胸部爆震伤后氧化应激反应对小鼠认知功能的影响

Effects of oxidative stress on cognitive function following chest blast injury in mice

目的探讨胸部爆震伤后氧化应激反应对小鼠认知功能的影响。方法选取60只C57BL/6雄性小鼠,按随机数字表法分为空白对照组(15只)和胸部爆震伤组(45只),胸部爆震伤组又分为伤后1、3、7 d进行后续实验。采用自主研发的爆震伤装置制备胸部爆震伤小鼠模型。采用Toklu评分评价行为学改变;Morris水迷宫试验评价空间记忆能力变化;HE染色观察额叶皮层和海马区病理学变化情况;组织活性氧(ROS)检测试剂盒检测额叶皮层和海马区ROS表达情况;Western blot法评估额叶皮层和海马区氧化应激丙二醛(MDA)和环氧化酶2(COX2)变化情况。结果胸部爆震伤组伤后1 d Toklu评分为(6.7±2.1)分,显著高于空白对照组的(2.0±0.0)分、胸部爆震伤组伤后3 d的(2.7±1.2)分和伤后7 d的(2.0±0.0)分(P均<0.01);胸部爆震伤组伤后3、7 d与空白对照组比较,差异无统计学意义(P均>0.05)。Morris水迷宫试验结果表明,胸部爆震伤组伤后1、3 d的潜伏期分别为60.1(60.1,60.1)s、60.1(56.3,60.1)s,显著长于空白对照组的10.1(3.9,18.3)s(P均<0.01);胸部爆震伤组伤后7 d的潜伏期为60.1(30.5,60.1)s,与空白对照组差异无统计学意义(P>0.05)。胸部爆震伤组伤后1、3、7 d比较,差异无统计学意义(P均>0.05)。空白对照组额叶皮层和海马区锥体细胞形态规则,细胞核呈深染并清晰可见,细胞质均匀。胸部爆震伤组伤后各时间点额叶皮层出现不同程度的锥体细胞坏死,海马区细胞质强烈嗜酸细胞增多。胸部爆震伤组伤后3 d额叶皮层ROS水平为(10.43±0.36)RFU/mg,伤后7 d为(2.91±0.35)RFU/mg,较空白对照组的(0.70±0.01)RFU/mg显著升高(P<0.05或0.01);胸部爆震伤组伤后3 d额叶皮层ROS水平显著高于伤后1 d的(2.13±0.65)RFU/mg和伤后7 d(P均<0.01),伤后7 d的额叶皮层ROS水平与伤后1 d差异无统计学意义(P>0.05)。胸部爆震伤组伤后3 d海马区ROS水平为(5.39±0.79)RFU/mg,伤后7 d为(5.65±1.17)RFU/mg,较空白对照组的(0.73±0.06)RFU/mg和胸部爆震伤组伤后1 d的(2.33±0.02)RFU/mg显著升高(P均<0.01);胸部爆震伤组伤后3 d的海马区ROS水平与伤后7 d比较、空白对照组海马区ROS水平与胸部爆震伤组伤后1 d比较,差异无统计学意义(P均>0.05)。额叶皮层与海马区之间ROS表达水平比较,胸部爆震伤组伤后3、7 d差异有统计学意义(P均<0.01),空白对照组与胸部爆震伤组伤后1 d差异无统计学意义(P均>0.05)。Western blot结果表明,胸部爆震伤组伤后1、3、7 d额叶皮层MDA水平分别为0.73±0.04、0.83±0.04、0.99±0.06,显著高于空白对照组的0.56±0.04(P<0.05或0.01);胸部爆震伤组伤后7 d额叶皮层MDA水平与伤后1、3 d比较,差异有统计学意义(P<0.05或0.01),伤后3 d与伤后1 d比较,差异无统计学意义(P>0.05)。胸部爆震伤组伤后1、3、7 d额叶皮层COX2水平分别为2.93±0.02、4.82±0.15、4.76±0.06,显著高于空白对照组的1.93±0.06(P均<0.01);胸部爆震伤组伤后3、7 d额叶皮层COX2水平与伤后1 d比较,差异有统计学意义(P均<0.01),伤后7 d与伤后3 d比较,差异无统计学意义(P>0.05)。胸部爆震伤组伤后1、3、7 d海马区MDA水平分别为0.92±0.11、0.83±0.03、0.68±0.03,显著高于空白对照组的0.49±0.03(P均<0.01);胸部爆震伤组伤后7 d海马区MDA水平与1 d和3 d比较,差异有统计学意义(P<0.05或0.01),伤后3 d与伤后1 d比较,差异无统计学意义(P>0.05)。胸部爆震伤组伤后1、3、7 d海马区COX2水平分别为0.88±0.06、0.87±0.06、0.80±0.06,显著高于空白对照组的0.37±0.04(P均<0.01);胸部爆震伤组伤后1、3、7 d比较,差异无统计学意义(P均>0.05)。额叶皮层与海马区之间的MDA表达水平比较,胸部爆震伤组伤后1、7 d差异有统计学意义(P均<0.01),空白对照组与胸部爆震伤组伤后1 d差异无统计学意义(P均>0.05)。额叶皮层与海马区之间的COX2表达水平比较,各组间差异有统计学意义(P均<0.01)。结论胸部爆震伤后小鼠短时期内会出现认知功能障碍,氧化应激反应是导致认知功能障碍发生的重要因素之一,并且认知相关的额叶皮层损伤重于海马区。

