氯胺酮对甲醛溶液致炎小鼠脊髓背角蛋白激酶Cγ和α表达的影响

Effect of ketamine on the expression of protein kinase C gamma and alpha in spinal dorsal horn of mice with formalin-induced inflammation pain

目的:观察氯胺酮对甲醛溶液致炎小鼠疼痛行为学的影响以及脊髓背角蛋白激酶Cγ和α表达的变化。方法:实验于2005-01/04在华中科技大学同济医学院附属同济医院进行。将成年健康昆明种小鼠25只随机分为正常组,盐水注射组,甲醛溶液组,甲醛溶液+20mg/kg氯胺酮组,甲醛溶液+40mg/kg氯胺酮组,后3组小鼠分别于右后足底注射体积分数为0.05的甲醛溶液100μL制成急性炎性痛模型,甲醛溶液+20mg/kg氯胺酮组,甲醛溶液+40mg/kg氯胺酮组小鼠再立即分别经腹腔注射20mg/kg或40mg/kg的氯胺酮,观察甲醛溶液注射后第一时相穴0～5min雪和第二时相穴15～60min雪各组大鼠摔腿,舔足等疼痛行为学变化,2h后处死小鼠,心内灌注固定,免疫组化方法检测各组小鼠脊髓背角蛋白激酶Cγ和α表达。结果:参加实验25只小鼠,灌注前意外死亡小鼠3只,随机补充3只,进入数据分析小鼠仍为25只。①甲醛溶液注射后1h内小鼠均出现明显的摔腿、舔足、跛行、致炎足不能着地等疼痛行为学改变,注射足呈高度肿胀。盐水注射组无任何异常行为学变化,20mg/kg氯胺酮注射后甲醛溶液致炎后第一时相和第二时相的疼痛行为学反应次数明显少于甲醛溶液组,差异具有极显著性(12±3.5,123±4.5;20±2.5熏80±2.5,P<0.01);而40mg/kg氯胺酮注射组可使小鼠翻正反射消失,并使小鼠疼痛行为学反应完全抑制。②正常组和盐水注射组脊髓背角的蛋白激酶Cγ和α的表达量极低,蛋白激酶Cγ主要局限于脊髓背角的Ⅱ层内侧,蛋白激酶Cα的表达主要位于脊髓背角的Ⅰ～Ⅱ层,甲醛溶液组蛋白激酶Cγ和α的表达明显高于正常组和盐水注射组(蛋白激酶Cγ:0.5740±0.0128熏0.4345±0.0172熏0.4323±0.0148;蛋白激酶Cα:0.5541±0.0151熏0.4325±0.0143熏0.4323±0.0151熏P均<0.001),而20mg/kg氯胺酮组脊髓背角蛋白激酶Cγ和α的表达穴0.4834±0.0137熏0.4722±0.0139雪明显低于甲醛溶液组(P<0.01),40mg/kg氯胺酮组脊髓背角蛋白激酶Cγ和α的表达穴0.4341±0.0146熏0.4331±0.0143雪明显低于20mg/kg氯胺酮组(P<0.05)。结论:在急性甲醛溶液炎性疼痛时,腹腔内注射氯胺酮在完全阻断小鼠疼痛行为学的同时,可减少脊髓背角蛋白激酶Cγ和α的表达。

AIM： To explore the effect of the ketamine on modulating the acute inflammation pain and changing the pain behavior and the expressions of protein kinase C （PKC） γ and α in spinal dorsal horn evoked by the formalin injection in mice. METHODS： The experiment was done in Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology from January to April 2005. Twenty-five health adult Kunming mice were randomly divided into five groups： Ⅰ normal group, Ⅱ saline group, Ⅲ formalin group, Ⅳ formalin＋20 mg/kg ketamine injection group, Ⅴ formalin＋40 mg/kg ketamine injection group. Formalin solution of 100 μL in the volume fraction of 0.05 was injected into right hindpaw of mice in Ⅲ, Ⅳ and Ⅴ groups respectively to establish acute inflammatory pain models, and then 20 mg/kg and 40 mg/kg ketamine were injected intraperitoneally in Ⅳ and Ⅴ group. The changes in the behavior of inflammatory pain mice following the injection of formalin were observed by calculating the times of licking foot and throwing legs at the first （0-5 minutes） and second time phases （15-60 minutes）. The mice were killed two hours after formalin-induced inflammation, and then fixed via infusion. Immunohistochemical ABC method was used to detect the expressions of PKC γ and α in spinal cord of mice. RESULTS： A total of 25 mice were involved in the result analysis after compensation of 3 unexpected deaths before the infusion. ① Changes of behavior in mice with inflammatory pain： After 1 hour of injection, the mice showed significant nociceptive pain behavior： throwing leg, licking foot, limping, and the inflammatory foot could not touch the ground. The injected foot was swelling highly. The reaction number of pain behavior at the first and second time phases in Ⅳ group was less obviously than that in Ⅲ group [（12±3.5）, （123±4.5） times; （20±2.5）, （80±2.5） times, P 〈 0.01]. Righting reflex disappeared immediately and the pain behavior was completely depressed in Ⅴ group. And there was no abnormal behavior change in Ⅱ group. ② The expressions of PKC γ and α were all increased significantly after the formalin injection, compared with control and saline groups （PKC γ： 0.574 0±0.012 8, 0.434 5±0.017 2, 0.432 3±0.014 8; PKC α： 0.554 1±0.015 1, 0.432 5±0.014 3, 0.432 3±0.015 1, P 〈 0.001）, but decreased significantly in Ⅳ group （0.483 4±0.013 7, 0.472 2±0.013 9, P 〈 0.01）, and the decrease was more significant in Ⅴ group （0.434 1±0.014 6, 0.433 1±0.014 3, P 〈 0.05）. PKC γ expressed in Ⅱ layer wall where PKC α expressed in Ⅰ - Ⅱ layers of spinal dorsal horn. CONCLUSION： lntraperitoneal injection of ketamine can completely depress the pain behavior and decrease the expressions of PKC γ and α in spinal cord of mice with formalin-induced acute inflammatory pain.