Abstract TP309: Cortical Extravasation of Neutrophils and Unexpected Persistence Following Ischemia/Reperfusion in a Murine Ischemic Stroke Model

Abstract

Background and Purpose: Neutrophils are the first recruited leukocytes following ischemia/reperfusion injury in the brain; however, when and where neutrophils enter the brain parenchyma to exert their effects is controversial. Understanding the spatiotemporal underpinnings of leukocyte extravasation is required to advance therapeutic interventions. Here we describe the complex evolution of neutrophil recruitment and extravasation following ischemic stroke. Methods: Circulating neutrophils were pulse-labeled with EdU to track specific cells recruited post stroke. The transient middle cerebral artery occlusion (tMCAO) model was used to simulate large vessel occlusion and reperfusion. Coronal sections were examined at various timepoints and the position of EdU-labeled neutrophils was precisely determined by fluorescence microscopy. PECAM antibody treatment was used to disrupt neutrophil extravasation at the cortical surface and alter migration kinetics. Results: At early timepoints, 12 h and 24 h, neutrophil recruitment was unexpectedly superficial. Over 120 h, neutrophils were found increasingly deeper into the subcortex. Timing of tMCAO surgery with EdU labeling allowed for specific labeling of the initial wave of neutrophils. Surprisingly, many EdU positive neutrophils were still observed at 72 h, indicating they had survived for several days. The number and distribution of EdU positive neutrophils confirms the majority of neutrophil recruitment occurs before 24 h across the infarct. Disrupting leukocyte extravasation with PECAM function-blocking antibodies restricted neutrophils to the cortical surface. Conclusions: Our findings demonstrate that neutrophil infiltration post stroke evolves over several days. This data suggests neutrophils are recruited to the infarct within the first 24 h, enter the brain at the cortical surface, distribute throughout the infarct, and persist over 72 h. Furthermore, disrupting extravasation with PECAM antibodies modulates neutrophil progression into the infarct. A better understanding of leukocyte spatiotemporal infiltration and its regulators will help inform future therapeutic interventions targeting leukocytes.