Role of Microthrombi and Inflammation in Delayed Deficits after SAH

项目总结/摘要 高达30%的蛛网膜下腔出血（aSAH）存活患者发生迟发性脑出血， SAH后4-10天的缺血和神经缺陷是发病率和死亡率的主要原因。 最近的临床研究表明，迟发性缺血和神经功能缺损的原因是多因素的， 包括微血栓和炎症。人类aSAH后血管内微血栓在术后1-2周达到峰值， SAH，反映了延迟的神经功能缺损发作，并且在患有SAH的aSAH患者的大脑中显著更高， 与无延迟性功能障碍的aSAH患者相比，蛛网膜下腔出血会引发炎症反应 这与aSAH患者的延迟神经功能衰退有关。炎性中性粒细胞 细胞外陷阱（NET，介导炎症和血栓形成的细胞外网状染色质支架） 在aSAH患者中已有报道，但尚未在SAH后迟发性功能障碍的背景下进行研究。 我们在人类和小鼠中的初步数据表明，微血栓和NET有助于发展。 SAH后的延迟性缺血和缺陷。因此，我们的中心假设是微血栓和NET是 延迟性脑梗死的病因（通过较低的脑灌注、小梗死和炎症） 蛛网膜下腔出血后的缺陷这将是第一个专门研究迟发性神经功能缺损的提案， SAH小鼠模型，以研究延迟性缺陷的潜在影响因素。 具体目标1：探讨微血栓在小鼠SAH后迟发性功能障碍中的作用。我们 假设是微血栓形成导致脑组织灌注持续减少， 迟发性梗死和迟发性功能缺陷。为了验证我们的假设，我们将使用原型抑制剂， 血小板活化以减弱SAH野生型和血小板荧光小鼠中的微血栓。各种 将使用成像模式来评估脑灌注、微血栓形成和梗塞。功能 在SAH后6天内，每天测试性能。该目的将确定血小板抑制/微血栓是否具有 对迟发性SAH损伤有实质性影响，包括迟发性梗死和迟发性功能缺陷。 具体目标2：研究中性粒细胞胞外陷阱对迟发性功能缺陷的作用 小鼠SAH后。我们的假设是NETs促进血管闭塞，导致延迟性梗死， 赤字为了测试这一点，我们将使用耗尽外周嗜中性粒细胞的SAH小鼠（用Ly 6 G抗体），或治疗SAH小鼠。 用Cl-脒（防止NET）或DNase-I（裂解NET）。将使用几种成像模式来评估 脑灌注、NET形成、梗塞和浸润免疫细胞。神经行为测试将持续6天。 这一目标将确定中性粒细胞胞外陷阱在SAH后迟发性缺血和功能障碍中的作用. 长期目标是确定微血栓和/或NET是否是发展的关键因素， 延迟性缺陷，并确定潜在的治疗靶点，以防止SAH后的延迟性缺血和缺陷。

Project Summary/Abstract Up to 30% of patients surviving aneurysmal subarachnoid hemorrhage (aSAH) develop delayed cerebral ischemia and neurological deficits 4-10 days post-SAH which are leading causes of morbidity and mortality. Recent clinical studies indicate that the cause of delayed ischemia and neurological deficits is multifactorial, and includes microthrombi and inflammation. Intravascular microthrombi after aSAH in humans peak 1-2 week post- SAH, mirroring delayed neurological deficit onset, and are significantly higher in the brains of aSAH patients who had delayed deficits vs aSAH patients who did not have delayed deficits. SAH triggers an inflammatory response which has been linked to delayed neurological decline in aSAH patients. Inflammation-induced neutrophil extracellular traps (NETs, extracellular web-like chromatin scaffolds which mediate inflammation and thrombosis) has been reported in aSAH patients, but have not been studied in the context of delayed deficits post-SAH. Our preliminary data in humans and mice suggest that microthrombi and NETs contribute to the development of delayed ischemia and deficits after SAH. So, our central hypothesis is that microthrombi and NETs are causative factors (via lower brain perfusion, small infarcts, and inflammation) for the development of delayed deficits after SAH. This will be the first proposal designed to specifically study delayed neurological deficits using a mouse model of SAH to investigate the potential contributing factors of delayed deficits. Specific Aim 1: Interrogate the role of microthrombi in delayed functional deficits after SAH in mice. Our hypothesis is that microthrombi formation causes a sustained reduction in brain tissue perfusion leading to delayed infarction and delayed functional deficits. To test our hypothesis, we will use prototypic inhibitors of platelet activation to attenuate microthrombi in wild-type and platelet-fluorescent mice subjected to SAH. Various imaging modalities will be used to assess brain perfusion, microthrombi formation, and infarction. Functional performance will be tested daily for 6 days post-SAH. This aim will identify if platelet-inhibition/microthrombi have a substantial influence on delayed SAH injury, including delayed infarction and delayed functional deficits. Specific Aim 2: Investigate the contribution of neutrophil extracellular traps to delayed functional deficits after SAH in mice. Our hypothesis is that NETs contribute to vessel occlusion leading to delayed infarction and deficits. To test this, we will use SAH mice depleted of peripheral neutrophils (with a Ly6G antibody), or treated with Cl-amidine (to prevent NETs) or DNase-I (to lyse NETs). Several imaging modalities will be used to assess brain perfusion, NET formation, infarction, and infiltrating immune cells. Neurobehavior will be tested for 6 days. This aim will determine the role of neutrophil extracellular traps in delayed ischemia and deficits post- SAH. The long-term goals are to determine if microthrombi and/or NETs are key contributors to the development of delayed deficits, and to identify potential therapeutic targets to prevent delayed ischemia and deficits after SAH.