Abstract 18: A Novel Neutrophil Subpopulation Mediates Neuroprotection In The Late Phases Of Cerebral Ischemia

Abstract

Cerebral ischemia triggers a powerful inflammatory reaction involving infiltration of peripheral leukocytes. Neutrophils, the first immune cells entering the ischemic brain, contribute to brain injury by limiting tissue perfusion and destabilizing the blood-brain barrier. However, recent evidence indicates that neutrophils can also participate to the repairing process. Nevertheless, the dynamics and complexity of neutrophil subsets are poorly understood. Here, we performed single-cell transcriptomics to obtain a picture of infiltrating neutrophil diversity in the mouse brain 2 (D02) and 14 days (D14) after transient middle cerebral artery occlusion (MCAo) or sham surgery (Sham). Clustering analysis of the transcripts revealed 5 neutrophil populations. In Sham, most neutrophils fell into clusters 1 and 4, characterized by expression of genes associated with neutrophil development such Retnlg and Mmp8 (cluster 1) and Camp, Ltf, Chil3 and Lcn2 (cluster 4). After MCAo, clusters 1 and 4 gradually decreased, whereas cluster 2, identified by type I response genes ( Isg15, Ifit3, Rsad2, Ifit1, Ifi204 ) and cluster 3, characterized by upregulation of chemokines/cytokines ( Ccl3 , Ccl4 , Csf1 ), increased progressively from D02 to D14. Interestingly, we found that the number of neutrophils in the ischemic brain was 5 times higher at D14 than at D02 (Sham, 0.2 ± 0.1 x10 3 ; D02: 3 ± 1.3 x10 3 ; D14: 14.5 ± 3.5x10 3 cells; p=0.02 D02 vs D14; n=10-14/group). Therefore, we next investigated if neutrophil infiltration at D14 has a beneficial or adverse effect on stroke recovery. To this end, 7 days after MCAo, neutrophils depletion was induced by systemic injection of both rat anti-Ly6G (50μg/mouse, i.p) and CXCR2-antagonist SB225002 (2 mg/kg, i.p), once per day, for 7 consecutive days. We found that neutrophil depletion increased brain tissue loss (-18.7 ± 2.9 vs -8 ± 2.6% IgG/vehicle mice; p=0.01; n=9/group). In addition, neutrophil depletion decreased ambulatory distance (p<0.05 vs vehicle; n=11/group) and stereotypic behavior (p<0.05 vs vehicle; n=11/group) without altering open field performance in sham mice. Our study unveils an unrecognized neutrophil subpopulation protecting the brain in the subacute stroke phase, opening new avenues for stroke treatment.