大鼠脊髓iNOS的过表达参与形成带状疱疹后神经痛

The overexpression of spinal iNOS contributes to postherpetic neuralgia in rat

目的:观察诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)在带状疱疹后神经痛(postherpetic neuralgia;PHN)大鼠脊髓的表达变化以及与痛行为的相关性。方法:SD大鼠随机分为水痘带状疱疹病毒感染组(varicella zoster virus,VZV组),假接种组(mock infected,Mock组)和空白对照组(Naive组);每组在接种前和接种后3、5、7、10、14、17、21 d先进行痛觉行为学检测,然后利用免疫组化染色和Western Blot检测脊髓iNOS以及NOS的其它两种同功酶nNOS和eNOS的表达变化;在痛觉阈值最低的时间点,鞘内注入各种NOS抑制剂进行行为药理学检测,同时在脊髓进行iNOS与NeuN,GFAP或OX-42的免疫荧光双重染色。结果:与Mock组和Naive组比较,VZV组在病毒注射后5 d形成了显著的机械性异常疼痛,14 d达到巅峰(P<0.05)。VZV组脊髓背角iNOS染色密度和表达量显著增加(P<0.05),免疫荧光双重染色显示iNOS由星形胶质细胞生成。iNOS的表达增加与痛觉阈值的降低显著相关(P<0.001,r=-0.89)。VZV组脊髓内nNOS和eNOS的表达没有变化。VZV组鞘内给予iNOS抑制剂L-NIL或NO清除剂PTIO能够有效镇痛,而nNOS或eNOS的抑制剂7-NINA,L-NIO则无镇痛作用。结论:脊髓背角iNOS的上调参与了PHN大鼠痛敏状态的形成。

Objective： To observe the expression change of inducible nitric oxide synthase （iNOS） in the spinal cord in a rat model of postherpetic neuralgia （PHN） and determine its correlation with pain behavioral performance. Methods： Adult male Sprague-Dawley rats were randomly divided into varicella zoster virus （VZV） group, mock infected group （ Mock group） and Naive group. At different time points （0,3,5,7,10,14,17,21 d after VZV infection） post-infection, immunostaining and Western Blot of iNOS, nNOS or eNOS were performed in each group after the detection of pain behavior. The inhibitors of iNOS, nNOS or eNOS was injected intrathecally in VZV group when the paw withdrawal threshold reached the lowest value. After injection, pain behavior was immediately measured. Meanwhile, double-labeling immuno-fluorescence staining of iNOS with NeuN, GFAP or OX- 42 was performed in the spinal cord sections. Results： Compared to Naive group and Mock infected group, the paw withdrawal threshold of VZV group significantly decreased at post-infection 5 d, reached the lowest value at 14 d （ n = 10/group ; P 〈 0.05 ）. The immunostaining and expression of iNOS was significantly increased in the spinal dorsal horn of VZV group （ P 〈 0.05 ）. Double immunofluorescent staining showed that iNOS-immunoreactivity was only localized in GFAP-immunopositive cells. The expression level of iNOS was found to be significantly correlated to the paw withdrawal threshold in VZV group （P 〈 0. 001, r = -0.89）. Intrathecal treatment with iNOS inhibitor, L-NIL or NO scavenger, PTIO exerted significant analgesic effect in VZV infected rats. However, intrathecal treatment with the inhibitors of nNOS and eNOS, 7-NINA and L-NIO, had no effect on pain behavior. Conclusion： The overexpression of iNOS contributes to PHN, and inhibiting spinal iNOS may represent a novel therapeu- tic strategy for clinical management of PHN.