Neonatal Stroke: The Role of Microglia

描述（申请人提供）：围产期动脉性中风是脑瘫的主要病因之一。产后早期大脑发育不成熟增加了其对缺血和炎症损伤的易感性。长期以来，小胶质细胞被认为是新生儿脑缺血后的纯损伤细胞。然而，我们和其他人的研究表明，新生儿损伤后的小胶质细胞表型是多样的，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CD36在脑血管损伤中起双重作用。CD36可以通过触发氧化应激和炎症造成损伤，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CD36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大脑，但新生儿大脑中缺乏CD36会加重急性大脑中动脉闭塞（MCAO）后的损伤，并对细胞内促炎症信号的影响与成人不同。cd36介导的效应是配体特异性和环境依赖性的，部分依赖于与特定toll样受体（TLR）的合作。我们假设CD36通过以tlr2依赖的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中风后的损伤。我们将首先确定新生儿MCAO后CD36信号通路中断（基因缺失、药理抑制）对小胶质细胞表型（细胞毒性和保护性）的影响，然后在活体损伤新生小鼠中使用双光子成像检测CD36抑制对小胶质细胞趋化活性的影响（目的1）。为了确定CD36是否通过TLR2作用于损伤，我们将在MCAO诱导的新生luc/GFP-TLR2和CD36ko-luc/GFP-TLR2小鼠中通过生物发光成像监测TLR2，或者通过TLR2依赖性和非依赖性方式刺激新生小鼠的小胶质细胞（目的2）。最后，我们将确定缺乏/抑制CD36对培养小胶质细胞中Lyn介导的细胞内信号传导的影响，以及CD36/Lyn信号传导改变对损伤培养神经元的影响（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).