Microglial Function and Neurologic Outcome in Rat Pups after Experimental Traumatic Brain Injury: Effects of Timing and Duration of Docosahexaenoic Acid Therapy

项目摘要创伤性脑损伤(TBI)是导致后天神经功能障碍的主要原因。 然而，目前还没有有效的治疗方法。炎症继发性损伤增加神经功能障碍 不受撞击后的损坏。小胶质细胞(大脑的常驻免疫细胞)介导炎症释放 颅脑损伤后早期活性氧(ROS)和细胞毒性因子的变化。成为修复性的小胶质细胞被移除 吞噬碎屑，消炎解毒。未成熟的大脑极易受到二次损伤。 由于其抗氧化剂储备低，炎症反应强烈。将小胶质细胞从 炎症性修复活动可能会减少脑外伤后的神经功能障碍，特别是在儿童中。 二十二碳六烯酸，或DHA，是治疗儿童脑外伤的候选药物。DHA是一种必需的脂肪酸 正常的大脑生长和功能，具有抗氧化和抗炎活性。在大鼠身上，DHA恢复 颅脑损伤后脑DHA丢失。在培养的小胶质细胞中，DHA促进修复活性。使用我们的儿科TBI 17天龄雄性大鼠受控皮质冲击(CCI)模型，CCI前DHA饮食降低学习能力 缺陷(Morris水迷宫，MWM)脑损伤体积和脑白质损伤(组织学和MRI，磁学 磁共振成像)。我们还表明，在CCI后给予DHA可以改善大鼠的结局。腹膜内 (Ip)CCI后30分钟的DHA，随后长达60天的DHA饮食，降低了ROS，小胶质细胞 损伤后第7天的炎症基因(PID7)和记忆功能障碍(新对象识别，NOR)。 DHA是否影响雌性幼犬的脑炎性标志物，或者是否影响小胶质细胞的活动(ROS (生产和吞噬)在脑外伤后，无论性别，是未知的。同样，是否延迟(晚于30分钟 在CCI后)或短期(7天疗程)DHA将保留急性和慢性神经保护，目前尚不清楚。最后，很少 已知DHA如何调节脑损伤后的小胶质细胞激活。我们假设要么30岁的DHA CCI后3分钟或3小时可减轻男性PID7小胶质细胞炎症，只有长期DHA才能 改善男女患者的PID45-60结果。我们假设DHA将减少PID7炎症 与TLR4降低和细胞膜上活化的STAT1蛋白及caspase活性有关。我们 将使用雄性和雌性P17大鼠幼鼠和CCI或假手术。我们将比较DHA的发病时间(30分钟或3小时) 持续时间为7天~60天。在CCI后的第一周，我们将使用细胞膜分析，组织 和小胶质细胞蛋白标记物，以及小胶质细胞活性来评估炎症。从PID45到60，我们将使用 NOR/MWM测试、组织学和MRI检查功能、解剖和炎症结果。这项建议是 意义重大，因为它关注的是严重脑外伤后的未成熟大脑，这是一种毁灭性的疾病，没有 存在有效的治疗方法。这将为性别和DHA对小胶质细胞功能的影响提供新的知识。 以及关于最佳DHA启动和持续时间的选择。结果将使DHA的未来临床试验成为可能 减轻儿童脑外伤后获得性神经损伤负担的潜力。

PROJECT SUMMARY Traumatic brain injury (TBI) is the leading cause of acquired neurologic disability in children; yet, no effective therapies exist. Secondary injury from inflammation increases neurologic disability from damage after impact. Microglia (the brain's resident immune cells) mediate inflammatory release of reactive oxygen species (ROS) and cytotoxic factors early after TBI. Microglia that become reparatory remove debris by phagocytosis and resolve inflammation. The immature brain is highly vulnerable to secondary injury due to its low antioxidant reserve and vigorous inflammatory response. Agents that move microglia away from inflammatory towards reparatory activity may decrease neurologic disability after TBI, particularly in children. Docosahexaenoic Acid, or DHA, is a candidate therapy for childhood TBI. DHA is a fatty acid essential for normal brain growth and function that has antioxidant and anti-inflammatory activity. In rats, DHA restores brain DHA losses after TBI. In cultured microglia, DHA promotes reparatory activity. Using our pediatric TBI model, controlled cortical impact (CCI) in male 17-day old (P17) rats, DHA diet before CCI reduced learning deficits (Morris Water Maze, MWM) brain lesion volume and white matter injury (histology and MRI, Magnetic Resonance Imaging). We also showed that DHA given after CCI improved rat pup outcomes. Intraperitoneal (IP) DHA at 30 minutes after CCI, followed by up to 60 days of DHA diet, decreased ROS, microglial inflammatory genes at post injury day 7(PID7) and memory dysfunction (Novel Object Recognition, NOR). Whether DHA affects brain inflammatory markers in female pups, or if DHA affects microglial activity (ROS production and phagocytosis) after TBI in either sex, is unknown. Similarly, whether delayed (later than 30 min after CCI) or short (7-day course) DHA will retain acute and chronic neuroprotection, is not known. Finally, little is known about how DHA may modulate microglial activation after TBI. We hypothesize that either DHA at 30 min or 3h after CCI will decrease PID7 microglial inflammation in males and that only long term DHA will improve PID45-60 outcomes in both sexes. We hypothesize that DHA will decrease PID7 inflammation associated with decreased TLR4 and activated STAT1 proteins on cell membranes and caspase activity. We will use male and female P17 rat pups and CCI or sham surgery. We will compare onset of DHA (30 min or 3h) and duration (7 days to 60 days) to control. During week 1 after CCI, we will use cell membrane assays, tissue and microglial protein markers, and microglial activity to assess inflammation. From PID 45 to 60 we will use NOR/ MWM testing, histology and MRI for functional, anatomic and inflammatory outcomes. This proposal is significant because it focuses on the immature brain after severe TBI, a devastating condition for which no effective therapies exist. It will yield new knowledge on the effects of sex and DHA on microglial function after TBI and on optimal DHA initiation and duration timing. Results will enable future clinical trials of DHA with the potential to decrease the burden of pediatric acquired neurologic injury after TBI.