Tongxinluo capsule inhibits no-reflow post-recanalization in experimental stroke by reducing circulating neutrophils.

ETHNOPHARMACOLOGICAL RELEVANCE: No-reflow post-recanalization is a challenge in ischemic stroke. Our prior study demonstrated that Chinese medicine Tongxinluo capsule (TXL) improves no-reflow in stroke by suppressing leukocyte-endothelium interactions. However, it remains unclear which leukocyte subpopulation plays a major role.

AIM OF THE STUDY: This study aimed to further explore the mechanisms of TXL improving no-reflow in stroke.

MATERIALS AND

METHODS

The mouse model of stroke with recanalization treatment was induced by transient middle cerebral artery occlusion (tMCAO). Neutrophil depletion was performed by injecting Ly6G-specific monoclonal antibody (anti-Ly6G). The mice were orally administered with TXL suspension. We examined the following parameters: cerebral blood flow (CBF) determined by laser speckle perfusion imaging, neutrophil percentages and apoptosis examined by flow cytometry, and granulocyte colony-stimulating factor (G-CSF) measured by cytometric bead array.

RESULTS

We confirmed our prior conclusion that TXL could significantly reduce blood neutrophil percentages (by up to 35 %) as well as improving no-reflow in stroke (increasing CBF in the ischemic area by 9 %). Interestingly, neutrophil depletion also suppressed no-reflow (increasing CBF by 15 %). These results suggest a causal role for neutrophil reduction in TXL's improvement of no-reflow. Subsequently, we found that tMCAO led to a 15 % increase in the percentage of bone marrow neutrophils but caused a 30 % decrease in the percentage of spleen neutrophils. However, TXL did not significantly affect the percentage of bone marrow neutrophils but interestingly increased the percentage of spleen neutrophils by up to 27 %. Importantly, tMCAO induced the increased granulocyte colony-stimulating factor (G-CSF) in the plasma and ischemic brain tissue, while both TXL and anti-Ly6G decreased the cytokine level. Moreover, TXL increased neutrophil apoptosis in the spleen without affecting their apoptosis in the blood and bone marrow.

CONCLUSIONS

Our study has elucidated an important mechanism of post-stroke no-reflow: the overproduction of G-CSF by ischemic brain triggers a massive release of neutrophils from the bone marrow and spleen into the circulation, consequently obstructing the cerebral microvasculature. Interestingly, TXL counteracts post-stroke no-reflow by targeting the pathogenic surge of circulating neutrophils via a dual mechanism: inhibiting cerebral G-CSF production to reduce splenic neutrophil release; and facilitating splenic neutrophil homing to promote their clearance.