目的 探讨丙泊酚对老年小鼠脾脏切除术后认知功能的影响及相关机制.方法 50只SPF级雄性老年小鼠按随机数字表法分为空白组、假手术组、手术组、假手术+丙泊酚组,手术+丙泊酚组,每组各10只.采用Morris水迷宫实验评估各组小鼠学习和记忆能力,HE染色和TUNEL染色观察脑组织神经元凋亡情况,ELISA法测定脑组织炎性因子的表达,透射电镜观察海马突触致密物结构,免疫荧光染色测定小鼠海马区小胶质细胞标志物离子化钙结合衔接子分子-1(ionized calcium-binding adapter molecule 1,IBA-1)/突触后致密物蛋白-95(postsynaptic density protein-95,PSD-95)的表达,免疫组化法测定皮质微管相关蛋白tau(microtubule-associated protein tau,tau)的表达,免疫组化法和Western blot法测定B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)、重组Bcl-2关联X蛋白(re-combinant Bcl-2 associated X protein,Bax)和半胱胺酸蛋白酶第三型(cysteine protease-3,Caspase-3)的表达.利用SPSS 21.0进行数据统计分析,Morris水迷宫实验数据多组间比较采用重复测量方差分析;其他研究数据组间比较采用单因素方差分析,两两比较采用LSD-t检验.结果 Morris水迷宫实验结果显示,时间和组别的交互效应对5组小鼠逃避潜伏期影响显著(F=95.17,P＜0.01).在定位航行实验4～6d,手术组小鼠的逃避潜伏期高于假手术组(P＜0.05),手术+丙泊酚组小鼠的逃避潜伏期低于手术组但高于假手术+丙泊酚组(均P＜0.05).空间探索实验中,5组小鼠穿越平台次数,目标象限路程百分比和目标象限滞留时间均差异有统计学意义(F=27.88,50.21,32.04,均P＜0.01).手术组小鼠各指标均低于假手术组(均P＜0.05),手术+丙泊酚组小鼠各指标均高于手术组[(2.60±0.66)次,(0.80±0.40)次;(40.56±1.51)％,(13.82±3.11)％;(19.25±1.31)s,(6.12±2.00)s](均P＜0.05).ELISA结果显示,5组小鼠脑组织肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)和白细胞介素-6(interleukin-6,IL-6)的水平均差异有统计学意义(F=1 017.11,583.55,185.35,均P＜0.01).手术+丙泊酚组小鼠脑组织TNF-α、IL-1β和IL-6的水平均低于手术组且高于假手术+丙泊酚组(均P＜0.05).透视电镜结果显示,5组小鼠海马突触后致密物的长度、宽度和突触数量均差异有统计学意义(F=137.06,55.36,19.93,均P＜0.01),手术+丙泊酚组小鼠海马突触后致密物的长度、宽度和突触数量均高于手术组(均P＜0.05).免疫荧光结果显示,5组小鼠海马区PSD95和IBA-1的相对荧光强度均差异有统计学意义(F=14.33,38.23,均P＜0.01).手术+丙泊酚组小鼠海马区PSD95的相对荧光强度高于手术组且低于假手术+丙泊酚组(均P＜0.05).手术+丙泊酚组小鼠海马区IBA-1的相对荧光强度低于手术组且高于假手术+丙泊酚组(均P＜0.05).TUNEL结果显示,5组小鼠海马凋亡细胞比率差异有统计学意义(F=101.94,P＜0.01),手术+丙泊酚组小鼠海马凋亡细胞比率低于手术组[(72.15±3.71)％,(82.24±4.40)％]且高于假手术+丙泊酚组[(50.64±2.36)％](均P＜0.05).免疫组化和Western blot结果显示,5组小鼠磷酸化tau蛋白和Bcl-2、Bax、Caspase-3相对表达量均差异有统计学意义(F=56.54,50.48,23.00,91.10,均P＜0.01),手术+丙泊酚组小鼠脑组织磷酸化tau蛋白、Bax和Caspase-3相对表达量均低于手术组(均P＜0.05),手术+丙泊酚组小鼠Bcl-2相对表达量高于手术组(P＜0.05).结论 丙泊酚预处理能够明显减轻老年小鼠术后学习记忆能力受损,缓解海马突触后致密物的结构损伤,可通过抑制神经细胞凋亡信号通路,改善老年小鼠术后认知功能下降.
