Abstract
Background and Purpose- A major process contributing to cell death in the ischemic brain is inflammation. Inflammasomes are multimolecular protein complexes that drive inflammation through activation of proinflammatory cytokines, such as IL (interleukin)-1β. Preclinical evidence suggests that IL-1β contributes to a worsening of ischemic brain injury. Methods- Using a mouse middle cerebral artery thrombosis model, we examined the inflammatory response after stroke and the contribution of the NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome to ischemic injury. Results- There was a marked inflammatory response after stroke characterized by increased expression of proinflammatory cytokines and NLRP3 and by recruitment of leukocytes to the injured tissue. Targeting NLRP3 with the inhibitor MCC950, or using mice in which NLRP3 was knocked out, had no effect on the extent of injury caused by stroke. Conclusions- These data suggest that the NLRP3 pathway does not contribute to the inflammation exacerbating ischemic brain damage, contradicting several recent reports to the contrary.
Highlights
Background and PurposeA major process contributing to cell death in the ischemic brain is inflammation
This study suggests that there is a marked inflammatory response after ischemic stroke, NLRP3dependent inflammation is not involved in exacerbation of the injury and does not represent a therapeutic target for ischemic brain injury.
We are grateful to Genentech for the NLRP3 (NACHT, LRR and PYD domains-containing protein 3)−/− mice
Summary
The data that support the findings of this study are available from the corresponding author on reasonable request. The experiments were performed on littermate controls except where stated and Figure I in the online-only Data Supplement which used mice maintained as homozygotes. Cells were incubated with Fc block (anti-CD16/CD32 and rat serum) and surface-stained with fluorescence-conjugated anti-CD11b (M1/70), anti-Ly6G (1A8), anti-CD45.2 (104), and anti-CD64 (X545/7.1). Brain sections from littermate WT-, NLRP3−/−-, and MCC950-treated WT mice perfused fixed as above were dried for 24 hours before undergoing heat-mediated antigen retrieval in Tris-EDTA pH 8.6 solution for 20 min in a water bath set to 97.5°C. Quantitative analysis was performed on 3 low magnification images of the lesion taken from 3 different coronal brain slices. Specific antibodies were used targeting mouse IL-1β (AF401; R&D), ASC (D2W8U, Cell Signalling Technology), caspase-1 p10 (EPR16883; Abcam), and β-actin (Sigma). Flow cytometry and lesion volumes were analysed using 2-tailed t test
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