110 Macrophage Cross Talk following Subarachnoid Hemorrhage: Contribution of Peripheral Immune Activation in the Development of Cerebral Vasospasm

Abstract

INTRODUCTION: Microglia respond to the initial insult of aneurysm rupture, but become overwhelmed by ferritin toxicity. These microglia release inflammatory cytokines that cause vasospasm to facilitate peripheral macrophage diapedesis. This process is dependent on the p-STAT3 pathway. METHODS: The autologous SAH pre-clinical model was utilized and compared to blood and CSF from human patients. Mouse blood was injected along the skull base to c57/bl6 and CCR2/Cx3CR1 mice. Microglia depletion was done with pexidartinib. Ferritin within microglia, TLR4, and NFKb were measured via immunohistochemistry. Vasospasm was measured via India Ink gelatin assay. The Modified Garcia Scale and right turn test was utilized. Clarity assay was utilized to determine association between macrophages and brain microglia. Cerebral doppler was used to measure spreading cortical depressions. p-STAT3 was measured via western blot. For all experiments, saline sham, subarachnoid hemorrhage, and subarachnoid hemorrhage with p-STAT3 inhibitor were utilized. RESULTS: Microglia recruitment occured within parenchyma along the skull base adjacent to subarachnoid blood. Ferritin toxicity was noted by day 5 (r-0.95, p < 0.05) and persisted to day 9 (r-0.98, p < 0.01). A shift in microglia occured following SAH with Cox-2 and INOS predominance (k1 0.76, k2 1.21). Blockade of p-STAT3 resulted in decreased vasospasm (p<0.01), improved modified Garcia scores (p < 0.05), and improved right turn test (p < 0.05) following SAH. Peripheral macrophage diapedesis occured in regions of microglia activation. P-STAT3 blockade improved cerebral blood flow and prevented spreading cortical depressions (p < 0.01). NFKb and TLR4 activation was markedly reduced by p-Stat3 inhibition (p < 0.05). Results correlated closely to human aneurysmal blood and CSF data. CONCLUSIONS: Microglia ferritin toxicity contributes to vasospasm to facilitate peripheral macrophage diapedesis. Targeting the p-STAT3 pathway leads to reduced vasospasm and improved behavioral outcomes.