图1 皮肤/肌肉牵拉引起大鼠50%机械刺激撤足阈值下降
Published:20 January 2024,
Received:26 August 2023,
Accepted:03 December 2023
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To investigate the role of proinflammatory cytokines tumor necrosis factor alpha (TNFα) and interleukin-1β (IL-1β) in rostral ventromedial medulla (RVM) in chronic postsurgical pain (CPSP) induced by skin/muscle incision and retraction (SMIR).
SD rats were randomly divided into 5 groups: ① Sham group; ② SMIR group; ③ SMIR+TNFα/IL-1β neutralizing antibody group; ④ SMIR+TNFα/IL-1β group and ⑤ SMIR+vehicle group. 50% paw mechanical withdrawal threshold (MWT) was measured by the up-down method, immunofluroscence was used to detect the TNFα and IL-1β expression and ELISA for the 5-Hydroxytryptamine (5-HT) level.
SMIR elicited persistent nociceptive sensitization, upregulated TNFα and IL-1β expression in RVM neurons and astrocytes. Microinjection of TNFα or IL-1β neutralizing antibody into RVM inhibited the development of nociceptive sensitization and decreased the level of 5-HT in both RVM and spinal dorsal horn. While microinjection of recombinant TNFα or IL-1β into RVM enhanced the development of nociceptive sensitization and increased the level of 5-HT in both RVM and spinal dorsal horn.
Up-regulation of proinflammatory cytokines in RVM may contribute to SMIR induced CPSP by promoting 5-HT release.
外科手术如腹股沟疝修补手术或开胸手术后,约10%~50%的患者会产生慢性术后疼痛(chronic postoperative pain, CPSP)。其表现为在手术过后2个月,手术切口部位仍持续性疼痛,且不存在慢性感染,皮肤切口愈合不良等情况。CPSP对身体健康,日常活动,心理健康,就业和经济情况具有显著有害影响。在所有的接受手术的病人中有 0.5%~1.5% 的病人患有严重的甚至是致残性的 CPSP[
成年Sprague-Dawley (SD) 大鼠,雌雄不限,体质量220 ~ 250 g,购于中山大学北校区实验动物中心(国家级动物实验中心)提供。动物的使用协议和动物处理程序获得了中山大学实验动物管理与使用委员会(IACUC)的批准。动物饲养于室温(22 ± 0.5)℃、湿度50%-60%、12 h 黑暗-光照循环照明的安静环境。单笼饲养,自由饮水摄食。动物随机分配到不同的实验组。实验动物适应环境一周后,随机将74只大鼠随机分为:假手术 (17只)、SMIR (22只)、SMIR+vehicle(5只)、 SMIR+TNFɑ(或IL-1β中和性抗体,10只)、vehicle (10只)、 TNFɑ(或IL-1β, 10只)组。我们没有使用统计方法,而是基于以前我们的经验[
将动物用异氟烷(1.5%-2.5%)、70%O2和30%N2O的混合物麻醉,在大鼠左侧后大腿内侧靠近隐静脉处大约4 mm远的皮肤表面做1.5-2 cm的切口,暴露大腿肌肉后,在肌肉浅层做7-10 mm的切口,插入微型解剖牵开器(Biomedical Research Instruments Inc.,USA)。将大腿的皮肤及浅表肌肉牵拉2 cm并持续1 h(如
图1 皮肤/肌肉牵拉引起大鼠50%机械刺激撤足阈值下降
Fig.1 Skin/muscle and incision (SMIR) decreased the 50% paw withdrawal threshold
A-D :surgery procedure E:Compared to the sham group, the 50% paw withdrawal threshold was significantly decreased for 21 days in SMIR rats but not in sham rats. day 1, Z=-3.252, P=0.001; day 3, Z=-3.255, P=0.001; day 5, Z=-3.249, P=0.001; day 7, Z=-3.216, P=0.001, day 14, Z=-3.252, P=0.001; day 21, Z=-2.674, P=0.001; day 35, Z=-1.854, P=0.007 2. **P<0.01,*** P<0.001 compared with sham group. n=7 /group.
