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

创伤性脑损伤（TBI）是脑外伤患者获得性神经功能障碍的主要原因。 儿童;然而，没有有效的治疗方法。炎症继发性损伤增加神经功能障碍 撞击后的损伤。小胶质细胞（大脑的常驻免疫细胞）介导炎症释放， TBI后早期活性氧（ROS）和细胞毒性因子。小胶质细胞修复性切除 通过吞噬作用清除碎屑并解决炎症。未成熟的大脑极易受到二次损伤 因为它的抗氧化储备低和炎症反应剧烈。将小胶质细胞从 炎症对修复活动可能会减少TBI后的神经功能障碍，特别是在儿童中。 二十二碳六烯酸（DHA）是儿童TBI的候选疗法。DHA是一种脂肪酸， 正常的大脑生长和功能，具有抗氧化和抗炎活性。在大鼠中，DHA 脑损伤后DHA的损失在培养的小胶质细胞中，DHA促进修复活性。利用我们的小儿创伤性脑损伤 模型，在17日龄（P17）雄性大鼠中控制皮质撞击（CCI），CCI前的DHA饮食减少了学习 缺陷（Morris水迷宫，MWM）脑损伤体积和白色物质损伤（组织学和MRI，磁共振成像） 共振成像）。我们还表明，CCI后给予DHA改善了大鼠幼崽的结局。腹膜内 (IP)CCI后30分钟的DHA，随后长达60天的DHA饮食，减少了ROS，小胶质细胞 损伤后第7天的炎性基因（PID 7）和记忆功能障碍（新物体识别，NOR）。 DHA是否影响雌性幼鼠的脑炎症标志物，或者DHA是否影响小胶质细胞活性（ROS 生产和吞噬作用），在任何性别的TBI后，是未知的。同样，无论延迟（晚于30分钟 CCI后）或短期（7天疗程）DHA是否能保留急性和慢性神经保护作用尚不清楚。最后，小 已知DHA如何调节TBI后小胶质细胞活化。我们假设要么是30岁时的DHA CCI后10分钟或3小时将减少男性PID 7小胶质细胞炎症，只有长期DHA才能 改善两性的PID 45 -60结果。我们假设DHA将减少PID 7炎症 与细胞膜上TLR 4和活化的STAT 1蛋白以及caspase活性降低相关。我们 将使用雄性和雌性P17大鼠幼仔和CCI或假手术。我们将比较DHA的起效时间（30分钟或3小时） 防治期7 ~ 60天。在CCI后第1周，我们将使用细胞膜测定、组织培养和免疫组织化学方法。 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PID 45到60，我们将使用 NOR/ MWM测试、组织学和MRI用于功能、解剖和炎症结局。这项建议是 意义重大，因为它关注的是严重TBI后未成熟的大脑，这是一种破坏性的情况， 存在有效的治疗方法。这将产生新的知识，性别和DHA对小胶质细胞功能的影响， TBI和最佳DHA起始和持续时间。结果将使未来的临床试验的DHA与 有可能减少TBI后儿童获得性神经损伤的负担。

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.