腺相关病毒在大鼠丘脑纹状体通路中的应用

Application of adeno-associated virus on thalamic striatum pathway in rats

目的探讨腺相关病毒(AAV2/9-GFP)在丘脑束旁核-纹状体通路中的神经元投射及其对组织的影响.方法成年雄性Wistar大鼠136只,立体定位实验随机分为丘脑束旁核(PF)组(n=64)、背外侧纹状体(DLS)组(n=64)、PF对照组(n=4)和DLS对照组(n=4);在立体定位实验的基础上进行行为学实验,分为PF行为学组(n=12)和行为学对照组(n=4);立体定位实验结束后进行神经元计数实验,分为PF计数组(n=16)、DLS计数组(n=16)、PF计数对照组(n=4)和DLS计数对照组(n=4).将不同剂量的AAV2/9-GFP立体定位注射至PF组和DLS组,转染不同时间,分析AAV2/9-GFP在PF和DLS内的转染效率,筛选出最佳剂量和转染时间.观察PF和DLS被转染的神经元以及两核团之间的神经元投射通路.分析大鼠行为学变化以及神经元的数量和形态变化.采用SPSS Statistics 21.0软件进行统计学分析.结果随着转染剂量和时间的增加,PF光密度值增大(F剂量=55.617,P<0.001;F时间=65.288,P<0.001)、转染面积增加(F剂量=202.443,P<0.001;F时间=224.064,P<0.001),呈上升趋势.随着转染剂量和时间的增加,DLS光密度值增大(F剂量=117.062,P<0.001;F时间=155.792,P<0.001)、转染面积增加(F剂量=240.422,P<0.001;F时间=304.571,P<0.001),呈上升趋势.病毒转染PF的最佳剂量为0.4μL,最佳时间为4周;转染DLS的最佳剂量为0.6μL,最佳时间为4周.AAV2/9-GFP注射至PF时,可见投射至背外侧纹状体、运动皮层、岛叶;注射至DLS时,可见投射至运动皮层、底丘脑核、束旁核、黑质.AAV2/9-GFP对神经元有损伤,表现为神经元数量减少(P<0.05),形态不规则,胞体变大,核仁及尼氏小体染色变浅.结论丘脑束旁核(PF)与背外侧纹状体(DLS)存在着密切的纤维联系,并且与皮层、底丘脑核和黑质等联系广泛.AAV2/9-GFP是一种有效的神经示踪剂但对神经元有一定程度的损伤,提示临床应用时应谨慎.

Objective To investigate the neuronal projection of adeno-associated virus(AAV2/9-GFP)in parafascicu-lar nucleus-striatum pathway and its effect on tissues.Methods A total of 136 adult male Wistar rats were randomly divided into parafascicular nucleus(PF)group(n=64),dorsolateral striatum(DLS)group(n=64),PF control group(n=4)and DLS control group(n=4)to conduct stereotactic localization experiments.Based on stereotactic localization experiments,behavior experiments were performed in PF behavior group(n=12)and behavior control group(n=4).Af-ter stereotactic localization experiments,the neuron counting experiments were conducted in PF counting group(n=16),DLS counting group(n=16),PF counting control group(n=4)and DLS counting control group(n=4).After dif-ferent doses of AAV2/9-GFP were injected into the PF group and DLS group for different durations,the transfection ef-ficiency was analyzed,and the optimal dose and transfection period were screened.The transfected neurons and neuronal projection pathways between the two nuclei were observed.The behavioral changes of rats and number and morphology of neurons were analyzed.SPSS Statistics 21.0 software was used for statistical analysis.Results With the increase of transfection doses and time,the PF optical density value increased(Fdoses=55.617,P<0.001;Ftime=65.288,P<0.001),and the transfection area increased(Fdoses=202.443,P<0.001;Ftime=224.064,P<0.001),showing an up-ward trend.With the increase of transfection doses and time,the DLS optical density value increased(Fdoses=117.062,P<0.001;Ftime=155.792,P<0.001),and transfection area increased(Fdoses=240.422,P<0.001;Ftime=304.571,P<0.001),showing an upward trend.The optimal dose of PF for viral transfection was 0.4μL and the optimal duration was 4 weeks.The optimal dose of DLS transfection was 0.6μL and the optimal duration was 4 weeks.When AAV2/9-GFP was injected into PF,it was projected to the dorsolateral striatum,motor cortex and insular lobe.When AAV2/9-GFP was injected to DLS,it was projected to the motor cortex,hypothalamic nucleus,parafascicular nucleus and sub-stantia nigra.AAV2/9-GFP had damage to neurons,which was characterized by a decreased number of neurons(P<0.05),irregular morphology,enlarged cell body,and shallower staining of nucleoli and Nissl bodies.Conclusion There is a close neuronal projection relationship between PF,DLS,motor cortex,hypothalamic nucleus and substantia nigra.AAV2/9-GFP is an effective neural tracer and harmful to neurons,so it should be cautiously used in clinical practice.