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中华普通外科学文献(电子版)

中华普通外科学文献(电子版) ›› 2018, Vol. 12 ›› Issue (01) : 9 -13. doi: 10.3877/cma.j.issn.1674-0793.2018.01.003

所属专题： 文献；

论著

炎性痛敏触发术后持久伤害性敏化模型的建立
李坤河1, 李毅1, 牛丽君1, 杨广1, 赵晴1, 文志双1, 张劲军1, 安珂1,†()   
  1. 1. 510080　广州，中山大学附属第一医院麻醉科
  • 收稿日期:2017-07-24 出版日期:2018-02-01
  • 通信作者: 安珂
  • 基金资助:
    国家自然科学基金面上项目(81171468); 教育部高等学校博士学科点专项科研金项目(200805581112); 广东省科技计划项目(2012B031800105)

Establishment of postoperative sustained injury sensitization model of inflammatory pain-sensitivity

Kunhe Li1, Yi Li1, Lijun Niu1, Guang Yang1, Qing Zhao1, Zhishuang Wen1, Jinjun Zhang1, Ke An1,†()   

  1. 1. Department of Anesthesiology, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China
  • Received:2017-07-24 Published:2018-02-01
  • Corresponding author: Ke An
  • About author:
    Corresponding author: An Ke, Email:
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引用本文：

李坤河, 李毅, 牛丽君, 杨广, 赵晴, 文志双, 张劲军, 安珂. 炎性痛敏触发术后持久伤害性敏化模型的建立[J]. 中华普通外科学文献(电子版), 2018, 12(01): 9-13.

Kunhe Li, Yi Li, Lijun Niu, Guang Yang, Qing Zhao, Zhishuang Wen, Jinjun Zhang, Ke An. Establishment of postoperative sustained injury sensitization model of inflammatory pain-sensitivity[J]. Chinese Archives of General Surgery(Electronic Edition), 2018, 12(01): 9-13.

目的

通过建立改良外周炎性痛敏触发切口痛后持久伤害性敏化的动物模型，动态观察术前痛敏对切口痛后持久伤害性敏化大鼠疼痛行为学及脊髓水平蛋白激酶Mζ（PKMζ）的表达变化。

方法

雄性SD大鼠28只，随机分为非触发组和触发组，各14只。非触发组大鼠脚底注射0.9%氯化钠溶液5 μl，触发组大鼠脚底注射1%角叉菜胶5 μl。两组分别于实验前、炎性痛敏后1~6 d以及切口痛后1~10 d观察大鼠对机械和伤害性热刺激的疼痛行为学变化；分别于切口痛前及后1、3、10 d观察脊髓PKMζ的表达。

结果

实验前所有大鼠机械刺激缩爪阈值（MWT）和热刺激缩足反射潜伏期值（TWL）差异无统计学意义。触发组大鼠在注射1%角叉菜胶5 μl后出现了短暂的痛阈降低，并于3 d后恢复到基础值；两组大鼠切口痛后均出现活动频繁、跛行、舔咬手术切口等异常行为，与术前比较，两组大鼠的MWT、TWL均明显降低，差异有统计学意义（P<0.05）。但在切口术后5 d，非触发组大鼠的MWT、TWL逐渐升高，于术后10 d恢复至术前水平，而触发组大鼠MWT、TWL在各时间点均低于非触发组，差异有统计学意义（P<0.05），并且这种差异一直持续到观察结束。与非触发组及术前相比，触发组大鼠脊髓背角PKMζ含量迅速升高（P<0.05），并一直维持较高水平至观察结束。非触发组大鼠脊髓背角中PKMζ含量与术前比较差异无统计学意义。术前大鼠脊髓背角中PKMζ免疫阳性细胞均有少量表达，主要分布于脊髓背角浅层。与非触发组及术前相比，术后10 d触发组大鼠脊髓背角浅层PKMζ的免疫阳性细胞数明显增加（P<0.05），非触发组脊髓背角浅层PKMζ的免疫阳性细胞数虽有所增加，但数量明显低于触发组（P<0.05）。

结论

术前外周炎性痛敏可以触发切口痛后大鼠持久伤害性的敏化状态；脊髓背角PKMζ特异性抑制剂ZIP可以抑制这一敏化状态，这一作用与其特异性抑制脊髓水平PKMζ的表达有关。

关键词: 蛋白激酶类, 脊髓, 模型，动物, 持久伤害性敏化
Objective

To investigate the effects of spinal PKMζ on the maintenance of persistent nociceptive sensitization after plantar incision following peripheral inflammation in rats, adapting a modified hyperalgesia priming model that produces a state of sensitization closely resembling clinical situations with increased risk of development of persistent postoperative pain.

