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

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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.