Microglia- Monocyte Interactions following Perinatal Brain Injury

 描述(申请人提供)：这份R21申请旨在确定感染敏感型围产期缺氧缺血损伤是否促进单核细胞入侵中枢神经系统，从而导致急性脑损伤并转变为小胶质细胞样亚炎症状态。这是一个意义重大的 由于对新生儿脑内单核细胞-小胶质细胞的相互作用/转化知之甚少，促炎性单核细胞衍生物可能会增加脑损伤后点燃和惊厥的易感性。此外，了解渗透的单核细胞是如何导致损伤的，可能会导致新的治疗策略。在这个项目中，我们将检验围产期感染/缺氧缺血损伤促进单核细胞入侵中枢神经系统并发挥炎症和破坏性作用的假设。后来，这些单核细胞中的一些采用了小胶质细胞的身份，但被锁定在促炎状态，以增加大脑对兴奋性毒素和点燃惊厥的敏感性。我们将在两个具体目标上检验这一假设。目的1：单核细胞侵袭在新生儿急性感染/缺氧缺血性脑损伤中的意义。我们将使用双基因CX3CR1-GFP：CCR2-RFP小鼠分别标记小胶质细胞和单核细胞，并将这些小鼠置于内毒素(LPS)敏化的缺氧缺血(HI)损伤中，以检测单核细胞在新生鼠脑中的内流及其促炎细胞因子的表达。接下来，我们将比较CCR2RFP/和纯合子CCR2RFP/RFP小鼠(相当于CCR2缺失，缺乏单核细胞趋化蛋白-1受体)对内毒素/HI损伤的反应，以及抗CCR2拮抗剂的好处，以评估单核细胞的致病作用。目的2：围产期感染/缺氧脑损伤后单核细胞内流的发育后果。我们将通过静脉将肌动蛋白-GFP/CCR2-RFP单核细胞转移到脂多糖/缺氧损伤的小鼠体内，并观察其发育结果。根据GFP显示的形态和抗细胞因子抗体的双重标记，我们将测试单核细胞是否转化为小胶质细胞并保持在成人脑内的促炎状态。接下来，我们将使用CCR2突变小鼠和选择性CCR2拮抗剂(RS102895)，测试单核细胞来源的小胶质细胞是否敏化大脑对红藻氨酸兴奋毒性和PTZ诱导的惊厥点燃的反应。

 DESCRIPTION (provided by applicant): This R21 application seeks to determine if infection-sensiztied perinatal hypoxic-ischemic injury promotes monocyte invasion of the CNS to induce acute brain injury and conversion to a microglia-like sub-inflammatory state. This is a significant subject because little is known about monocyte-micorglia interactions/transformation in the neomnatal brain and pro-inflammatory monocyte-derivatives may increase the brain susceptibility to kindling and convulsion after injury. Further, understanding how infiltrating monocytes contribute to injury may lead to novel therapeutic strategies. In this project, we will test the hypothesis that perinatal infection/hypoxic-ischemic injury promotes monocytes to invade the CNS and exert inflammatory and destructive actions. Later, some of these monocytes adopt the microglial identity but are locked-in in a pro-inflammatory state to raise the brain susceptibility to excitotoxins and the kindling to convulsions. We will test this hypothesis n two specific aims. Aim 1: Significance of invading monocytes in acute infection/HI brain injury of newborns. We will use bitransgenic CX3CR1-GFP: CCR2-RFP mice to label microglia and monocytes respectively, and subject these mice to endotoxin (LPS)-sensitized hypoxia-ischemia (HI) injury to test the influx of monocytes in neonatal brains and their expression of pro-inflammatory cytokines. Next, we will compare the responses by hemizygous CCR2RFP/+ and homozygous CCR2RFP/RFP mice (equivalent to CCR2-null and lacking the receptor for Monocyte Chemoattractant Protein-1) to LPS/HI insult, as well as the benefits of anti-CCR2 antagonists to assess the pathogenic role of monocytes. Aim 2: Developmental consequences of monocyte influx after perinatal infection/HI brain injury. We will intravenously transfer actin-GFP/CCR2-RFP monocytes into LPS/HI-injured murine pups and follow their developmental outcomes. Based on the GFP-revealed morphology and double labeling with anti-cytokine antibody, we wiil test whether monocytes convert to microglia and maintain a pro-inflammatory state in adult brains. Next, we will use CCR2-mutant mice and a selective CCR2 antagonist (RS102895), to test whether or not monocyte-derived microglia sensitize the brain response to the kainic acid excitotoxicity and PTZ-induced kindling to convulsions.