Department of Neurology, University Hospital Würzburg, Josef-Schneider-Straße 11, 97080 Würzburg, Germany.
Department of Neurology, Klinikum Main-Spessart, Grafen-von-Rieneck-Str. 5, 97816 Lohr, Germany.
Brain Behav Immun. 2021 Feb;92:223-233. doi: 10.1016/j.bbi.2020.12.009. Epub 2020 Dec 9.
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.
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.
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.
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.
脑缺血诱导强烈的神经炎症反应，但潜在的分子机制尚不清楚。炎性小体（NLRP1、NLRP3、NLRC4、AIM2）是细胞内的多蛋白复合物，可诱导一系列促炎细胞因子和趋化因子，从而指导炎症反应。我们在此评估了 NLRP3 在短暂性脑缺血模型中的缺血再灌注（I/R）损伤中的功能作用。
采用 60 分钟短暂性大脑中动脉闭塞（tMCAO）和 3、7 或 23 小时再灌注的方法，在 C57Bl/6 小鼠中诱导缺血性中风，这是 I/R 损伤的模型。通过半定量实时 PCR 和 Western Blot 分析评估缺血半球中炎性小体的表达模式，并通过免疫细胞化学进行蛋白定位。最后，动物在 tMCAO 再通前或再通后直接用炎性小体抑制剂 Sulforaphane、Genipin、MCC950 或载体进行处理。在 tMCAO 后 1 天评估中风结果，包括梗塞面积和功能缺陷、局部炎症反应、神经元存活以及血脑屏障功能。
tMCAO 后，缺血半球中 NLRP3 的相对基因表达水平在 1 天内增加了 20-30 倍，这转化为神经元中 NLRP3 的表达增加。相应地，NLRP3 调节剂 Bruton 的酪氨酸激酶（BTK）和 NLRP3 诱导的细胞因子 IL-1β的基因表达水平显著升高。其他炎性小体的变化较小或不显著。在 tMCAO 前或后给予覆盖所有炎性小体或特异性 NLRP3 的炎性小体抑制剂可显著减少梗塞体积，并伴有 caspase 1 明显激活的证据。这种减轻中风的作用与缺血半球中免疫细胞浸润减少和血脑屏障完整性的保持一致。
我们的数据表明，神经元中 NLRP3 炎性小体的诱导驱动急性缺血性中风中的神经炎症。早期阻断 NLRP3 通过减轻炎症和稳定血脑屏障来保护免受 I/R 损伤。
