Hemorrhagic transformation associated with delayed reperfusion in perinatal and childhood ischemic stroke: brain maturation-dependent role of leukocytes

摘要 脑动脉阻塞后脑微循环的再灌注可以发挥双重作用- 如果再灌注是早期的神经保护，但是如果再灌注是不良事件和增加的神经功能障碍， 被耽搁了机械血栓切除术彻底改变了成人卒中的护理， 多项随机临床试验，时间窗从4.5小时逐渐增加到24小时或更长时间。在 动脉缺血性卒中儿童，回顾性病例系列研究表明 血栓切除术，然而，选择偏倚和缺乏未经治疗的对照结局引起了人们的担忧， 儿童可以安全地受益于血管内血栓切除术。缺乏科学依据， 在小儿缺血性卒中后清除血栓是安全的。围产期动脉缺血性卒中（PAIS） 和儿童动脉缺血性卒中（CAIS）是具有不同发生率和复发率的疾病， 由于年龄相关的CNS成熟差异， 和免疫系统。在这个探索性的R21赠款中，我们将开始填补知识空白， 使用实验性PAIS和CAIS模型安全地清除血栓。 中心假设：单核细胞-小胶质细胞-中性粒细胞相互作用介导不同的时间窗， PAIS和CAIS中晚期再通的不良影响。 在长时间短暂性大脑中动脉闭塞（tMCAO）后的“晚期再灌注”模型中， 新生儿和青少年男性和女性，我们将确认再灌注，并检查出血的程度 与血脑屏障（BBB）完整性、水肿和短期组织学结果相关的转化。 每个年龄组中炎症反应的程度将通过以下方式确定： 血液和脑中的炎性细胞因子/趋化因子（多重）、出血性标志物（D-二聚体 测定）和浸润的中性粒细胞和单核细胞的表型（流式细胞术）（目的1）。然后我们将 检查CCR 2或Cx 3CR 1信号传导的遗传破坏是否影响出血性脑梗死的程度。 晚期再灌注后的转化、BBB渗漏和短期组织学和功能结局 在两种性别的P9和P21小鼠中，并确定脑成熟对浸润的表型的作用。 白细胞和活化的小胶质细胞/巨噬细胞（流式细胞术，免疫荧光）， 神经炎症（细胞因子复合物）（目的2）。拟议的研究将作为以下方面的原则证明： 定义出生后大脑成熟如何影响晚期脑损伤后出血性转化的易感性 再通，并帮助定义延迟再通的安全指南， 中风

Abstract Reperfusion of cerebral microcirculation following cerebral artery blockage can have dual roles — neuroprotection if reperfusion is early but adverse events and increased neurological disability if reperfusion is delayed. Mechanical thrombectomy has revolutionized care in adult stroke and is of proven benefit in multiple randomized clinical trials with time windows gradually increasing from 4.5 to 24 or more hours. In children with arterial ischemic stroke, retrospective case series suggest safety and possible benefit of thrombectomy, however selection bias and a lack of non-treated control outcomes raise concerns on whether children can safely benefit from endovascular thrombectomy. There is lack of science-based evidence on when it is safe to remove thrombus after pediatric ischemic stroke. Perinatal arterial ischemic stroke (PAIS) and childhood arterial ischemic stroke (CAIS) are diseases with distinct occurrence and recurrence rates and many non-overlapping injury mechanisms due to the age-related differences in the maturation of the CNS and the immune system. In this exploratory R21 grant we will begin filling the knowledge gap of when it is safe to remove thrombus using experimental PAIS and CAIS models. Central Hypothesis: Monocyte-microglia-neutrophil interactions mediate distinct time windows for adverse effects of late recanalization in PAIS and CAIS. In models of ‘late reperfusion’ following long transient middle cerebral artery occlusion (tMCAO) in neonatal and juvenile males and females we will confirm reperfusion and examine the extent of hemorrhagic transformation in relation to blood-brain barrier (BBB) integrity, edema and short-term histological outcomes. The magnitude of the inflammatory response in each age group will be determined by accumulation of inflammatory cytokines/chemokines (multiplex) in the blood and in the brain, hemorrhagic markers (d-dimer assay) and the phenotypes of infiltrated neutrophils and monocytes (flow cytometry) (Aim 1). We will then examine whether genetic disruption of CCR2 or Cx3CR1 signaling affects the magnitude of hemorrhagic transformation, BBB leakage, and short-term histological and functional outcomes following late reperfusion in P9 and P21 mice of both sexes, and determine the role of brain maturation on the phenotypes of infiltrated leukocytes and activated microglia/macrophages (flow cytometry, immunofluorescence) and neuroinflammation (cytokine multiplex) (Aim 2). The proposed studies will serve as proof-of-principle in defining how postnatal brain maturation affects the susceptibility to hemorrhagic transformation after late recanalization and help define safety guidance for delayed recanalization in infants of children who suffer stroke.