剖腹探查术对创伤性脑损伤大鼠认知功能的影响及与钙超载的关系

Effects of laparotomy on cognitive function in rats with traumatic brain injury and the realationship with calcium overload

目的评价剖腹探查术对创伤性脑损伤大鼠认知功能的影响及与钙超载的关系。方法健康雄性Wistar大鼠150只,2~3月龄,体质量190~220 g,采用随机数字表法分为5组(n=30):空白组(B)、空白大鼠手术组(BS)、假模型大鼠手术组(SS)、脑损伤大鼠组(T)、脑损伤大鼠手术组(TS)。T和TS组大鼠采用Feeney法制备创伤性脑损伤大鼠,SS组大鼠仅做颅骨骨窗,不进行颅脑撞击,B组及BS组均为正常大鼠。20 d后BS、SS、TS组大鼠在七氟醚麻醉下行剖腹探查术(手术时间约为3 h),而B组和T组吸入纯氧3 h。于术前1d和术后3 d、7 d时,每组分别随机取10只大鼠行Morris水迷宫实验。水迷宫实验结束后取该10只大鼠的海马组织,采用流式细胞术测定海马神经元凋亡率和钙离子浓度,采用Western blot法测定海马组织的cleaved caspase-3的表达水平。结果术前1 d时,与B组逃避潜伏期[(9.8±0.8)s],穿越平台次数[(5.8±0.8)次],海马神经元凋亡率[(2.5±0.9)%],钙离子浓度[(2.3±0.2)],cleaved caspase-3的表达[(0.22±0.07)]比较,T组和TS组的逃避潜伏期延长[T组:(25.5±0.7)s,P<0.05;TS组:(25.1±1.1)s,P<0.05],穿越平台次数减少[T组:(2.7±0.8)次,P<0.05;TS组:(2.8±0.6)次,P<0.05],海马神经元凋亡率[T组:(5.3±0.6)%,P<0.05;TS组:(5.2±1.0)%,P<0.05],钙离子浓度升高[T组:(3.7±0.4),P<0.05;TS组:(3.6±0.5),P<0.05],海马组织cleaved caspase-3表达上调[T组:(0.45±0.07),P<0.05;TS组:(0.44±0.05),P<0.05],差异均有统计学意义;与SS组比较,TS组术后各时点逃避潜伏期延长[术后3 d:SS组:(23.8±1.3)s,TS组:(56.4±2.5)s,P<0.05;术后7 d:SS组:(16.6±1.8)s,TS组:(38.1±2.1)s,P<0.05],穿越平台次数减少[术后3 d:SS组:(2.9±0.6)次,TS组:(1.1±1.1)次,P<0.05;术后7 d:SS组:(4.2±1.2)次,TS组:(1.7±1.3)次,P<0.05],海马神经元凋亡率[术后3 d:SS组:(4.8±0.6)%,TS组:(14.4±0.6)%,P<0.05;术后7 d:SS组:(3.8±1.1)%,TS组:(9.6±1.3)%,P<0.05]和钙离子浓度升高[术后3 d:SS组:(3.1±0.3),TS组:(6.4±0.5),P<0.05;术后7 d:SS组:(2.6±0.3),TS组:(4.8±0.4),P<0.05],海马组织cleaved caspase-3的表达上调[术后3 d:SS组:(0.42±0.03),TS组:(0.88±0.08),P<0.05;术后7 d:SS组:(0.33±0.05),TS组:(0.63±0.06),P<0.05];与T组比较,TS组术后各时点逃避潜伏期延长[术后3 d:T组:(18.6±2.0)s,TS组:(56.4±2.5)s,P<0.05;术后7 d:T组:(13.8±2.6)s,TS组:(38.1±2.1)s,P<0.05],穿越平台次数减少[术后3 d:T组:(3.4±0.5)次,TS组:(1.1±1.1)次,P<0.05;术后7 d:T组:(4.3±1.2)次,TS组:(1.7±1.3)次,P<0.05],海马神经元凋亡率[术后3 d:T组:(4.4±0.7)%,TS组:(14.4±0.6)%,P<0.05;术后7 d:T组:(3.3±1.3)%,TS组:(9.6±1.3)%,P<0.05]和钙离子浓度升高[术后3 d:T组:(3.4±0.4),TS组:(6.4±0.5),P<0.05;术后7 d:T组:(3.0±0.3),TS组:(4.8±0.4),P<0.05],海马组织cleaved caspase-3的表达上调[术后3 d:T组:(0.40±0.04),TS组:(0.88±0.08),P<0.05;术后7 d:T组:(0.35±0.02),TS组:(0.63±0.06),P<0.05]。结论剖腹探查术可加重创伤性脑损伤大鼠认知功能损伤,引起术后认知障碍,其机制可能与加重钙超载的程度进而增加海马神经元凋亡率有关。

