Neonatal Stroke: The Role of Microglia

描述（由申请人提供）：围产期动脉卒中是脑瘫的主要原因之一。出生后早期大脑的不成熟增加了其对缺血性和炎症性损伤的易感性。长期以来，小胶质细胞被认为是新生儿脑缺血后的纯粹损伤。然而，我们和其他人的工作表明，新生儿损伤后的小胶质细胞表型是多种多样的，细胞毒性和保护性小胶质细胞之间的平衡可能会调节损伤。清道夫受体CD 36在脑血管损伤中起双重作用。CD 36可以通过触发氧化应激和炎症来损伤，但可以通过吞噬作用清理神经元碎片或通过改变损伤后的巨噬细胞表型来保护。虽然CD 36的基因缺失通过减少大脑中动脉闭塞（MCAO）后的局部炎症来保护成人脑，但新生儿脑中CD 36的缺乏加重了急性MCAO后的损伤，并且与成人相比差异地影响细胞内促炎信号传导。CD 36介导的作用是配体特异性的和环境依赖性的，并且部分取决于与特定Toll样受体（TLR）的结合。我们推测，CD 36通过TLR 2依赖性方式改变细胞毒性和保护性小胶质细胞之间的平衡来改善新生儿卒中后的损伤。我们将首先确定新生儿MCAO后CD 36信号传导（基因缺失、药理学抑制）中断对小胶质细胞表型的影响，包括细胞毒性和保护性，然后在活体损伤新生小鼠中使用双光子成像检查CD 36抑制对小胶质细胞趋化活性的影响（目的1）。为了确定CD 36是否通过TLR 2作用来修饰损伤，我们将通过生物发光成像来监测遭受MCAO的活新生luc/GFP-TLR 2和CD 36 ko-luc/GFP-TLR 2小鼠的TLR 2，或者通过以TLR 2依赖性和非依赖性方式刺激活新生小鼠的小胶质细胞（目标2）。最后，我们将确定缺乏/抑制CD 36对培养的小胶质细胞中的Lyn介导的细胞内信号传导的影响，以及改变的CD 36/林恩信号传导对损伤的培养神经元的影响（目的3）。

DESCRIPTION (provided by applicant): Perinatal arterial stroke is one of the main causes of cerebral palsy. The immaturity of the brain in the early postnatal period increases its susceptibility to ischemic and inflammatory damage. For a long time, microglial cells were considered to be purely injurious after neonatal brain ischemia. However, work by us and others has shown that the microglial phenotypes are diverse after neonatal injury and that the balance between the cytotoxic and protective microglia may modulate injury. The scavenger receptor CD36 plays a dual role in cerebrovascular injury. CD36 can injure by triggering oxidative stress and inflammation but can protect by cleaning up neuronal debris through phagocytosis or by altering the macrophage phenotype after injury. While genetic deletion of CD36 protects the adult brain by reducing local inflammation after middle cerebral artery occlusion (MCAO), the lack of CD36 in the neonatal brain aggravates injury after acute MCAO and affects intracellular pro- inflammatory signaling differentially than in the adult. CD36-mediated effects are ligand- specific and context-dependent, and in part depend on partnering with particular Toll-like receptors (TLR). We hypothesize that CD36 ameliorates injury after neonatal stroke by altering the balance between the cytotoxic and protective microglia in a TLR2-dependent manner. We will first determine the effect of disrupted CD36 signaling (genetic deletion, pharmacological inhibition) after neonatal MCAO on the microglial phenotypes, both cytotoxic and protective and then examine the effects of CD36 inhibition on the chemotactic activity of microglia using two-photon imaging in living injured neonatal mice (Aim 1). To determine if CD36 acts through TLR2 to modify injury, we will monitor TLR2 by bioluminescence imaging in living neonatal luc/GFP-TLR2 and CD36ko-luc/GFP-TLR2 mice subjected to MCAO or, alternatively, by stimulating microglia in living neonatal mice in both a TLR2-dependent and -independent manner (Aim 2). Finally, we will determine the effects of a lack/inhibition of CD36 on Lyn-mediated intracellular signaling in cultured microglia and the effects of altered CD36/Lyn signaling on injured cultured neurons (Aim 3).