摘要
目的探讨颅脑冲击伤(bTBI)后非致命性淹溺大鼠认知功能变化。方法80只SD大鼠按随机数字表法分为正常组、冲击伤组、淹溺组和冲击伤+淹溺组,每组20只。正常组不做任何处理;冲击伤组采用BST-Ⅰ型生物激波管(4.0 MPa驱动段压力)构建bTBI模型;淹溺组水面上1 m处自由落下(水温18℃、水深30 cm),待游泳力竭后捞出;冲击伤+淹溺组通过生物激波管致伤后,即刻同法淹溺。伤后3 d,采用高架十字迷宫试验和Morris水迷宫试验评估大鼠神经行为学变化;尼氏染色观察海马组织CA1、CA3区神经元病理学变化并统计其存活数量;ELISA法检测海马组织神经递质谷氨酸、γ-氨基丁酸(GABA)、甘氨酸和内质网应激(ERS)相关蛋白葡萄糖调节蛋白(GRP78)、半胱氨酸蛋白水解酶(caspase)-12水平;Western blot检测海马凋亡相关蛋白B淋巴细胞瘤-2(Bcl-2)、B淋巴细胞瘤-2相关X蛋白(Bax)和caspase-3的表达,并计算Bcl-2/Bax比率。结果高架十字迷宫试验显示,伤后3 d冲击伤+淹溺组开臂进入次数百分比和探索行为次数低于正常组和冲击伤组(P<0.05或0.01),与淹溺组差异无统计学意义(P均>0.05),且淹溺组探索行为次数低于冲击伤组(P<0.05)。Morris水迷宫试验显示,冲击伤+淹溺组在定位航行试验第3,4天寻靶潜伏时间高于正常组,空间探索试验穿越靶区次数和靶象限游泳时间百分比低于正常组(P<0.05或0.01);而冲击伤组、淹溺组和正常组差异无统计学意义(P均>0.05)。尼氏染色显示,伤后3 d正常组海马CA1、CA3区神经元排列整齐、尼氏体清晰,其他组均出现不同程度损伤,与正常组相比,其他各组海马CA1、CA3区神经元数目呈不同程度减少,其中冲击伤+淹溺组神经元数目最少,低于冲击伤组和淹溺组(P<0.05或0.01)。ELISA法检测显示,伤后3 d冲击伤+淹溺组海马谷氨酸水平高于其他各组,且冲击伤组和淹溺组高于正常组(P<0.05或0.01);冲击伤+淹溺组甘氨酸水平低于正常组(P<0.05),冲击伤组、淹溺组和正常组差异无统计学意义(P均>0.05);各组GABA水平差异无统计学意义(P均>0.05)。此外,冲击伤组、淹溺组和冲击伤+淹溺组GRP78表达高于正常组(P<0.05或0.01),但三组间差异无统计学意义(P均>0.05);淹溺组和冲击伤+淹溺组caspase-12表达高于正常组(P<0.05或0.01),但两组间差异无统计学意义(P>0.05),而与冲击伤组相比,冲击伤+淹溺组表达显著升高(P<0.05)。Western blot结果显示,伤后3 d冲击伤+淹溺组Bcl-2表达低于其他各组,且冲击伤组和淹溺组低于正常组,淹溺组低于冲击伤组(P<0.05或0.01);冲击伤+淹溺组Bax表达高于其他各组,且淹溺组高于正常组(P<0.05或0.01),冲击伤组和淹溺组差异无统计学意义(P>0.05);冲击伤+淹溺组Bcl-2/Bax比率低于其他各组,且冲击伤组和淹溺组低于正常组,淹溺组低于冲击伤组(P<0.05或0.01)。淹溺组、冲击伤+淹溺组caspase-3表达高于正常组和冲击伤组(P<0.05或0.01),冲击伤+淹溺组和淹溺组差异无统计学意义(P>0.05)。结论非致命性淹溺可加重bTBI大鼠海马神经元损伤,引起记忆、情绪等认知功能障碍,其机制可能涉及海马组织神经递质谷氨酸和甘氨酸失衡引起细胞稳态破坏,并在损伤早期通过ERS激活下游促凋亡途径,诱导海马神经元凋亡。
Objective To investigate the changes of cognitive function in non-fatal drowning rats after blast-induced traumatic brain injury(bTBI).Methods Eighty SD rats were divided into normal group,bTBI group,drowning group and bTBI plus drowning group according to the random number table,with 20 rats per group.Rats in normal group were not injured.In bTBI group,bTBI was established in a BST-I biological shock tube with a pressure of 4.0 MPa in the driving section.In drowning group,rats were subjected to non-fatal drowning by falling into the water with temperature of 18℃and depth of 30 cm from the height of 1 m and were taken out quickly after swimming to exhaustion.After being injured in a biological shock tube,rats in bTBI plus drowning group were immediately forced to drowning using the same method.On day 3 post-injury,the neurocognitive function was evaluated by elevated plus maze and Morris water maze tests.Morphological changes of neurons in CA1 and CA3 regions of hippocampus were observed by Nissl staining,and the number of surviving neurons were counted.The concentrations of hippocampal neurotransmitters glutamate,γ-aminobutyric acid(GABA),glycine and endoplasmic reticulum stress(ERS)related glucose-regulated protein 78(GRP78)and caspase-12 were examined by ELISA analysis.Levels of B-cell lymphoma-2(Bcl-2),Bcl-2 associated protein(Bax)and caspase-3 were detected by Western blotting.The ratio of Bcl-2 to Bax was calculated as well.Results In elevated plus maze test,the percentage of open arm entry and number of head-dipping behaviour were decreased in bTBI plus drowning group compared with normal and bTBI groups at 3 days after injury(P<0.05 or 0.01),with no statistical difference from those in drowning group(P>0.05).The number of head-dipping behaviour in drowning group was lower than that in bTBI group(P<0.05).In Morris water maze test,bTBI plus drowning group showed increased target latency on the third and fourth days of spatial acquisition training and decreased number of crossing the target area and percentage of swimming time in the target quadrant during probe trials as