Transient ischemic stroke induces substantial leukocyte recruitment to both the ischemic and non‐ischemic cerebrovasculature as visualized through live animal imaging.

Abstract

Ischemic stroke is a leading cause of a death and disability worldwide. Current treatment strategies rely on early restoration of blood flow; however, injury persists despite successful reperfusion. The innate immune response plays a pivotal role in reperfusion injury, but the mechanisms and time‐course of neutrophil and monocyte recruitment to the cerebrovasculature (CBV) during ischemic stroke remain unclear. We sought to characterize the real‐time leukocyte response at the CBV in a mouse stroke model with reperfusion.To visualize leukocyte recruitment induced by ischemia, Ly6G‐tdTomato mice (which have fluorescent red neutrophils) were subjected to 90 minutes of transient middle cerebral artery occlusion (tMCAO) followed by reperfusion. Real‐time spinning‐disc confocal intravital microscopy of the CBV was performed at 24 and 72 hours following reperfusion. Intracranial windows were placed in both ischemic and non‐ischemic hemispheres; and tMCAO treated mice were compared to mice that underwent sham surgery. Neutrophil recruitment (adhesion, rolling) and extravasation were quantified in vivo on both the ischemic and contralateral non‐ischemic hemispheres. Postmortem staining with 2,3,5‐triphenyltetrazolium chloride was used to confirm induction of the stroke and to quantify infarct volume. In addition, wide‐field microscopy was used to examine neutrophil extravasation across the parenchyma.Animals subjected to sham surgery showed negligible leukocyte recruitment to the CBV. However, marked increases in leukocyte rolling, adhesion, and extravasation were observed at 24 and 72 hours following the tMCAO procedure. Interestingly, non‐ischemic hemispheres also demonstrated increased leukocyte rolling and adhesion, but not extravasation tMCAO group. Also, substantially more neutrophil extravasation was observed in the core of the infarct at 72 hours. These findings are consistent with an early recruitment of neutrophils to the cortical surface at early time points and a delayed infiltration into the core, as reported by others.These results show that a significant leukocyte response persists after reperfusion following tMCAO. This response is present both on the ischemic and, to a lesser extent, the non‐ischemic cortical CBV. Acute leukocyte recruitment observed on the non‐ischemic hemisphere suggests that ischemic stroke triggers a global inflammatory response within the CBV. Further reducing inflammation through immunomodulatory therapy in conjunction with reperfusion therapy may be beneficial in not only reducing stroke volume but potentially mitigating inflammation mediated secondary neuronal damage.Support or Funding InformationNIH R01 HL046849 and R37 HL064774 to W.A.M.