摘要
目的探讨缝隙连接功能异常在左旋多巴诱发异动症（ LID）发生机制中的作用。方法对6-羟多巴胺制备的偏侧帕金森病大鼠模型重复腹腔注射左旋多巴（20 mg/kg ）和卞丝肼（10 mg/kg）共21 d,以建立左旋多巴诱发异动症模型。将实验动物分为3组：LID组、帕金森病组、正常对照组。通过腹腔注射甘珀酸和侧脑室注射奎宁来观察缝隙连接阻断剂对异动症大鼠行为学的影响。免疫双标法分析脑啡肽阳性纹状体传出神经元及小清蛋白（ parvalbumin）阳性中间神经元缝隙连接蛋白36（connexin 36,Cx36）的表达情况。蛋白质印迹检测纹状体及运动皮质Cx36蛋白水平的表达。结果行为学研究显示大剂量（〉60 mg/kg ）的甘珀酸腹腔注射和奎宁侧脑室注射（0.5、1.0、2.0μmol/L,〉2.5μmol/L）能减少异动症大鼠的不自主运动评分,蛋白质印迹检测显示异动症大鼠损毁侧纹状体和运动皮质Cx36表达水平分别为219.56%依18.12%、226.03%依16.33%,高于正常对照组（104.05%依3.82%,t=15.389,P〈0.01；105.27%依2.82%,t=8.074,P〈0.01）和帕金森病组（119.31%依8.92%,t=13.356,P〈0.01；138.20%依17.88%, t=5.872,P〈0.01）。免疫双标显示异动症大鼠损毁侧纹状体脑啡肽阳性神经元 Cx36表达（57.59%依5.36%）较正常对照组（32.67%依4.22%）和帕金森病组（37.24%依0.86%）增强（F=78.060,P〈0.01）,小清蛋白阳性中间神经元 Cx36表达（68.49%依11.60%）也较正常对照组（40.43%依2.30%）和帕金森病组（31.92%依5.68%）增强（F=39.567, P〈0.01）。结论左旋多巴诱发的异动症大鼠损伤侧纹状体及运动皮质的Cx36蛋白表达水平增强,纹状体脑啡肽阳性传出神经元Cx36表达水平上调,且小清蛋白阳性中间神经元Cx36表达水平增高,缝隙连接阻断剂能有效减少大鼠的异动行为,提示缝隙连接异常可能参与了异动症大鼠脑皮质-基底节环路功能紊乱的发生机制。
Objective To explore whether gap junction disturbances are involved in the pathogenesis of levodopa-induced dyskinesia （ LID ）. Methods The hemi-parkinsonian （ PD ） rat was treated intraperitoneally with L-dopa methylester （20 mg/kg） and benserazid （10 mg/kg） for 21 days and abnormal involuntary movement was evaluated to establish LID rat model. The experimental animals were divided into three groups： LID group, PD group and normal control group, respectively. The behavior responses of intraperitoneal injection of different doses of carbenoxolon and intracerebroventricular injection of quinine were observed to estimate the effects of gap junctional blockade on the abnormal involuntary movement （ AIM ） in the rat model of LID. Double immunofluorescence labeling was used to analyze the expression of connexin 36 （ Cx36 ） in enkephalin positive medium spiny neurons and parvalbumin （ PV ） positive interneurons in the striatum. Western blottings was used to observe the expression of Cx36 in the striatum and moter cortex. Results Behavioral characteristics indicated that high dose of carbenoxolone （ 〉60 mg/kg） intraperitoneal injection and intracerebroventricular injection of quinine （ 0.5, 1.0, 2.0 μmol/L, 〉 2.5 μmol/L ） could decrease the AIM score of LID rats. Western blotting indicated that expression of Cx36 in lesioned striatum and motor cortex of LID rat model was 219.56% ±18.12% and 226.03% ±16.33%, respectively, which induced a significant upregulation in comparison with the normal control group （104.05% ±3.82%, t=15.389, P〈0.01;105.27% ±2.82%,t=8.074, P〈0.01） and untreated PD group （119.31% ±8.92%, t=13.356, P〈0.01; 138.20% ±17.88%, t=5.872, P〈0.01）. Double immunofluorescence labeling staining revealed that Cx36 expression was increased in Enk-positive striatum neurons in LID model （ 57.59% ±5.36%） compared with that in normal control group （32.67% ±4.22%） and PD group （37.24% ±0.86%, F=78.060, P〈0.01）. The expression of Cx36 in PV-positive interneurons was also elevated in LID group （68.49% ±11.60%） in comparison with normal control group （ 40.43% ± 2.30%） and PD group （ 31.92% ± 5.68%, F = 39.567, P 〈 0.01 ）.Conclusions The Cx36 expression is generally increased in lesioned striatum and motor cortex of LID rat model. In the striatum, the up-regulation of Cx36 is specifically observed in Enk-positive striatum neurons and in PV-positive interneurons. The dyskinesia behavior of LID rats can be significantly reduced by treatment with gap junction blockade. All these results suggest that gap junction dysfunction may play an important role in the pathogenesis of LID.
出处
《中华神经科杂志》
CAS
CSCD
北大核心
2014年第6期375-381,共7页
Chinese Journal of Neurology
基金
安徽省科技厅年度科研计划项目（10021303036）
