Abstract TMP26: Age Exacerbates the Pathogenicity of Neutrophils in Ischemic Stroke

Abstract

Introduction: Ischemic stroke is a major cause of disability and mortality worldwide. Neutrophils may exacerbate stroke-related injury via the release of reactive oxygen species (ROS) and granule proteins. As age is the strongest risk factor for poor outcomes after ischemic stroke, we hypothesized that age exacerbates the deleterious functions of neutrophils after brain ischemia. Methods and Results: Young (3 month) and aged (20-22 month) mice were subjected to middle cerebral artery (MCAO) stroke. Aged mice had higher mortality than young mice at 7 days (0% young, 60% aged, p<0.05, n=5/group). Aged mice also had greater neurodeficits than young mice (p<0.05). Flow cytometry showed that aged animals display an increase in neutrophil proportions in the blood, spleen, lung and bone marrow (p<0.001) after ischemic stroke compared to young. Furthermore, flow cytometry demonstrated that neutrophils from the blood of aged animals produced ROS at higher levels than neutrophils from young animals (p<0.05). Aged stroke animals had higher serum IL-6 (p<0.0001) and CXCL1 (p<0.01), two powerful neutrophil activating cytokines, at 24 hours after stroke. Follow-up serum measurements in human ischemic stroke patients (n=143) demonstrated that increased age was associated with higher levels of IL-6 (p=0.014) and IL-8 (p<0.0001) at 24 hours. We then used anti-Ly6G, a neutrophil specific monoclonal antibody, to deplete circulating neutrophils as a potential treatment for ischemic stroke. Mice were randomized to receive 500ug anti-Ly6G IP or control at 4, 24 and 48 hours after stroke (n=6-7/group). Treatment conferred no benefit to young mice, but resulted in a significant improvement in behavioral recovery in aged mice as measured by adhesive test at 7 days (p<0.01), corner test at 14 days (p<0.001) and hangwire test at 14 days (p<0.05). Conclusions: Neutrophils from aged animals exhibit altered organ trafficking and a higher reactive oxygen species generation. Elevated levels of neutrophil-activating cytokines were seen in both aged animals and aged ischemic stroke patients at 24 hours. Finally, anti-neutrophil depletion therapy had no effect in young animals, but resulted in significantly enhanced behavioral recovery in aged animals.