胶质细胞系源性神经营养因子保护帕金森模型大鼠黑质多巴胺能神经元机制的研究

The protective mechanism of GDNF on dopaminergic neurons in substantia nigra of Parkinson disease rats

目的探讨胶质细胞系源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)在保护黑质多巴胺(dopamine,DA)能神经元过程中,钙结合蛋白D28K(calbindin 28K,CB)和星形胶质细胞的可能作用。方法采用大鼠右侧纹状体内注射6-羟多巴胺(6-OHDA)制备帕金森病(Parkinson disease,PD)模型。注射后第7天,根据行为学分析筛选模型鼠,将模型鼠随机分为3组:空白对照组,PBS组(右侧黑质注射PBS)和GDNF组(右侧黑质注射GDNF)。空白对照组动物分别于6-OHDA注射后第7天1、4天2、1天和28天时,PBS组和GDNF组动物分别于6-OHDA注射后第14天、21天和28天时,行黑质节段连续冠状石蜡切片,以酪氨酸羟化酶(tyrosinehydroxylase,TH)、CB和胶质原纤维酸性蛋白(glial fibrous acid protein,GFAP)免疫组织化学染色,分别显示DA能神经元、含CB神经元和星形胶质细胞,光镜观察以及细胞计数,统计学处理。结果①TH免疫组织化学染色结果:6-OHDA注射后第28天,GDNF组大鼠右侧黑质TH表达阳性神经元数量显著多于空白对照组和PBS组;②CB免疫组织化学染色结果:6-OHDA注射后第7天,空白对照组大鼠右侧黑质尚可观察到CB表达阳性神经元,之后的时间点空白对照组和PBS组均观察不到,而GDNF组大鼠右册黑质处在所检测的各时间点均可观察到CB表达阳性神经元;③GFAP免疫组织化学染色结果:空白对照组在6-OHDA注射后第7天时有少量的GFAP表达阳性细胞,于注射后第14天时GFAP表达阳性细胞数达高峰,之后逐渐减少,注射后第28天时已基本检测不到;PBS组GFAP表达阳性细胞数量与空白对照组的表现一致,而GDNF组大鼠黑质在所检测的各时间点均可观察到大量反应性增生的星形胶质细胞。结论CB和(或)星形胶质细胞可能参与GDNF保护并促进PD模型大鼠黑质DA能神经元存活的过程。

Objective To explore the possible roles of astrocytes and calbindin D28K （CB） in the process for glial cell line - derived neurotrophic factor （GDNF） to protect dopaminergic neurons （DA neurons） in rat substantia nigra （SN） against 6 - hydroxydopamine （ 6- OHDA） neurotoxicity. Methods Rat model of Parkinson disease （PD） was established by stereotaxlcal injection of 6- OHDA into the right striatum, with the qualified models screened by passing the behavioral tests. The model rats were randomly divided into three groups： PD control, PBS treatment control and GDNF treatment groups, to undergo varied treatments. The animals were decapitated 1 - 4 weeks after 6 - OHDA injection to proceed with the histologic study on the continuous coronal sections of the rat midbrain by means of immunohistochemistry using tha antibodies against tyrosine hydroxylase （TH）, CB and glial fibrillary acidic protein （GFAP） to detect the DA neurons, CB - containing neurons and astrocytes respectively. Results （ 1 ） The density of TH positive cells was significantly higher in the GDNF treatment group than that in the control groups. （2） The CB positive neurons could only be seen on the 7th day after 6 - OHDA injection in the control groups, but they persisted in the GDNF treatment group through the whole 28 - day course of experiment. （3） In the two control groups, the GFAP positive cells emerged on the 7th day after 6 - OHDA injection, reached the climax staining and desity on the 14th day, began to decline on the 21th day and almost disapeared on the 28th day. However, in the GDNF treatment group, the phenomenon of activation of astrocytes could be evidenced throughout the experimental course. Conclusion CB and/or astrocytes may be involved in the mechanism for GDNF to protect DA neurons against 6- OHDA neurotoxicity.