Abstract Body: Introduction: Polymorphonuclear leukocytes (PMNs) are among the first leukocytes recruited to the infarct following reperfusion in ischemic stroke. Although transendothelial migration (TEM) blockade in preclinical studies was found to reduce infarct size, TEM blockade in clinical trials has been unsuccessful. Studying PMN recruitment and lifespan in ischemic stroke may offer insight into these failures in clinical trials and advance therapeutic development.
Methods: PMNs were briefly pulse-labeled with 5-Ethynyl-2’-deoxyuridine (EdU) to determine PMN lifespan and migration pattern following ischemia/reperfusion in stroke. Large vessel occlusion and reperfusion were simulated using the transient middle cerebral artery occlusion model (tMCAO, 90 minutes of ischemia followed by reperfusion). Infarct size was determined via triphenyl tetrazolium chloride staining. The percentage of EdU+ PMN in circulation was assessed each day following tMCAO and compared to the percentage of EdU+ PMNs in the ischemic hemisphere at sacrifice by flow cytometry. The position of PMN in coronal sections was examined by wide field fluorescence microscopy.
Results: Large cortical ischemic strokes involving over 20% of cerebral volume were induced in mice. A majority of PMN recruitment and extravasation was localized to the cortical surface of the infarct at early time points (12 and 24 h post stroke), contrasting other vascular beds where there is PMN migration deep into the infarcted tissue by 24 h. EdU+ PMNs persisted in the ischemic hemisphere until 96 h post stroke despite their clearance from circulation at least 48 h prior, indicating PMN can survive for days in the infarct. Additionally, the percentage of EdU+ PMN in the ischemic hemisphere was similar to the percentage of EdU+ PMN in circulation at 24 h post stroke, suggesting PMN recruitment centers around 24 h.
Conclusions: Our findings demonstrate PMN recruitment and infiltration post stroke evolves over several days. This data suggests that the majority of PMNs are recruited to the infarct within the first 48 hours and likely can persist within the infarct for days. Modulating the timing of PMN labeling via EdU can lead to a better understanding of PMN spatiotemporal infiltration and persistence and will help inform future therapeutic interventions targeting leukocytes.