纹状体注射不同剂量6-OHDA制备帕金森病小鼠模型的评价

Establishment and evaluation of the striatal 6-OHDA lesioned mice model of Parkinson's disease

目的纹状体内注射6-OHDA可用于制备帕金森病(Parkinson’s disease,PD)模型,其在小鼠模型上的行为学、形态学及多巴胺水平的变化需要进行系统评价。方法在小鼠右侧纹状体背外侧部立体定位分别注入4、6、8及10μg的6羟基多巴胺(6-hydroxydopamine,6-OHDA),动态观察各剂量组小鼠的基本生理状况。旋转行为和转棒行为可评价PD小鼠的运动症状;酪氨酸羟化酶(tyrosine hydroxylase,TH)免疫组化染色可反映黑质-纹状体内多巴胺能系统的功能改变;高效液相色谱法能检测多巴胺(dopamine,DA)及其代谢产物的含量并分析DA代谢率。结果除了10μg 6-OHDA组的小鼠体质量呈一过性下降,其他剂量对小鼠体质量无显著影响。在6-OHDA注射一周后,各组小鼠均出现了持续性的异常旋转行为增加。此外,高剂量组(8μg及10μg)小鼠的转棒时间较低剂量组(4μg及6μg)小鼠明显缩短。TH组织化学染色显示,低剂量组小鼠损伤侧丢失了约60%的多巴胺神经元及40%的多巴胺神经纤维;而高剂量组小鼠则有超过80%的多巴胺神经元丢失和70%的多巴胺神经纤维减少。各组小鼠损伤侧纹状体内的DA水平及其代谢产物含量也显著降低,与6-OHDA注射量具有剂量依赖性;而DA代谢率却出现了代偿性增加改变。结论纹状体内注射不同剂量6-OHDA均能引起一定程度的PD病理表型,其损伤程度与6-OHDA剂量相关,为研究PD不同病理阶段及其干预策略提供了新的模型。

Objective To establish different 6-hydroxydopamine （ 6-OHDA ） lesioned mice model, we evaluated the behavioral, neurochemical and histological effect of different dosage injection of 6-OHDA on motor dysfunction and dopamine denervation. Methods Different doses of 6-OHDA（4 μg, 6 μg, 8 μg, 10 μg） were injected into the right dorsal striatum under the facilitation of stereotactic equipment. The body weight after 6-OHDA injection was observed. Apomorphine-induced rotation and rota-rod test were used to estimate motor impairment. Tyrosine hydroxylase（TH） immunostaining was performed to determine the dopaminergic cell loss. Dopamine（DA） content was determined by HPLC. Results 10 μg 6-OHDA injection induced a temporary weight loss, while as other dose of 6-OHDA had no obvious effect on the body weight of mice. At the 1st week after the injection of 6-OHDA, the positive apomorphine induced rotation was observed and this rotational performance was stable for at least 5 weeks. The mice of 8 μg and 10 μg 6-OHDA injection decreased the latency time on the rota-rod. TH-immunolabeling indicated that the 4 μg and 6 μg 6-OHDA injections reduced the number of 60% DA neurons and 40% fibers, as well as 8 μg and 10 μg 6-OHDA injections reduced the number of 80% DA neurons and 70%fibers. Besides, 6-OHDA injection leads to the significant decrease of striatal DA and its metabolites. The decreased of DA content showed a positive correlation with the injection dosage of 6-OHDA, and a negative correlation with dopamine availability（ DOPAC＋HVA/DA） . Conclusion 4 μg and 6 μg 6-OHDA injection induced partial lesions of the nigrostriatal DA system to mimic the early phases of Parkinson’ s disease（ PD） . 8 μg and 10 μg 6-OHDA injection induced completely lesions of the nigrostriatal DA system to mimic the late phases of PD.