Methods

Twenty-eight healthy adult male Sprague-Dawley rats weighing 220-250 gram were randomly divided into two groups (n=14): NS+Surgery group which was induced by intraplantar injection of carrageenan (1%, 5 μl), and Car.+Surgery group with NS (0.9%NaCl, 5 μl) into one hindpaw in rats. Changes of pain behavior were assessed by withdrawal threshold to von Frey filament stimulation intensity, response latency of the hindpaw to radiant the thermal and a cumulative pain scores 1-6 d before incision as well as 1-10 d after incision. The expression of PKMζ was measured in the spinal cord by Western blotting and immunohistochemical analysis at 1, 3, 10 d after incision.

Results

There were no significant differences in the value of MWT and TWL before treatment and surgery. Compared with NS+ Surgery group, the MWT and TWL of Car.+Surgery group decreased significantly (P<0.05), which lasted about 3 d and returned to normal. All rats showed allodynia in response to innocuous mechanical stimulation. Compared with pre-incision, the MWT and TWL of two groups decreased significantly (P<0.05). While the MWT and TWL of NS+Surgery group increased thereafter and returned to normal by 6 d. Compared with pre-incision and NS+Surgery group, the MWT and TWL of Car.+Surgery group decreased significantly, lasting for 10 d (P<0.05). Compared with NS+Surgery group, the number of positive cells and the expression of spinal PKMζ after incision remarkably increased in Car.+Surgery group (P<0.05).

Conclusion

Spinal PKMζ involves in the development and maintenance of persistent nociceptive sensitization after plantar incision following peripheral inflammation pain in rats.

Key words: Protein kinases, Spinal cord, Models, animal, Persistent nociceptive sensitization
表1 术后持续性疼痛大鼠术前及术后MWT的变化（g，±s）
时间点（d） 非触发组 触发组 t值 P值
1 14.4±2.2 14.4±3.4 0.000 1.000
2 14.1±4.1 7.3±4.5 4.179 0.000
3 14.9±3.7 9.8±3.4 3.798 0.001
4 14.3±6.4 13.9±4.9 0.186 0.854
5 14.5±7.1 14.5±5.6 0.000 1.000
6 14.7±1.1 14.4±4.5 0.242 0.810
7 4.4±4.1 4.3±4.5 0.062 0.951
8 6.9±4.9 3.8±3.4 1.945 0.063
9 12.8±6.4 3.7±4.9 4.224 0.000
11 13.8±7.1 3.8±5.2 4.252 0.000
13 14.0±4.9 4.1±4.9 5.345 0.000
16 14.5±4.5 4.8±5.6 5.052 0.000
图1 术后持续性疼痛大鼠术前及术后MWT的变化（±s，n=14）与NS+Surgery非触发组比较，*P<0.05, **P>0.05
表2 术后持续性疼痛大鼠术前及术后TWL的变化（s，±s）
时间点（d） 非触发组 触发组 t值 P值
1 15.5±4.5 15.5±1.9 0.000 1.000
2 15.5±4.5 8.8±3.7 4.303 0.000
3 14.8±3.7 10.9±3.4 2.904 0.007
4 15.1±5.2 15.0±6.0 0.047 0.963
5 14.8±4.9 15.1±5.6 -0.151 0.881
6 15.4±4.9 15.5±3.4 -0.063 0.950
7 8.2±6.4 7.8±5.6 0.176 0.862
8 12.6±5.2 5.5±6.4 3.222 0.003
9 14.2±5.6 6.1±7.1 3.352 0.002
11 14.9±3.7 6.2±6.4 4.403 0.000
13 14.6±6.7 6.9±6.0 3.203 0.004
16 14.8±6.4 7.2±6.0 3.241 0.003
图2 术后持续性疼痛大鼠术前及术后TWL的变化（±s，n=14）与NS+ Surgery组比较，*P<0.05, **P>0.05
表3 外周炎性痛敏触发切口痛后持续伤害性敏化模型脊髓PKMζ含量的变化（±s, n=14）
组别 0 d 1 d 3 d 10 d
非触发组 0.92±0.24 0.80±0.25 0.91±0.23 0.92±0.34
触发组 0.93±0.12 1.24±0.11 1.32±0.32 1.38±0.34
t值 -0.139 -6.236 -3.893 -3.579
P值 0.890 0.000 0.001 0.001
图3 Western blotting显示各组脊髓PKMζ蛋白质的表达　A为NS+ Surgery非触发组；B为Car+Surgery触发组
图4 外周炎性痛敏触发切口痛后持续伤害性敏化大鼠脊髓中PKMζ表达的变化（±s，n=14，与NS+ Surgery非触发组比较，*P<0.05
表4 外周炎性痛敏触发切口痛后持续伤害性敏化模型脊髓PKMζ免疫阳性细胞的变化（±s, n=14）
组别 0 d 1 d
非触发组 50.4±8.8 48.3±9.4
触发组 49.8±9.9 105.6±9.0
t值 0.170 -16.440
P值 0.866 0.000
图5 非触发组术前及术后10 d大鼠脊髓背角PKMζ免疫阳性细胞（光学显微镜　×200）
图6 触发组术前及术后10 d大鼠脊髓背角PKMζ免疫阳性细胞（光学显微镜　×200）
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