Effect of propofol preconditioning on cognitive function and hippocampal neuronal apoptosis in aged mice after splenectomy
Objective To evaluate effects of propofol on cognitive dysfunction and underlying mech-anisms after splenectomy in aged mice.Methods Fifty SPF grade male aged mice were divided into five groups:control group,sham operation group,surgery group,sham operation+propofol group,and surgery+propofol group,with 10 mice in each group.The Morris water maze test was used to assess the learning and memory abilities of mice in each group.The hematoxylin-eosin(HE)staining and TUNEL staining were used to observe the apoptosis of brain neurons.ELISA was performed to measure the expression of inflamma-tory factors in brain tissues.Transmission electron microscopy was used to observe the structure of hippocam-pal post-synaptic density.Immunofluorescence was employed to determine the expression of ionized calcium-binding adapter molecule 1(IBA-1)and postsynaptic density protein-95(PSD-95)in the hippocampus.Immunohistochemistry was used to measure the expression of microtubule-associated protein tau(tau)in the cortex.The expression of B-cell lymphoma-2(Bcl-2),recombinant Bcl-2-associated X protein(Bax),and cysteine protease-3(Caspase-3)was determined using both immunohistochemistry and Western blot.Data were statistically analyzed using SPSS 21.0.For comparisons among multiple groups,the results of Morris wa-ter maze experiment were compared by repeated measures ANOVA,while other data comparison was conduc-ted by one-way ANOVA,and pairwise comparisons were conducted using the LSD-t test.Results The re-sults of the Morris water maze test showed that the interaction effect of time and group on the escape latency of the five groups of mice was significant(F=95.17,P＜0.01).During the spatial navigation test of day 4-6,the escape latency of the surgery group was significantly higher than that of the sham operation group(P＜0.05),while the escape latency of the surgery+propofol group was significantly lower than that of the sur-gery group and higher than that of sham operation+propofol group(both P＜0.05).In the spatial exploration test,the number of platform crossings,the percentage of the distance traveled in the target quadrant out of the total exploration distance,and the duration spent in the target quadrant were all significantly different among the five groups of mice(F=27.88,50.21,32.04,all P＜0.01).These values were all lower in the surgery group compared to the sham operation group(all P＜0.05),and higher in the surgery+propofol group com-pared to the surgery group((2.60±0.66)vs(0.80±0.40);(40.56±1.51)％vs(13.82±3.11)％;(19.25±1.31)s vs(6.12±2.00)s)(all P＜0.05).ELISA results showed that the levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in the brain tissues of the five groups of mice were significantly different(F=1 017.11,583.55,185.35,all P＜0.01).The levels of TNF-α,IL-1β and IL-6 in the brain tissues of the surgery+propofol group were all lower than those in the surgery group and higher than that of sham operation+propofol group(all P＜0.05).Transmission electron microscopy re-sults showed that the length,width,and number of synapses in the hippocampal postsynaptic density of the five groups of mice were significantly different(F=137.06,55.36,19.93,all P＜0.01),and these values were all higher in the surgery+propofol group compared to the surgery group(all P＜0.05).Immunofluores-cence results showed that the relative fluorescence intensity of PSD95 and IBA-1 in the hippocampus of the five groups of mice were significantly different(F=14.33,38.23,both P＜0.01).The relative fluorescence intensity of PSD95 in the hippocampus of the surgery+propofol group was higher than that of the surgery group and lower than that of sham operation+propofol group(both P＜0.05),while the relative fluorescence intensity of IBA-1 in the hippocampus of the surgery+propofol group was lower than that of the surgery group and higher than that of sham operation+propofol group(both P＜0.05).TUNEL results showed that the proportion of positive apoptotic cells in the dentate gyrus(DG)region of the brain tissue of the five groups of mice was significantly different(F=101.94,P＜0.01),and this proportion was lower in the surgery+propo-fol group compared to the surgery group((72.15±3.71)％vs(82.24±4.40)％)and was higher than that of sham operation+propofol group((50.64±2.36)％)(both P＜0.05).Immunohistochemical and Western blot results showed that the relative expression levels of phosphorylated tau protein,Bcl-2,Bax,and Caspase-3 in the brain tissues of the five groups of mice were significantly different(F=56.54,50.48,23.00,91.10,all P＜0.01).The relative expression levels of phosphorylated tau protein,Bax,and Caspase-3 in the brain tissues of the surgery+propofol group were all lower than those in the surgery group(all P＜0.05),while the relative expression level of Bcl-2 was higher in the surgery+propofol group compared to the surgery group(P＜0.05).Conclusion Propofol pretreatment can significantly alleviate postoperative learning and memory impairments in aged mice,mitigate structural damage to the hippocampal postsynaptic density,and improve postoperative cognitive dysfunction by inhibiting the neuronal apoptosis signaling pathway.
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