Objective: To evaluate the role of thrombin-activated microglia in the neurodegeneration of nigral dopaminergic neurons in the rat substantia nigra (SN) in vivo.
Methods: After stereotaxic thrombin injection into unilateral SN of rats, immunostaining, reverse transcription polymerase chain reaction (RT-PCR) and biochemical methods were used to observe tyrosine hydroxylase (TH) immunoreactive positive cells, microglia activation, nitric oxide (NO) amount and inducible nitric-oxide synthase (iNOS) expression.
Results: (1) Selective damage to dopaminergic neurons was produced after thrombin injection, which was evidenced by loss of TH immunostaining in time-dependent manner; (2) Strong microglial activation was observed in the SN; (3) RT-PCR demonstrated the early and transient expression of neurotoxic factors iNOS mRNA in the SN. Immunofluorescence results found that thrombin induced expression of iNOS in microglia. The NO production in the thrombin-injected rats was significantly higher than that of controls (P < 0.05).
Conclusion: Thrombin intranigral injection can injure the dopaminergic neurons in the SN. Thrombin-induced microglia activation precedes dopaminergic neuron degeneration, which suggest that activation of microglia and release of NO may play important roles in dopaminergic neuronal death in the SN.
目的: 探讨凝血酶 (Thrombin) 诱导小胶质细胞 (Micoglia) 激活与黑质多巴胺能神经元变性的关系.
方法: 采用立体定向术注射凝血酶至大鼠黑质,在不同时间点观察酪氨酸羟化酶 (tyrosine hydroxylase, TH) 神经元的表达及小胶质细胞的激活情况; 同时检测黑质NO量及iNOS mRNA表达.
结果: (1) 凝血酶注入大鼠黑质导致明显的黑质多巴胺能神经元变性,呈时间依赖性,TH 阳性细胞数在第3 d 开始下降,第7 d 有大量的TH 阳性细胞丢失,与对照侧相比下降达约53% (P < 0.01); 高倍镜下可见胞体皱缩、突起明显缩短或减少; 14 d 时细胞数下降至21%,30 d 时下降至12% (P < 0.01). (2) 凝血酶注射入黑质4 h 后小胶质细胞开始呈现为“灌木丛样”或少量呈现“阿米巴样”;12 h 后小胶质细胞数目明显增加且绝大部分呈现“阿米巴样”; 24 h 后细胞已完全激活, “阿米巴样”细胞达高峰; 3 d 维持高峰; 14 d 后小胶质细胞染色变淡, 体积变小, “阿米巴样”细胞数目下降. (3) 与对照组相比,iNOS mRNA表达明显上调及NO合成增加 (P < 0.05),并且有iNOS在小胶质细胞表达.
结论: 凝血酶对多巴胺能神经元具有一定的损毁作用,小胶质细胞的激活先于多巴胺能神经元变性,其激活后释放的NO有可能参与多巴胺能神经元变性.