亚甲蓝治疗椎间盘源性下腰痛机制的动物实验研究

EXPERIMENTAL STUDY OF THE MECHANISM ABOUT TREATING DISCOGENIC LOW BACK PAIN BY INTRADISCAL METHYLENE BLUE INJECTION

目的:研究亚甲蓝椎间盘内注射对退变椎间盘内神经纤维的灭活作用,探讨亚甲蓝治疗椎间盘源性下腰痛的机制。材料与方法:通过采用18只SD雄性大鼠建立椎间盘退变动物模型。将损伤尾4、5全层纤维环作为实验阳性对照椎间盘组;损伤尾5、6全层纤维环作为实验椎间盘组;尾6、7作为无损伤完整对照椎间盘组。所有大鼠均在术前,术后进行X线和MR I的检查,分别于术后第2周,术后第6周,术后第11周,随机取2只动物处死后进行Masson染色,观察纤维环愈合状况,到了第11周实验时间点,对实验组椎间盘进行亚甲蓝的注射,并在12周处死余下所有12只实验动物,切取大鼠尾椎椎间盘,进行免疫组化染色。检测SP(P物质)、PGP9.5(神经纤维);参考Madsen描述的免疫组化半定量方法,根据切片的神经在实验椎间盘的分布情况进行评分;评分标准如下:椎间盘中没有免疫阳性细胞和免疫阳性反应,0分;在椎间盘观察区有一个阳性细胞,1分;在椎间盘外部的2个或几个观察区包括几个阳性细胞,2分;在椎间盘几个观察区内包括几个甚至更多的阳性细胞,并延伸到椎间盘中央区,3分。记录各组数据,进行组间等级资料秩和检验,在软件SPSS14.0上进行,以P<0.05为有统计学意义。结果:X线可见椎间盘损伤前椎间隙正常,损伤后11周鼠尾椎有较明显的椎间隙变窄和骨质增生的反应间隙变窄和骨质增生的反应。MR I可见损伤前大鼠尾椎序列良好,椎间盘未见退变征象,高度基本一致。损伤后11周MR I复检可见SD大鼠尾椎序列紊乱,穿刺损伤的椎间盘出现高度丢失,体积缩小,椎体边缘骨桥形成等退变征象。在损伤后12周,在尾4/5椎间盘的前1/4区检测到SP和PGP9.5免疫阳性细胞分布。尾5/6椎间盘在损伤后11周,并注射亚甲蓝1周后神经出现率很低(P<0.01)。结论:亚甲蓝椎间盘内注射对椎间盘内神经纤维有灭活作用,可能是治疗椎间盘源性下腰痛的主要机制。

Objective：To explore the mechanism about treating diseogenie low back pain by intradigeal methylene blue injection through the study on intradiscal methylene blue injection to disc degeneration nerve fiber destroy. Methods：Using 18 SD maleness rats created intevertebral degeneration animal model. Injured tail 4/5 annular fibrosus as positive control group;Injured 5/6 as experiment group ;6/7 as normal disc control group. All rats before and after operation performed X ray and MRI, after 2 week, 6 week, 11 week, random used 2 rats to perform Masson dyeing, observing annular fibrosus heal condition, at the 11 week, to experiment group performed intradiscal methylene blue injection and at 12 week sacrifice left 1 2 rats , excised intervertebral disc and dyeing by inmmunohistochemistry. Detected substance P （ SP ） ,PGP9.5 ; Acroding to Maden discripe semi-quantitation methods, Scoring according to seetiones never in experiment intetwertebral disc distribution; Scoring standard： in disc without inmmuno-positive ,0 score; in intervertebral disc 1 positive cell, 1 score; in extra disc 2 or several district positive cells, 2 score; in intervertebral disc several observe districts including more positive cells, and extending to central district, 3 score. Recorded every group data and performed grade data rank sum test by SPSS14.0 software, if P 〈 0.05 be consided having significance. Result ： At two times of before and after operation, rat tail intervertebral disc became obvious narrow and hyperostetosis reaction by X-ray. Before injured rat tail intervenebral disc, MRI displayed Sequence good,intervertebral disc without degeneration sign and heigh lost. Injured after 11 weeks, MRI displayed degeneration sign of SD rat tail vertebral sequnce disarangement, high lost, volume decreasing, the edge of vertebral bone bi＇ige formation et al. After 12 weeks,at tail 4/5 disc SP and PGP9.5 imuno-positive cell distribution were checked and found tail 5/6 disc low positive rate （P 〈 0.01 ）. Conclution ：Methylene blue intradisc injection can destroy never fibers, which may be the main mechanism to treat discogenic low back pain.