compared with normal group(P<0.05 or 0.01),but there was no statistical difference among bTBI,drowning and normal groups(all P>0.05).Nissl staining showed that the neurons in the CA1 and CA3 regions of hippocampus in normal group were arranged neatly with clear Nissl bodies at 3 days after injury,while the other groups showed different degrees of injury.In contrast with normal group,the neurons in the CA1 and CA3 regions of hippocampus in all other groups were decreased with the lowest number in bTBI plus drowning groups(P<0.05 or 0.01).In ELISA analysis,the level of hippocampal glutamate in bTBI plus drowning group was higher than that in all other groups at 3 days after injury and the level in bTBI injury and drowning groups was higher than that in normal group(P<0.05 or 0.01);the level of hippocampal glycine in bTBI plus drowning group was lower than that in normal group(P<0.05),but there was no statistical difference among bTBI,drowning or normal groups(all P>0.05);the concentration of hippocampal GABA had no statistical difference among all groups(all P>0.05).In addition,the concentration of GRP78 in bTBI injury,drowning and bTBI injury plus drowning groups were increased compared with normal group(P<0.05 or 0.01),but did not statistically differ from each other(all P>0.05).The concentration of caspase-12 in drowning and bTBI plus drowning groups were increased compared with normal group(P<0.05 or 0.01),but was not statistically different from each other(P>0.05),and its concentration in bTBI plus drowning group was increased compared with bTBI group(P<0.05).In Western blotting,the level of Bcl-2 in bTBI plus drowning group was decreased compared with all other groups at 3 days after injury,and the level in bTBI and drowning groups were decreased compared with normal group,but a much lower level was observed in drowning group than that in bTBI group(P<0.05 or 0.01);the level of Bax in bTBI plus drowning group was increased compared with all other groups at 3 days after injury,and the level in drowning group was increased compared with normal group(P<0.05 or 0.01),with no statistical difference between bTBI and drowning groups(P>0.05).The ratio of Bcl-2 to Bax in bTBI plus drowning group was decreased compared with all other groups,while the ratio in bTBI and drowning groups were decreased compared with normal group,showing a much lower level in drowning group than that in bTBI group(P<0.05 or 0.01).Also,the level of caspase-3 in drowning and bTBI plus drowning groups were increased compared with normal and bTBI groups(P<0.05 or 0.01),but there was no statistical difference between drowning and bTBI plus drowning groups(P>0.05).Conclusions Non-fatal drowning can aggravate hippocampal neuron damage in bTBI rats and cause memory,emotion and other cognitive dysfunction.The mechanism may involve the imbalance of hippocampal neurotransmitters glutamate and glycine,which activates the downstream pro-apoptotic pathway through ERS in the early stage of injury to induce hippocampal neuron apoptosis.
作者
李森
龙在云
王海燕
余静
廖志康
高洁
刘媛
伍亚民
杨策
Li Sen;Long Zaiyun;Wang Haiyan;Yu Jing;Liao Zhikang;Gao Jie;Liu Yuan;Wu Yamin;Yang Ce(Department of Special War Wound,Research Institute of Surgery,State Key Laboratory of Trauma,Burns and Combined Injury,Daping Hospital,Army Medical University,Chongqing 400042,China)
出处
《中华创伤杂志》
CAS
CSCD
北大核心
2022年第12期1132-1140,共9页
Chinese Journal of Trauma
基金
“十三五”军队“双重”建设科研创新项目(4142Z63)
陆军军医大学大坪医院人才创新能力培养计划(2019CXJSB007)。
关键词
爆震伤
颅脑损伤
认知功能障碍
内质网应激
溺水
Blast injuries
Craniocerebral trauma
Cognitive dysfunction
Endoplasmic reticulum stress
Drowning