Objective To explore the effect of oxidative stress on cognitive function following chest blast injury in mice.Methods Sixty male C57BL/6 mice were divided into control group(n=15)and chest blast group(n=45)according to a random number table.The chest blast group was subgrouped at 1,3,7 days after injury for subsequent experiments.A self-developed blast injury device was used to prepare the mouse model of chest blast injury.Toklu score was used to evaluate the behavior changes in mice.Morris water maze test was used to evaluate the changes in spatial memory.HE staining was used to observe the pathological changes in the frontal cortex and hippocampus.Tissue reactive oxygen species(ROS)assay kit was used to detect ROS expression in the frontal cortex and hippocampus.Western blotting was used to assess changes of malondialdehyde(MDA)and cyclooxygenase-2(COX2)in the frontal cortex and hippocampus.Results The Toklu score of the chest blast group at 1 day after injury was(6.7±2.1)points,significantly higher than that of the control group[(2.0±0.0)points],as well as those of the chest blast group at 3 and 7 days after injury[(2.7±1.2)points and(2.0±0.0)points](all P<0.01).There was no significant difference in the Toklu score between the control group and the chest blast group at 3 and 7 days after injury(all P>0.05).The Morris water maze test showed that the latency periods at 1 and 3 days after injury were 60.1(60.1,60.1)seconds and 60.1(56.3,60.1)seconds,significantly longer than that of the control group[10.1(3.9,18.3)seconds](all P<0.01).The latency period of the chest blast group at 7 days after injury was 60.1(30.5,60.1)seconds,with no difference from the control group(P>0.05).No significant differences were found in the latency periods of the chest blast group at 1,3 and 7 days after injury(all P>0.05).In the control group,the pyramidal cells in the frontal cortex and hippocampus were regular in shape,with intensely-stained and clearly visible nuclei as well as uniform cytoplasm.In the chest blast group,diflerent degree of necrosis of pyramidal cells in the frontal cortex and strong cytoplasmic eosinophilia in the hippocampus were observed at different time points after injury.The levels of ROS in the frontal cortex of the chest blast group were(10.43±0.36)RFU/mg and(2.91±0.35)RFU/mg at 3 and 7 days after injury,which were significantly higher than that of the control group[(0.70±0.01)RFU/mg](P<0.05 or 0.01).The level of ROS in the frontal cortex of the chest blast group at 3 days after injury was significantly higher than that at 1 day[(2.13±0.65)RFU/mg]and that at 7 days after injury(all P<0.01).There were no statistical differences in the levels of ROS in the frontal cortex of the chest blast group at 1 and 7 days after injury(P>0.05).The levels of ROS in the hippocampus of the chest blast group were(5.39±0.79)RFU/mg and(5.65±1.17)RFU/mg at 3 and 7 days after injury,which were significantly higher than those of the control group and of the chest blast group at 1 day after injury[(0.73±0.06)RFU/mg and(2.33±0.02)RFU/mg](all P<0.01).No significant differences were found between the levels of ROS in the hippocampus of the chest blast group at 3 and 7 days after injury and between the ROS levels of the control group and of the chest blast group at 1 day after injury(all P>0.05).The levels of ROS in the frontal cortex and hippocampus showed significant differences between the chest blast group at 3 and 7 days after injury(all P<0.01)but no significant differences between the control group and the chest blast group at 1 day after injury(all P>0.05).Western blotting showed that the levels of MDA in the frontal cortex of the chest blast group were 0.73±0.04,0.83±0.04 and 0.99±0.06 at 1,3 and 7 days after injury,which were significantly higher than that of the control group(0.56±0.04)(P<0.05 or 0.01).The level of MDA in the frontal cortex of the chest blast group was significantly higher at 7 days after injury compared with that at 1 and 3 days after injury(P<0.05 or 0.01),but there was no statistical difference between 1 day and 3 days after injury(P>0.05).The levels of COX2 in the frontal cortex of the chest blast group were 2.93±0.02,4.82±0.15 and 4.76±0.06 at 1,3 and 7 days after injury,which were significantly higher than that of the control group(1.93±0.06)(all P<0.01).There were statistical differences in the levels of COX2 in the frontal cortex of the chest blast group at 3 and 7 days after injury compared with that at 1 day after injury(all P<0.01),but no statistical significance was found between 3 and 7 days after injury(P>0.05).The levels of MDA in the hippocampus of the chest blast group were 0.92±0.11,0.83±0.03 and 0.68±0.03 at 1,3 and 7 days after injury,which were significantly higher than that of the control group(0.49±0.03)(all P<0.01).There was a significant difference in the level of MDA in the hippocampus of the chest blast group at 7 days after injury compared with those at 1 and 3 days after injury(P<0.05 or 0.01),but the difference was not statistically significant among other groups(all P>0.05).The levels of COX2 in the hippocampus of the chest blast group were 0.88±0.06,0.87±0.06 and 0.80±0.06 at 1,3 and 7 days after injury,which were significantly higher than that of the control group(0.37±0.04)(all P<0.01).There were significant differences in the levels of COX2 of the chest blast group among 1,3 and 7 days after injury(all P>0.05).Statistically significant differences were found between the levels of MDA in the frontal cortex and hippocampus of the chest blast group at 1 and 7 days after injury(all P<0.01),but no statistical significant difference between the control group and the chest blast group at 1 day after injury(P>0.05).The levels of COX2 in the frontal cortex and hippocampus were significantly different among all groups(all P<0.01).Conclusions In the short term after chest blast injury,there will be cognitive dysfunction in mice.Oxidative stress is one of the important contributing factors,and the cognitive damage in the frontal cortex is more serious than that in the hippocampus.