右美托咪定后处理对大鼠创伤性脑损伤后脑保护作用及其机制

Protective effect of dexmedetomidine post-treatment on traumatic brain injury in rats and possible mechanism

目的观察右美托咪定对大鼠脑外伤后行为学、脑含水量及程序性神经元死亡的影响,探讨右美托咪定减轻脑外伤的作用机制。方法健康雄性Wistar大鼠280只,由青岛市实验动物中心提供,按随机数字法分为4组:假手术组(Sham组)、脑外伤组(TBI组)、小剂量右美托咪定后处理组(DEX1组)和大剂量右美托咪定后处理组(DEX2组),每组70只。按Feeney法建立自由落体脑损伤动物模型,假手术组仅作开窗处理。右美托咪定在致伤后1 h经尾静脉5 min内以3μg/kg的剂量静脉推注,然后以3μg/(kg·h)(DEX1组)或6μg/(kg·h)(DEX2组)的剂量静脉输注2 h。每组随机抽取10只大鼠作水迷宫实验,剩余60只大鼠按伤后不同时间点分为4个亚组:伤后1、3、5、7 d,每个亚组15只。分别在伤后1、3、5、7 d行改良的神经功能缺损评分(mNSS)、干湿重法测定大脑半球含水量、苏木精-伊红(HE)染色观察损伤周边区及海马神经元形态变化、原位缺口末端标记法(TUNEL)法检测细胞凋亡、蛋白质印迹法(Western blot)检测微管相关蛋白1轻链3(LC3)和p62蛋白表达。水迷宫资料采用重复测量方差分析,其余资料采用单因素方差分析或Kruskal-Wallis非参数检验(不满足方差分析条件时),组间两两比较采用LSD检验(方差齐性时)或Dunnett’s T3检验(方差不齐时)。结果与TBI组比较,DEX1组、DEX2组伤后各时间点神经功能评分降低[伤后3 d:(8.00±1.07)、(6.87±1.95)分,F=126.203,P<0.01];水迷宫实验逃避潜伏期缩短[伤后8 d:(38.73±5.06)、(39.73±4.11)s,F=51.209,P<0.01]、目标象限内搜索时间延长[(51.75±14.37)、(53.27±11.45)s,F=14.104,P<0.01]及经过目标平台位置次数增加[(13.75±6.69)、(14.57±4.85)次,F=12.304,P<0.01];脑含水量减少[伤后3 d:(79.32±1.42)%、(79.33±2.86)%,F=18.142,P<0.01];损伤周围区及海马凋亡神经元减少[伤后3 d:(37.87±4.12)、(39.39±6.56)个,F=64.782,P<0.01];损伤周围区及海马LC3Ⅱ/LC3Ⅰ和p62蛋白表达减少(伤后3 d:0.38±0.12比0.45±0.14、0.36±0.16比0.32±0.14,F=12.587、17.354,P<0.01)。DEX1、DEX2组之间上述指标差异无统计学意义(P>0.05)。结论右美托咪定后处理可减轻大鼠脑外伤后学习及记忆功能障碍,与减轻脑水肿、减少神经元凋亡和自噬有关;增大右美托咪定剂量上述脑保护作用并不增强。

Objective To observe the effects of dexmedetomidine on behaviors,brain water content and programmed neuronal death after traumatic brain injury in rats,and to explore the mechanism of dexmedetomidine in alleviating brain trauma.Methods Two hundreds and eighty healthy male Wistar rats were divided into 4 groups according to a random number method:sham operation group(Sham group),brain trauma group(TBI group),low-dose dexmedetomidine post-treatment group(DEX1 group)and high-dose dexmedetomidine post-treatment group(DEX2 group),70 rats in each group.According to the Feeney method,an animal model of free-falling brain injury was established.Dexmedetomidine was injected intravenously at a dose of 3μg/kg within 5 min through the tail vein 1 h after injury,and then infused continuously at a dose of 3μg/(kg·h)(DEX1 group)or 6μg/(kg·h)(DEX2 group)for 2 h.Ten rats were randomly selected from each group for the water maze experiment.The remaining 60 rats in each group were divided into 4 subgroups according to different time points:1st,3rd,5th,7th day after injury,and 15 rats in each subgroup.On the 1st,3rd,5th and 7th day after injury,the degree of neurological impairment was evaluated by modified neurological deficit score(MNSs),the water content of cerebral hemisphere was measured by wet and dry weight method,the morphological changes of neurons in peripheral area around the injury and hippocampus were observed by hematoxylin and eosin(HE)staining,apoptosis was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)method,and the expression of microtubule-associated protein 1 light chain 3(LC3)and p62 proteins was detected by Western blotting.Results As compared with the TBI group,the mNSS scores in the DEX1 and DEX2 groups decreased at various time points after injury(at 3rd day after injury:8.00±1.07,6.87±1.95,F=126.203,P<0.01);the escape latency of the water maze experiment was shortened[at 8th day after injury:(38.73±5.06),(39.73±4.11)s,F=51.209,P<0.01],search time in the target quadrant was prolonged[(51.75±14.37),(53.27±11.45)s,F=14.104,P<0.01]and the times of passing the target platform position[(13.75±6.69),(14.57±4.85)times,F=12.304,P<0.01]increased;brain water content decreased[at 3rd d after injury:(79.32±1.42)%,(79.33±2.86)%,F=18.142,P<0.01];apoptotic neurons in the peripheral area and hippocampus decreased(at 3rd d after injury:37.87±4.12,39.39±6.56,F=64.782,P<0.01);the expression of LC3Ⅱ/LC3Ⅰand p62 protein in peripheral area around the injury and hippocampus decreased(at 3rd day after injury:0.38±0.12 vs.0.45±0.14,0.36±0.16 vs.0.32±0.14,F=12.587,17.354,P<0.01).There was no statistically significant difference in the above indicators between DEX1 and DEX2 groups(P>0.05).Conclusion Dexmedetomidine post-treatment can alleviate learning and memory dysfunction in rats after TBI,which may be related to alleviation of brain edema,and reduced neuronal apoptosis and autophagy.Increasing the dose of dexmedetomidine does not enhance the brain protection.