Ischemic Injury in a Dmcao Model of Stroke Demonstrates Long-Term Immune Cell Diapedesis Into the Brain Parenchyma.

Background: Stroke injury following a middle cerebral artery occlusion (MCAo) induces a rapid migration of leukocytes into the injured brain that lasts for weeks. The current study focuses on whether a focal cortical stroke using the distal MCAo (dMCAo) model induces similar long-term immune cell diapedesis into the brain parenchyma as seen following transient (t)MCAo stroke. Methods: Cells were isolated from spleens and brain hemispheres of adult 1-year old male C57BL/6J (B6; Jackson Labs) mice 30 days after a dMCAo (n=10). Sham animals (n=5) received a craniotomy without the distal MCA ligation. Peripheral migration was assessed in the spleen and brain using flow cytometry (FACSymphony) to identify viable (Ghost dye 780) CD3, CD4, CD8b, CD11b, Ly6C/Ly6G, CD19, CD45, and NK1.1 leukocytes. Populations were analyzed with FlowJo and assessed via repeated measures two-way ANOVA, Sidak post-hoc test (Graphpad Prism). Significance was p<0.05. Results: CD45 + leukocytes were elevated in the ipsilesional (ipsi) hemisphere compared to the contralesional hemisphere (p=0.01) after dMCAo, though a group hemispheric effect (F(1,13)=5.4; p=0.04) suggests long-term inflammation in sham-treated mice. Hemispheric effects also occurred for CD8 + T cells (p=0.046), B cells (p=0.03), monocytes (p=0.01), and macrophages (p=0.06), with elevations in both dMCAo and sham-treated mice in the ipsi vs. contralesional hemispheres. Only monocyte populations were significantly elevated (p=0.03) in dMCAo vs. sham mice. Conclusions: Our study shows that immune cells remain elevated in the injured hemisphere at 30 days after a focal stroke confined to the neocortex, but inflammation occurred in both sham and dMCAo-treated animals. Only monocytes were differentially affected by dMCAo, and infiltrating cell numbers are not as robust as after tMCAo. This demonstrates a long-term injury response from the craniotomy in the dMCAo model that should be considered for long-term studies using this model.