丰富环境对神经病理性疼痛大鼠焦虑抑郁样行为及突触可塑性的影响

Effects of enriched environment on anxiety-and depressive-like behavior and synaptic plasticity in neuropathic pain rats

目的探究丰富环境对坐骨神经分支选择性损伤模型(selective nerve injury,SNI)大鼠神经病理性疼痛及焦虑、抑郁样行为的作用及其潜在机制。方法清洁级健康雄性SD大鼠36只,6~8周龄,采用随机数字表法分为3组(每组n=12):假手术组+标准环境组(假手术组)、SNI+标准环境组(标准环境组)、SNI+丰富环境组(丰富环境组)。采用坐骨神经分支选择性损伤模型构建大鼠神经病理性疼痛模型。丰富环境从术前7 d开始,持续至术后21 d。测定机械缩足阈值(paw withdraw threshold,PWT)及热缩足潜伏期(paw withdraw latency,PWL)以评估痛觉超敏及痛觉过敏行为,测试旷场实验、高架十字迷宫实验、新环境进食抑制实验以及强迫游泳实验以评估焦虑、抑郁样行为。采用免疫蛋白印迹(Western blot)法检测脊髓及前扣带皮质(anterior cingulate cortex,ACC)突触可塑性相关分子环磷酸腺苷反应元件结合蛋白(cAMP response element binding protein,CREB)及其磷酸化形式(p-CREB)、脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)、突触后致密物-95(postsynaptic density-95,PSD-95)以及神经连接蛋白2(neuroligin 2,NLGN2)的表达。采用免疫荧光测定CREB、BDNF在ACC中表达含量的改变。采用GraphPad prism 8.0和SPSS 23.0进行数据分析,组间比较采用单因素方差分析,PWT、PWL结果采用双因素重复测量方差分析,进一步两两比较采用Tukey检验。结果(1)重复测量方差分析结果显示,在PWT、PWL实验中,各组大鼠PWT的分组与时间的交互作用、组别主效应与时间主效应均显著(F=13.4,39.6,369.6,均P<0.05),PWL的分组与时间的交互作用、组别主效应与时间主效应均显著(F=3.8,10.3,58.8,均P<0.05)。与假手术组相比,标准环境组PWT[(8.0±3.5)g,(2.4±1.4)g,(2.3±1.1)g,(2.2±1.6)g,(1.6±0.5)g]与PWL[(8.6±1.3)s,(7.3±1.5)s,(7.9±1.0)s,(6.6±1.1)s,(7.7±1.4)s]各个时间点均较低(均P<0.05)。(2)与假手术组相比,标准环境组旷场实验中进入中央区次数[(1.3±1.7)次]及停留时间[(1.6±1.3)s]显著减少(t=4.585,5.423,均P<0.05),高架十字迷宫实验中进入开放臂次数的比率[(8.0±7.8)%]及停留时间的比率[(1.3±1.2)%]也明显减少(t=4.682,5.202,均P<0.05),新环境进食抑制实验中进食潜伏期[(365.2±94.4)s]及强迫游泳实验不动时间[(127.6±24.3)s]显著增长(t=6.008,14.290,均P<0.05)。丰富环境组大鼠开放臂进入次数和开放臂停留时间均高于标准环境组(均P<0.05),进食潜伏期和强迫游泳不动时间均低于标准环境组(均P<0.05)。(3)与假手术组相比[CREB:(1.6±0.2),(0.8±0.5);BNDF:(0.8±0.5),(1.0±0.4)],标准环境组脊髓和ACC中CREB[(1.8±0.1),(1.5±0.2)],BDNF[(0.9±0.6),(1.4±0.3)]表达增多(脊髓:t=3.283,4.989;ACC:t=5.502,4.257,均P<0.05),ACC中突触标志物PSD-95[(1.6±0.2),(1.0±0.2)]以及NLGN2[(1.5±0.5),(1.1±0.2)]表达增多(t=4.257,2.214,均P<0.05)。与标准环境组相比,丰富环境组脊髓中CREB(1.3±0.3)、BDNF(0.7±0.4)、PSD-95(1.0±0.3)以及NLGN2(1.1±0.4)表达降低(t=5.007,2.166,2.358,2.322,均P<0.05)。与标准环境组相比,丰富环境组ACC中PSD-95(1.2±0.3)和NLGN2(1.1±0.2)表达水平也显著降低(t=2.674,2.944,均P<0.05),而ACC中p-CREB(1.7±0.6),BDNF(2.4±0.2)表达水平却显著升高(t=4.180,7.610,均P<0.05)。结论丰富环境可以改善SNI大鼠神经病理性疼痛及其所致焦虑、抑郁样行为,这可能与干预脊髓及ACC中突触可塑性相关。