具体操作方法参照我们之前的操作[
为了将药物显微注射入RVM,我们使用戊巴比妥钠麻醉大鼠,并将其置于脑立体定位仪上。具体操作方法参照我们之前的操作[
具体操作方法参照我们之前的操作[
脑冰冻切片用0.01 mol/L PBS洗片3次,每次5 min,室温(25 ℃)下加入封闭液以封闭非特异性结合位点,1 h后弃去封闭液,加入含有待检测蛋白抗体(TNF-α抗体或IL-1β抗体,1 : 400或 1 : 200)的一抗稀释液,置于4 ℃摇床过夜。吸去一抗,洗片3次,每次10 min, 随后加入荧光二抗,室温(25 ℃)下避光摇床上慢摇1 h。弃去二抗, 洗片3次,每次10 min。随机挑选切片贴于载玻片上,封片后立即于荧光显微镜(LEICA DFC350 FX Camera,Germany)下观察并拍照保存。半定量分析使用Image J 软件,每组每只动物随机挑选5张切片,每组动物数n = 5, 计算出每张切片RVM区域的免疫荧光阳性面积,再比上RVM的总面积,得到免疫荧光阳性面积百分比。
迅速提取脊髓背角L3处和脑组织RVM部分,具体操作方法参照我们之前的操作[
统计分析使用 SPSS 16.0统计分析软件,实验结果以均数 ± 标准误(Mean± SEM)表示,p<0.05则认为有统计学差异。行为学测试结果因不符合正态分布,均采用非参数检验进行分析。所有定量资料满足正态分布,且通过方差齐性检验后采用单因素方差分析,否则采用非参数检验。两个不同实验组同一时间点的数据比较采用Mann-Whitney U检验。免疫荧光光密度,ELISA测得的5-HT 浓度数据在满足正态分布,且通过方差齐性检验后采用单因素方差分析(one-way ANOVA),组间差异用Tukey法(Tukey post hoc test)检测。
行为学测试发现,与手术前基础值或与假手术组比较,隐神经支配部位的皮肤及肌肉牵拉持续1 h后(
TNF-α是神经损伤以及神经炎症过程最重要和最早释放的致炎细胞因子[
免疫组化实验结果表明,SMIR可引起RVM内TNFα表达上调,如
图2 皮肤/肌肉牵拉对RVM TNFα及IL-1β 表达的影响
Fig.2 The effect of SMIR on the expression of TNFα及IL-1βin RVM
Compared to the sham group, SMIR upregulated the expression of TNFα(A-D)及IL-1β(E-H) at day 1 and 7 (n=5/group).D: F (2, 12)=22.09, P<0.001, *** P =0.000 2 at SMIR 1d and 7 d compared with sham group. H: F(2, 12)=20.14, P=0.000 1, ***P =0.000 3 at SMIR 1 d,***P=0.000 2 at SMIR 7 d compared with sham group.
图3 SMIR 后7 d RVM内TNFα及IL-1β表达的细胞类型
Fig. 3 The cell types that expressed TNFα及IL-1β 7 days after SMIR
TNFα was expressed mostly in NeuN labeled neurons (A-C), and to a much lesser extent in GFAP marked astrocyte (D-F) and Iba1 marked microglia (G-I) in RVM at day 7 after SMIR. IL-1β was expressed mostly in NeuN labeled neurons (J-L), and to a much lesser extent in GFAP marked astrocyte (M-O) and Iba1 marked microglia (P-R) in RVM at day 7 after SMIR. n=3/group.
上述结果表明SMIR手术导致了RVM内TNFα及IL-1β表达的上调,那么致炎细胞因子的上调与疼痛产生之间的关系又是怎样的呢?为此我们在RVM内分别微量注射了TNFα中和性抗体(100 pg,1 μL,每天1次,SMIR术前30 min开始,共4次)或IL-1β中和性抗体(100 pg,1 μL,SMIR术前30 min开始,每天1次,共4次),结果发现其均可阻断SMIR引起的机械痛敏,而给予对照溶剂则无作用,SMIR后50%机械刺激撤足阈值仍然出现下降(
图4 RVM内注射TNFα或IL-1β中和性抗体对SMIR引起的机械痛敏及5-HT水平的影响
Fig.4 The effect of TNFα or IL-1β neutralizing antibody on the nociceptive sensitization and 5-HT level in RVM and spinal cord induced by SMIR
A: Compared to the vehicle group, microinjection of TNFα neutralizing antibody into RVM inhibited the development of nociceptive sensitization .day -1, Z=-8.383, P=0.421; day 1, Z=-2.41, P=0.016; day 3, Z=-2.611, P=0.09;day 5, Z=-2.611, P=0.09; day 7, Z=-2.643, P=0.008, day 9, Z=-Z=-2.643, P=0.008. B : Compared to the vehicle group , microinjection of IL-1β neutralizing antibody into RVM inhibited the development of nociceptive sensitization. C-D :the effect of TNFα or IL-1β neutralizing antibody on the 5-HT level in RVM (C) and spinal dorsal horn(D) C: F(4, 20)=69.25, P<0.0001, ***P<0.001, SMIR+TNF antibody group ( the 5-HT level was significantly decreased ) compared with SMIR+vehicle group.***P<0.001, SMIR+IL-1 antibody group ( the 5-HT level was significantly decreased ) compared with SMIR+vehicle group . **P<0.01,***P<0.001 compared with the related group. n=5/group.
