuating effect coincided with less immune cell infiltration in the ischemic hemisphere and preservation of the blood-brain barrier integrity. CONCLUSIONS: Our data show that induction of the NLRP3 inflammasome in neurons drives neuroinflammation in acute ischemic stroke. Early blockade of NLRP3 protects from I/R injury by mitigating inflammation and stabilizing the blood-brain barrier.
Authors: Yuli Han; Xuewang Li; Liu Yang; Duoduo Zhang; Lan Li; Xianan Dong; Yan Li; Sen Qun; Weizu Li Journal: J Ginseng Res Date: 2021-08-10 Impact factor: 5.735