Objective To explore the effect of enriched environment on pain sensitivity,anxiety-and depressive-like behavior in selective nerve injury(SNI)rats model and its potential mechanism.Methods A total of 36 male clean grade SD rats aged 6-8 weeks were randomly divided into three groups(n=12 in each group):sham operation+standard environment group(sham group),SNI+standard environment group(standard environment group),SNI+enriched environment group(enriched environment group).The rat model of neuropathic pain was established by SNI.The rats in the enriched enviroment group were placed in an enriched enviroment 7 days before operation until 21 days after operation.The paw withdraw threshold(PWT)and paw withdraw latency(PWL)were performed to assess hyperalgesia.The open field test,elevated plus maze test,novelty suppressed feeding test and forced swimming test were used to assess anxiety and depression like behavior.The expressions of cAMP response element binding protein(CREB),p-CREB,brain-derived neurotrophic factor(BDNF),postsynaptic density-95(PSD-95)and neuroligin 2(NLGN2)were detected by Western blot.The expression of CREB and BDNF in contralateral ACC were measured by immunofluorescence.GraphPad prism 8.0 and SPSS 23.0 were used for data analysis.One way ANOVA was used for inter group comparison,repeated measurement ANOVA was used to analyze PWT and PWL results,and Tukey test was used for pairwise comparison.Results(1)In PWT and PWL experiments,the interaction effect between group and time,group main effect and time main effect of PWT were significant(F=13.4,39.6,369.6,all P<0.05),and the interaction effect between group and time,group main effect and time main effect of PWL were significant(F=3.8,10.3,58.8,all P<0.05).Compared with sham group,PWT((8.0±3.5)g,(2.4±1.4)g,(2.3±1.1)g,(2.2±1.6)g,(1.6±0.5)g)and PWL((8.6±1.3)s,(7.3±1.5)s,(7.9±1.0)s,(6.6±1.1)s,(7.7±1.4)s)in standard environment group decreased at each time point(all P<0.05).(2)Compared with sham group,the number of entrying into the central area(1.3±1.7),the time of entrying into the central area((1.6±1.3)s),the proportion of entering open arms((8.0±7.8)%)and the proportion of time in the open arms((1.3±1.2)%)all significantly decreased in standard environment group(t=4.585,5.423,4.682,5.202,all P<0.05).The eating latency((365.2±94.4)s)and immobility time((127.6±24.3)s)dramatically increased(t=6.008,14.290,both P<0.05).The number and time of entrying into central area of enriched environment group were both higher than those of standard environment group(both P<0.05),while the eating latency and immobility time of enriched environment group were both lower than those of standard environment group(both P<0.05).(3)Compared with sham group(CREB:(1.6±0.2),(0.8±0.5);BDNF:(0.8±0.5),(1.0±0.4)),the expression of CREB((1.8±0.1),(1.5±0.2)),BDNF((0.9±0.6),(1.4±0.3))in spinal cord and ACC of standard environment group increased(spinal:t=3.283,4.989;ACC:t=5.502,4.257,all P<0.05).The expression of PSD-95((1.6±0.2),(1.0±0.2))and NLGN2((1.5±0.5),(1.1±0.2))also increased in ACC of standard enviroment group(t=4.257,2.214,both P<0.05).Compared with standard environment group,the expression of CREB(1.3±0.3),BDNF(0.7±0.4),PSD-95(1.0±0.3)and NLGN2(1.1±0.4)in spinal cord of enriched environment group decreased(t=5.007,2.166,2.358,2.322,all P<0.05).The expression of PSD-95(1.2±0.3)and NLGN2(1.1±0.2)also decreased in ACC of enriched environment group(t=2.674,2.944,both P<0.05).However,the expression of p-CREB(1.7±0.6)and BDNF(2.4±0.2)increased in ACC(t=4.180,7.610,P<0.05).Conclusion Enriched environment can improve neuropathic pain and anxiety-and depressive-like behavior in SNI rats,which may be related to the change of synaptic plasticity in spinal cord and ACC.