我们进一步观察了RVM内TNFα或IL-1β是否是引起机械痛敏的充分条件。发现RVM内微量注射TNFα或IL-1β均可引起机械痛敏(
图5 RVM 内微量注射TNFα或IL-1β对正常动物50%机械刺激撤足阈值及RVM 内和脊髓背角神经递质5-HT含量的影响
Fig.5 The effect of microinjection of TNFα or IL-1β into RVM of naive rats on the 50% paw withdrawal threshold and the level of 5-HT in RVM and spinal dorsal horn
A-B:The effect of microinjection of TNFα or IL-1β into RVM of naïve rats on the 50% paw withdrawal threshold A: day -1, Z=-2.643, P=0.008; day 1 and 3, Z=-2.611, P=0.008; day 5, Z=-2.627, P=0.008;day 7, Z=-2.611, P=0.009; day 14, Z=-2.619, P=0.008, day 21, Z=-2.227, P=0.051.B: day -1, Z=-4.88, P=0.639; day 1, Z=-2.847, P=0.003; day 3 and 5, Z=-2.842, P=0.003; day 7, Z=-2.847, P=0.003;day 14, Z=-2.842, P=0.003; day 21, Z=-2.632, P=0.008. C-D: The effect of microinjection of TNFα or IL-1β into RVM of naïve rats on the level of 5-HT in RVM and spinal dorsal horn。C: 5-HT level in RVM.F(3, 16)=143.4, P<0.001. ***P<0.001, TNFα group compared with vehicle group;***P<0.001, IL-1β group compared with vehicle group。D: 5-HT level in SP.F(3, 16)=29.14, P<0.001. ***P<0.001, TNFα group compared with vehicle ;***P<0.001, IL-1β group compared with vehicle group.
在本研究中,我们发现SMIR可引起大鼠后肢产生持续性痛觉敏感,且RVM中致炎细胞因子TNFα和IL-1β显著上调。此外,将TNFα和IL-1β中和性抗体微量注射入RVM中可防止SMIR引起机械痛敏的产生,并抑制SMIR引起的RVM及脊髓背角5-HT水平的升高。而在正常动物RVM内微量注射TNFα或IL-1β均可引起持续性机械痛敏,并引起RVM及脊髓内5-HT的水平升高。这些结果表明,RVM致炎细胞因子增多通过引起下行性易化作用参与了CPSP。
以前的观点认为慢性术后痛主要由手术期间外周神经的损伤产生。然而,研究表明,皮肤/肌肉切开和牵拉(SMIR模型)导致CPSP产生的同时并没有引起外周神经损伤[
内源性痛觉调制系统主要由中央导水管周围灰质和RVM构成。过度疼痛可激活内源性痛觉调制系统,从而起控制疼痛的作用。RVM内 50% 左右的神经元为 5-HT能神经元,可对伤害性信息传递产生紧张性抑制作用。与急性痛时不同,机体处于病理性疼痛状态时,内源性痛觉调制系统自身发生了显著的变化[
我们的前期工作已经报道SMIR可引起DRG和脊髓背角致炎细胞因子的上调[
RVM内预防性给予TNFα或IL-1β中和性抗体可缓解机械痛敏并降低RVM和脊髓背角中的5-HT水平。而在正常动物,RVM内注射微量的TNFα或IL-1β可引起机械痛敏,同时RVM和脊髓背角5-HT水平升高。RVM的神经元根据对μ受体激动剂的敏感性分成两类,一类对μ受体激动剂不敏感,而另外一类细胞膜上表达μ受体(MOR)且对μ阿片受体激动剂产生超级化反应,即抑制反应[
既往研究表明,TNFɑ和IL-1β 的受体,即TNFR1和IL-1R, 主要表达在含有NMDAR亚基NR1的RVM 神经元[
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