CD200R对HIBD后脑内MC及PBMCs突触修剪的调控机制研究

Hypoxia and ischemia brain damage (HIBD) is the major cause of long-term behavior deficit in neonates. Microglial cell (MC) activation and its related inflammatory response are believed as a key mechanism of HIBD. CD11b is a marker of MC, interestingly, our previously study found that peripheral blood monocyte cells (PBMCs) infiltrated into brain and expressed with CD11b in a rodent model of HIBD. MC-mediated synapse pruning plays fundamental contributes in normal brain developing. However, whether and how MC responses to HIBD and modulates synapses in HIBD-affected subjects remains uncovered. CD200R plays key roles in modulate brain inflammation but its role in synapses pruning is unclear. In this proposal, we focus on CD200R’s modulatory mechanism in MC- and PBMCs-induced synaptic pruning after HIBD. In order to distinguish MC and PBMCs, we will apply Cx3cr1GFP/+Ccr2RFP/+ double transgenic mice to develop HIBD model. In an in vivo study, inflammatory protein expression, synapse number, and phagocytic ability of MC or PBMCs will be measured after HIBD. In the in vitro system, we will use CD11b magnetic beads to isolate CD11b+ cell from HIBD mice brain. Microfluidic will be adopted to establish MC and neuron co-culture or PBMCs and neuron co-culture systems. Within these in vitro co-cultures, MC or PBMCs synapse pruning function will be tested via axon clearance and axon growth. CD200R antibody will be used to test CD200R’s role in modulating MC- or PBMCs-mediated synaptic pruning after HIBD. The completion of this proposal will shield lights on synaptic pruning after HIBD and may help to develop potential therapeutic strategy for HIBD treatment.Inflammatory protein, synapse number and phagocytic ability of MC or PBMCs will be used to evaluate inflammation and synaptic pruning function change of MC or PBMCs after HIBD in vivo. Along with it, CD11b magnetic beads will be applied to isolate CD11b+ cell from HIBD mice brain. Microfluidic will be adopted to establish MC or PBMCs and neuron co-culture system and axon clearance and axon growth experiment will be used to test MC or PBMCs synapse pruning function in vitro. CD200R blockage antibody will be applied in order to test CD200R function in modulate MC/PBMCs synaptic pruning function in the brain after HIBD. The successful completion of this proposal will shield lights on synaptic pruning after HIBD and may help to develop potential therapeutic strategy for HIBD treatment.

缺氧缺血性脑损伤（HIBD）是新生儿远期行为异常的主要原因，小胶质细胞（MC）活化及炎症反应为其关键。CD11b系MC标记物，前期研究发现HIBD后MC活跃、外周血单核细胞（PBMCs）入侵脑内并表达CD11b但功能不清。MC对突触的修剪系脑发育所必需，但其在HIBD中的变化不明。CD200R能调控脑内炎症反应但对突触修剪的影响有待研究。项目拟揭示CD200R对HIBD后MC及PBMCs突触修剪的作用。采用Cx3cr1GFP/+Ccr2RFP/+小鼠构建模型，以区分MC及PBMCs，对二者炎症反应蛋白、吞噬功能、突触修剪细胞数进行分析，揭示它们在HIBD炎症反应及突触修剪中的贡献；采用CD11b磁珠提取结合流式分选出MC及PBMCs，分别与神经元共培养观察对轴突清除和生长的作用，并采用CD200R阻断抗体阐明CD200R对MC及PBMCs突触修剪的调控，为HIBD远期行为障碍的机制提供证据

新生儿缺氧缺血性脑损伤（HIBD）是新生儿致死致残率最高的疾病，其远期行为异常为家庭和社会带来极大的社会负担。突触是信息传递的中心，突触损伤是造成HIBD患儿远期行为异常的关键，但其损伤及可能的调控机制不清。脑损伤后，突触被吞噬是其丢失的主要原因，但HIBD后突触被吞噬的机制不清。HIBD后脑内将出现两种具有吞噬能力的细胞，小胶质细胞（MC）和由外周入侵脑内的单核细胞（PBMCs）。项目的研究揭示，HIBD后外周血白细胞活跃并与认知功能障碍成正相关，流式细胞和免疫荧光多重染色揭示脑内MC和PBMCs的吞噬分子表达存在差异；HIBD后脑内突触出现丢失，激光共聚焦结合三维重建技术揭示MC而非PBMCs是HIBD后脑内吞噬吞噬的细胞，HIBD后TREM2的表达显著上升，并与MC出现特异性的共定位现象，可见TREM2是介导MC进行吞噬的重要分子。CD200R是调控髓系细胞炎症的关键分子，项目发现HIBD后它的表达上调，并与MC和PBMCs出现共定位，这是HIBD后炎症活跃的结果。免疫荧光和流式细胞技术，以及体外MC原代培养OGD模型均证实，脑内MC是白细胞介素1β（IL-1β）的主要来源，PBMCs则是白细胞介素6（IL -6）的主要来源，据此，项目在体外进一步构建了原代MC的吞噬刺激模型，结果发现OGD后MC的吞噬能力增加显著，IL-1β和IL-6均能提高MC的吞噬能力，如果给予CD200R的激动剂，能够使得MC的吞噬能力出现下调。CD200R具有抑制炎症介质表达的作用，这说明CD200R可能通过下调IL-1β和IL-6使MC的吞噬能力降低。项目的结论，MC是HIBD后吞噬突触的细胞，它通过分泌IL-1β，PBMCs通过分泌IL-6经TREM2调控MC吞噬突触，这为HIBD后突触的挽救提供了新的依据。