PD模型大鼠纹状体中等多棘神经元树突棘运动依赖可塑性研究
Study on the Motor-dependent Plasticity of Dendritic Spines of Striatal Medium Spiny Neurons in PD Model Rats
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摘要: 目的:揭示帕金森病(Parkinson’s disease,PD)模型大鼠纹状体中等多棘神经元(medium spiny neurons,MSNs)树突棘运动依赖可塑性发生的细胞靶点。方法:清洁级SD大鼠随机分为3组:假手术组(Con组)、PD组和PD运动组(PD+Ex组),采用神经毒素6-羟基多巴胺(6-Hydroxydopamine,6-OHDA)注射于大鼠右脑内侧前脑束(me-dial forebrain bundle,MFB)建立偏侧损毁PD模型大鼠,假手术组于相同部位给予同等剂量的生理盐水作为对照组。阿扑吗啡(apomorphine,APO)诱导旋转行为测试并结合黑质和纹状体酪氨酸羟化酶(tyrosine hydroxxylase,TH)免疫组织化学染色评价PD模型的可靠性。PD+Ex组于手术后1周开始进行跑台训练干预(11 m/min,30 min/d,5 d/w,共4周)。采用爬杆实验评价模型大鼠的四肢协调能力;采用逆行神经示踪结合荧光素标记方法区分D1-MSNs和D2-MSNs;采用免疫印迹技术检测纹状体突触连接蛋白突触后致密物-95(postsynaptic density-95,PSD-95)和突触素(synaptophysin,Syn)的表达水平。结果:APO诱导的旋转行为测试和TH免疫组织化学检测结果表明,PD大鼠模型可靠,成模率为75%。爬杆实验结果表明,与Con组相比,PD组大鼠爬杆延迟时间显著延长(P<0.01);与PD组相比,PD+Ex组大鼠爬杆延迟时间显著缩短(P<0.05)。逆行神经示踪结合荧光素标记结果表明,与Con组相比,PD组和PD+Ex组大鼠D1-MSNs树突棘密度无显著改变(P>0.05);PD组大鼠D2-MSNs树突棘密度显著降低(P<0.01);与PD组相比,PD+Ex组大鼠D2-MSNs树突棘密度显著增加(P<0.05)。免疫印迹结果表明,与Con组相比,PD组大鼠纹状体PSD-95、Syn表达水平显著下调(P<0.01);与PD组相比,PD+Ex组大鼠纹状体PSD-95、Syn表达水平显著上调(P<0.05)。结论:PD模型大鼠纹状体D2-MSNs树突棘丢失,跑台运动干预可选择性降低PD模型大鼠纹状体D2-MSNs树突棘丢失。运动通过上调突触连接蛋白表达促进PD模型大鼠纹状体MSNs形态结构重塑。纹状体MSNs树突棘发生运动依赖可塑性变化为PD模型大鼠运动功能障碍的改善提供了必要的解剖学基础。Abstract: Objective:To reveal the target cells of striatum MSNs plasticity depending on exercise in PD model rats.MethodsHeanth adult SD rats were randomly divided into three groups:sham operation group (Con),PD group (PD) and PD with exercise group(PD+Ex).6-Hydroxydopamine (6-OHDA) was injected into the medial forebrain bundle (MFB) of the right brain to induce PD model of unilateral injury in rats.The sham-operation group was received the same dose of saline as the control group.The reliability of the model was evaluated by apomorphine (APO)-induced rotational behavior test combined with immunohistochemica staining of the substantial nigra and tyrosine 3-monooxygenase striatum.In the exercise group,treadmill training (11 m/min,30 min day,5days/week,4weeks) was performed at 1 week after operation.Free locomotor activity was evaluated by open field test,D1-MNs and D2-MSNs of striatum were labeled by retrograde neural tracing and fluorescein labeling the expression of streptolysin(Syn) and streptolysin (PSD-95) in striatum were detected by Western Blotting.Results:The results of APO-induced rotation behavior test and TH immunohistochemical test showed that PD model rats were reliable and the molding rate was 75%.In the pole test,the delay time of climbing from pole top to bottom was longer in PD group compared with control group (P<0.01);compared with PD group,the delay time of climbing from pole top to bottom was decreased significantly in PD+Ex group (P<0.05).The results of retrograde nerve tracing combined with fluorescein labeling showed that the density of dendritic spines in D1-MSNs was not changed obviously in PD and PD+Ex group than that of control group,but the density of dendritic spines in D2-MSNs of PD group was decreased compared with control group (P<0.01);in addition,the density of dendritic spines in D2-MSNs of PD+Ex group was increased compared with PD group (P<0.05).Western Blotting results showed that the expression level of PSD-95 and Syn protein in striatum of PD group were decreased compared with control group (P<0.01).The expression level of PSD-95 and Syn protein of PD+Ex group were increased compared with PD group (P<0.05).Conclusion:The loss of D2-MSNs dendritic spine in striatum of PD model rats were selectively alleviated by treadmill exercise intervention.Exercise promotes the morphological and structural remodeling of MSNs in the striatum of PD model rats by up-regulating the expression of synaptophysin.The movementdependent plasticity of striatal MSNs dendritic spines provides a necessary anatomical basis for the improvement of motor dysfunction in PD model rats.
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