Neutrophil Distribution in the Post Stroke Brain Evolves Dramatically Over Time and can be Modulated by Disrupting PECAM Function

Abstract

Current therapies for ischemic stroke focus on reperfusion but do not address the acute inflammatory response. Previous clinical trials aimed at modulating the inflammatory milieu by disrupting leukocyte infiltration failed to show clinical efficacy. One possible explanation for this unexpected shortcoming is an incomplete understanding of the precise spatio-temporal underpinnings of leukocyte extravasation and infiltration. Here we describe the pattern of the acute inflammatory response in a mouse transient middle cerebral artery occlusion (tMCAO) stroke model at several time points after reperfusion. We used widefield and confocal immunofluorescence microscopy to examine the distribution of neutrophils with precise examination of the leukocyte position relative to the cerebrovasculature and adjacent perivascular space. The recruitment of neutrophils varied dramatically across the infarcted tissue and surrounding penumbra, especially at early time points. At 12 and 24 hours, neutrophil recruitment and extravasation are predominantly observed at the cortical surface. Over the next few days (48 and 72 hours), neutrophils are increasingly found deeper into the subcortex. Disrupting leukocyte transendothelial migration with PECAM function-blocking antibodies caused a marked redistribution of neutrophils away from the subcortex with a corresponding increase at the cortical surface at 72 hours. Our findings suggest that the vast majority of infiltrating neutrophils are initially recruited to cortical venules. Furthermore, neutrophils rapidly escape the perivascular compartment and enter the parenchyma. In addition, this initial recruitment to the cortical surface was highly regionalized. In spite of this, over the course of several days neutrophils did redistribute more evenly across the entire infarcted hemisphere. Taken together our findings demonstrate that the infiltration of neutrophils dynamically evolves over several days following reperfusion. The initial heterogeneous pattern of neutrophil recruitment does not correlate well with traditional markers of cellular dysfunction but does become more homogenous as the pathology evolves. A better understanding of the precise spatio-temporal infiltration of inflammatory cells could help inform the next generation of therapeutic interventions.