Abstract WP267: 9-Tert-Butyl Doxycycline Inhibits Neuronal Injury and Neutrophil Trafficking After Global Cerebral Ischemia

Abstract

Introduction: Cardiac arrest induces a robust systemic inflammatory response that exacerbates CNS injury and is characterized in part by neutrophil (PMN) migration to the brain. Tetracyclines have been shown to reduce inflammation after stroke, and here we test the effects of the tetracycline derivative 9-tert-butyl doxycycline (9TB) on PMN activation, trafficking, and ischemic neuroprotection in a mouse model of global ischemia-reperfusion. Hypothesis: Inhibition of PMN activation by 9TB will decrease PMN extravasation in the CNS and mitigate ischemia-reperfusion injury. Methods: Transient global cerebral ischemia was induced in LysM-EGFP mice by 3-vessel occlusion (3VO). Mice were given 50 μg/kg lipopolysaccharide (LPS) to simulate endotoxemia observed after cardiac arrest and daily injections of 25 mg/kg 9TB or vehicle control. Blood was collected 4 hours after 3VO/LPS for fluorescence-activated cell sorting (FACS) analysis of PMN activation. Brain and spleen immunohistochemistry were performed after 3 days to assess injury and PMN burden. Results: Expression of the PMN activation marker CD11b was increased 4 hours after 3VO/LPS compared to sham (mean fluorescence intensity = 55,516 vs. 6,537) and lower in 3VO/LPS animals treated with 9TB (MFI = 36,445). While sham-treated groups showed little PMN infiltration into the brain (7.5 PMN/mm2 ± 2.2 vs. 1.9 PMN/mm2 ± 1.3; NS), 9TB treatment of shams increased splenic PMN sequestration (81.4 ± 8.3 vs. 38.0 ± 7.5; p<0.001). This effect was enhanced with 3VO/LPS (112.2 ± 8.9 vs. 37.3 ± 0.1; p<0.0001). 3VO/LPS animals receiving 9TB exhibited lower cortical PMN counts (19.3 PMN/mm2 ± 13.6) compared to those receiving vehicle (46.5 PMN/mm2 ± 5.5; p<0.05). Daily 9TB after 3VO/LPS also decreased cortical injury compared to vehicle-treated controls (9.7% ± 6.8 vs. 32.2% ± 6.4; p<0.05) with no difference between sham groups (5.5% ± 0.9 vs. 6.8% ± 5.8; NS). PMN burden was positively correlated with neuronal injury (Pearson’s r=0.8621, p<0.01). Conclusions: 9TB treatment rapidly inhibits PMN activation and reduces PMN migration and CNS injury when delivered acutely following cerebral ischemia-reperfusion. Future studies are required to link ischemic neuroprotection with the observed effects on PMN activity.