Objective To evaluate the effect of laparotomy on cognitive function in rats with traumatic brain injury and the relationship with calcium overload. Methods One hundred and fifty healthy male Wistar rats, aged 2~3 months, weighing 190~220 g, were assigned into 5 group (n=30 each) using a random number table: blank group(group B), surgery group of blank rats(group BS), surgery group of sham rats(group SS), traumatic brain injury rats group(group T), surgery group of traumatic brain injury rats(group TS). TBI model was made in rats of group T and TS by Feeney method.Rats in group SS were only treated with cranial bone window without crainocerebral impact.Both group B and BS were normal rats. Then the rats in group BS, SS and TS group underwent laparotomy under sevoflurane anesthesia (the operation time was about 3 hours) and the rats in group B and T inhaled pure oxygen for 3 hours after 20 days. One day before surgery, 3 and 7 d after surgery, 10 rats in each group were randomly selected for Morris water maze test. The hippocampus tissue of 10 rats were taken after the water maze test. The apoptosis rate and calcium concentration of hippocampal neurons were measured by flow cytometry, and the expression level of cleaved caspase-3 in hippocampal tissues was determined by Western blot. Results One day before surgery, compared with group B(the escape latency(9.8±0.8)s, the number of crossing platform (5.8±0.8), the apoptosis rate of hippocampal neurons (2.5±0.9)%, calcium ion concentration (2.3±0.2), the expression of cleaved caspase-3 (0.22±0.07)), the escape latency of group T and group TS were prolonged (group T:(25.5±0.7)s, P<0.05;group TS:(25.1±1.1)s, P<0.05), the number of crossing platform decreased (group T:(2.7±0.8), P<0.05;group TS:(2.8±0.6), P<0.05), the apoptosis rate of hippocampal neurons increased (group T:(5.3±0.6)%, P<0.05;group TS:(5.2±1.0)%, P<0.05), calcium ion concentration increased (group T:(3.7±0.4), P<0.05;group TS:(3.6±0.5), P<0.05) and the expression of cleaved caspase-3 increased (group T:(0.45±0.07), P<0.05;group TS:(0.44±0.05), P<0.05), the differences were statistically significant. Compared with the group SS, the escape latency (3 d after surgery: group SS:(23.8±1.3)s, group TS:(56.4±2.5)s, P<0.05;7 d after surgery: group SS:(16.6±1.8)s, group TS:(38.1±2.1)s, P<0.05)of the rats in group TS were prolonged, the number of crossing platform decreased (3 d after surgery: group SS:(2.9±0.6), group TS:(1.1±1.1), P<0.05;7 d after surgery: group SS:(4.2±1.2), group TS:(1.7±1.3), P<0.05), the apoptosis rate of hippocampal neurons (3 d after surgery: group SS:(4.8±0.6)%, group TS:(14.4±0.6)%, P<0.05;7 d after surgery: group SS:(3.8±1.1)%, group TS:(9.6±1.3)%, P<0.05), calcium ion concentration (3 d after surgery: group SS:(3.1±0.3), group TS:(6.4±0.5), P<0.05;7 d after surgery: group SS:(2.6±0.3), group TS:(4.8±0.4), P<0.05), the expression of cleaved caspase-3 (3 d after surgery: group SS:(0.42±0.03), group TS:(0.88±0.08), P<0.05;7 d after surgery: group SS:(0.33±0.05), group TS:(0.63±0.06), P<0.05) in the hippocampus increased after surgery (P<0.05). Compared with the T group, the escape latency (3 d after surgery: group T:(18.6±2.0)s, group TS:(56.4±2.5)s, P<0.05;7 d after surgery: group T:(13.8±2.6)s, group TS:(38.1±2.1)s, P<0.05) of the rats in group TS were prolonged, the number of crossing platform (3 d after surgery: group T:(3.4±0.5), group TS:(1.1±1.1), P<0.05;7 d after surgery: group T:(4.3±1.2), group TS:(1.7±1.3), P<0.05) decreased, the apoptosis rate of hippocampal neurons (3 d after surgery: group T:(4.4±0.7)%, group TS:(14.4±0.6)%, P<0.05;7 d after surgery:(3.3±1.3)%, group TS:(9.6±1.3)%, P<0.05), calcium ion concentration (3 d after surgery: group T:(3.4±0.4), group TS:(6.4±0.5), P<0.05;7 d after surgery: group T:(3.0±0.3), group TS:(4.8±0.4), P<0.05), the expression of cleaved caspase-3 (3 d after surgery: group T:(0.40±0.04), group TS:(0.88±0.08), P<0.05;7 d after surgery:(0.35±0.02), group TS:(0.63±0.06), P<0.05) in the hippocampus increased after surgery(P<0.05). Conclusion Laparotomy can aggravate the cognitive impairment of rats with traumatic brain injury and cause postoperative cognitive impairment, which may be related to the increased degree of calcium overload and the increased rate of hippocampal neuron apoptosis.