选择性神经损伤大鼠发生疼痛和抑郁样行为的机制以及米诺环素的干预作用

Mechanism of developing pain and depression-like behaviors in rats with spared nerve injury and intervention effect of minocycline

目的探讨选择性神经损伤(SNI)模型大鼠发生疼痛、抑郁样行为的可能机制,以及侧脑室注射米诺环素对其疼痛、抑郁样行为的干预效果。方法将120只成年雄性SD大鼠随机分为假手术组(Sham组)、SNI组、SNI+1μg Mino组和SNI+10μg Mino组,每组30只。SNI组、SNI+1μg Mino组和SNI+10μg Mino组大鼠在建立SNI模型后,分别给予侧脑室注射磷酸缓冲盐溶液、1μg/μL和10μg/μL米诺环素溶液,Sham组只进行假手术,不损伤及结扎神经,并于术后给予侧脑室注射磷酸缓冲盐溶液。于实验前1 d及鞘内置管术后(即术后)1 d、3 d、7 d、14 d,通过测定热缩足反射潜伏期(PWTL)评估大鼠的疼痛程度;于实验前1 d及术后1 d、7 d通过转棒试验评估大鼠的运动功能。于实验前1 d及术后1 d、7 d、14 d,采用强迫游泳实验及糖水偏好实验评估大鼠的抑郁样行为。采用免疫荧光法及蛋白免疫印迹法分析术后14 d大鼠海马组织离子钙接头蛋白1(Iba-1)的表达量,采用ELISA检测术后3 d、7 d、14 d大鼠海马组织肿瘤坏死因子α(TNF-α)表达水平及白细胞介素6(IL-6)含量。结果在术后各时间点,与Sham组相比,SNI组的PWTL缩短,强迫游泳不动时间延长,糖水偏好百分比降低,海马组织中小胶质细胞明显增殖,Iba-1蛋白的表达量、TNF-α表达水平和IL-6含量均升高(均P<0.05)。与SNI组相比,SNI+1μg Mino组、SNI+10μg Mino组大鼠在术后各时间点的PWTL延长,术后14 d强迫游泳不动时间缩短、糖水偏好百分比增加,海马组织中小胶质细胞增殖受到抑制,Iba-1蛋白的表达量、TNF-α表达水平和IL-6的含量均降低(均P<0.05)。4组大鼠在疲劳转棒上所停留的时间差异无统计学意义(均P>0.05)。结论SNI大鼠出现的神经病理性疼痛及抑郁样行为,均与海马区小胶质细胞的激活和神经炎症有着密切的联系,而侧脑室注射小胶质细胞抑制剂米诺环素可改善上述情况。

Objective To investigate the possible mechanism of developing pain and depression-like behaviors in rat models of spared nerve injury(SNI),as well as the intervention effect of an injection of minocycline in lateral ventricle on pain and depression-like behaviors of the rats.Methods One hundred and twenty male adult SD rats were randomly divided into sham operation group(Sham group),SNI group,SNI+1μg Mino group and SNI+10μg Mino group,with 30 rats in each group.Rats in the SNI group,SNI+1μg Mino group and SNI+10μg Mino group were given injections of phosphate buffered saline,minocycline saline 1μg/μL,and minocycline saline 10μg/μL in lateral ventricle,respectively,following the establishment of SNI models.The Sham group underwent sham operation alone without nerve injury and ligation and was given an injection of phosphate buffered saline in lateral ventricle after the operation.One day before the experiment and one,three,seven and 14 days after the intrathecal catheterization(i.e.,after the operation),the pain intensity of rats was assessed by determining the paw withdrawal thermal latency(PWTL);one day before the experiment and one and seven days after the operation,the motor function of rats was evaluated by the rotarod test.One day before the experiment and one,seven and 14 days after the operation,the forced swim test and the sucrose preference test were employed to evaluate the depression-like behaviors of rats.Immunofluorescence method and Western blotting assay were used to analyze the expression of ionized calcium binding adaptor molecule 1(Iba-1)in hippocampal tissues 14 days after the operation,and ELISA to detect the expression levels of tumor necrosis factorα(TNF-α)and the contents of interleukin 6(IL-6)in hippocampal tissues three,seven and 14 days after the operation.Results At each time point after the operation,as compared with the Sham group,the SNI group exhibited a decreased PWTL,prolonged immobility time in the forced swim test,a lower percentage of rats preferring to sucrose,a significant increase in hippocampal microglia proliferation,elevated expression of Iba-1 protein,a higher expression level of TNF-α,and a higher IL-6 content(all P<0.05).Compared with the SNI group,rats in the SNI+1μg Mino and SNI+10μg Mino groups yielded a prolonged PWTL at each time point after the operation,a reduced immobility time in the forced swim test 14 days after the operation,an increased percentage of rats preferring to sucrose,reduced proliferation of microglia in hippocampal tissues,lower expression of Iba-1 protein,a lower expression level of TNF-α,and a lower IL-6 content(all P<0.05).There was no statistically significant difference in the length of rats′stay on the rotating rod among the four groups(all P<0.05).Conclusion Neuropathic pain and depression-like behaviors occur in rats with SNI,and both closely correlate with microglia activation and neuroinflammation in the hippocampus,whereas an injection of minocycline,a microglia inhibitor,in lateral ventricle can improve the aforementioned conditions.