The NLRP3 inflammasome drives inflammation in ischemia/reperfusion injury after transient middle cerebral artery occlusion in mice
Abstract
Purpose:
Cerebral ischemia triggers a strong neuroinflammatory response, yet the molecular mechanisms behind this process remain poorly understood. Inflammasomes—such as NLRP1, NLRP3, NLRC4, and AIM2—are intracellular protein complexes that initiate the production of pro-inflammatory cytokines and chemokines, thereby orchestrating inflammatory signaling. This study investigates the specific role of the NLRP3 inflammasome in ischemia/reperfusion (I/R) injury using a mouse model of transient cerebral ischemia.
Methods:
Transient middle cerebral artery occlusion (tMCAO) was performed on C57Bl/6 mice for 60 minutes, followed by reperfusion periods of 3, 7, or 23 hours to model I/R injury. Expression of inflammasomes in the ischemic brain hemispheres was analyzed using semiquantitative real-time PCR, Western blotting, and immunocytochemistry for protein localization. To evaluate therapeutic potential, mice received inflammasome inhibitors—Sulforaphane, Genipin, or MCC950—or vehicle control either before or at the time of reperfusion. Stroke outcomes were assessed 24 hours post-tMCAO, including infarct volume, neurological deficits, inflammation, neuronal survival, and blood-brain barrier (BBB) integrity.
Results:
NLRP3 gene expression in the ischemic hemisphere increased 20–30-fold within 24 hours after tMCAO, accompanied by elevated NLRP3 protein levels in neurons. Corresponding upregulation of Bruton’s Tyrosine Kinase (BTK), a modulator of NLRP3, and IL-1β, a downstream cytokine, was also observed. In contrast, other inflammasomes showed minimal or no significant changes. Treatment with broad-spectrum or NLRP3-specific inflammasome inhibitors significantly reduced infarct size and caspase-1 activation when administered before or after stroke. These effects were associated with reduced immune cell infiltration and preserved BBB integrity in the affected brain region.
Conclusions:
Our findings demonstrate that neuronal activation of the NLRP3 inflammasome plays a key role in Atuzabrutinib driving neuroinflammation during acute ischemic stroke. Early inhibition of NLRP3 mitigates I/R injury by dampening inflammation and preserving blood-brain barrier function.