Inhibition of PECAM‐1 Significantly Delays Leukocyte Extravasation into the Subcortex Post‐Stroke

Abstract

Background and PurposeCurrent therapies for ischemic stroke focus on reperfusion but do not address the acute inflammatory response that results in significant reperfusion injury. To advance future therapies, a thorough understanding of the precise spatiotemporal underpinnings of leukocyte extravasation and infiltration is necessary. We describe the evolution of the inflammatory response in a mouse transient middle cerebral artery occlusion (tMCAO) stroke model at several time points after reperfusion and the modulation of this response with PECAM blockade.MethodsThe transient Middle Cerebral Artery Occlusion model (90 minutes of ischemia followed by reperfusion) was used to simulate large vessel occlusion stroke and recanalization. We used wide field and confocal immunofluorescence microscopy to examine the exact distribution of neutrophils with close examination of the leukocyte position with regard to the brain vasculature and the perivascular space. Flow cytometry of single cell suspensions was used to confirm cell identity at different time points post‐stroke.ResultsLarge ischemic strokes involving both the subcortex and cortex (over 20% of the ischemic hemisphere) were induced in mice. At 12 and 24 hours, leukocyte recruitment and extravasation was primarily localized to the cortical surface. This contrasts with other organs where there is considerable migration of neutrophils deep into the inflamed tissue by 24 hours. Flow cytometry showed at 24 hours a majority of leukocytes were neutrophils. Over 48 to 72 hours, leukocytes were increasingly found deeper into the subcortex. Throughout the infarct (determined with triphenyl tetrazolium chloride staining), leukocyte recruitment was not uniform but rather organized in clusters. Disrupting leukocyte diapedesis with PECAM function‐blocking monoclonal antibody restricted leukocytes to within 500 microns of the surface when compared to control; and this was still evident at 72 hours (n=3 mice per group, p<0.01, Control 46% ± 4.0 %; PECAM‐1 Ab 62% ± 5.0%). High‐resolution wide‐field microscopy confirmed inhibition of TEM by PECAM‐1 blockade at 24 hours. Flow cytometry showed approximately equal numbers of monocytes and neutrophils at 72 hours.ConclusionsOur findings demonstrate that leukocyte infiltration into a stroke evolves over several days following reperfusion. The use of PECAM blockade modulates the natural progression of leukocytes into the infarcted stroke bed. A better understanding of leukocyte spatiotemporal infiltration and its regulators could help inform the next generation of therapeutic interventions.

A year in review: brain barriers and brain fluids research in 2022

“…Multiple approaches have been used to try and reduce such extravasation. Recently, Arias et al [ 152 ] found that a PECAM-1 neutralizing antibody reduces leukocyte influx and affects their spatial distribution within brain. BBB disruption occurs after stroke and it contributes to neuroinflammation and edema formation.…”

Emerging diagnostic markers and therapeutic targets in post-stroke hemorrhagic transformation and brain edema

“…It facilitates the final steps of leukocyte migration through the 10.3389/fnmol. 2023.1286351 endothelial cell junctions post-stroke (Arias et al, 2022). PECAM-1 plays a role in transmitting survival signals in endothelial cells, preventing apoptosis (Gao et al, 2003).…”

“…Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM-1/CD31) is an immunoglobulin-like cell adhesion molecule expressed on the surface of endothelial cells, platelets, neutrophils, and some subsets of lymphocytes ( Arias et al, 2022 ; Fu et al, 2023 ). It facilitates the final steps of leukocyte migration through the endothelial cell junctions post-stroke ( Arias et al, 2022 ).…”

“…Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM-1/CD31) is an immunoglobulin-like cell adhesion molecule expressed on the surface of endothelial cells, platelets, neutrophils, and some subsets of lymphocytes ( Arias et al, 2022 ; Fu et al, 2023 ). It facilitates the final steps of leukocyte migration through the endothelial cell junctions post-stroke ( Arias et al, 2022 ). PECAM-1 plays a role in transmitting survival signals in endothelial cells, preventing apoptosis ( Gao et al, 2003 ).…”

Serum EpCAM or PECAM levels and risk of ischemic stroke: a two-sample Mendelian randomization study

“…7 Blocking PECAM after the onset of a stroke has been shown in various studies to reduce severity in murine models, pointing to PECAM's potential as a therapeutic target to mitigate neuroin ammation and aid in cerebral recovery post-IS. 8,9 While elevated levels of EpCAM and PECAM post-IS are documented, the direct causal relationship between these increased serum levels and the stroke event remains to be fully unraveled. Mendelian randomization (MR), employing genetic variations as instrumental variables, is a robust approach for ascertaining the causative impact of risk factors on disease.…”