鱼藤酮对体外培养的大鼠中脑脑片多巴胺能神经元的毒性作用

NEUROTOXICITY OF ROTENONE ON DOPAMINERGIC NEURONS IN CULTURED SLICES OF THE MIDBRAIN IN THE RAT

目的研究鱼藤酮(rotenone)对多巴胺(DA)能神经元的早期毒性作用,并探索一种较理想的组织模型。方法采用界面组织培养法建立SD乳鼠的中脑脑片长期培养体系。加入鱼藤酮作用一定的时间,并用测定培养基中乳酸脱氢酶(LDH)活力、组织中DA含量以及进行酪氨酸羟化酶(TH)染色等技术观察它对整个脑片及脑片上DA能神经元的毒性效应。结果不同浓度的鱼藤酮作用24 h后,组织中DA含量随浓度增加明显下降,TH阳性神经元突起呈串珠样改变,数量减少甚至消失。低浓度鱼藤酮作用14 d后,脑片组织中的DA水平显著降低,但未见明显的DA能神经元形态学变化。结论建立了长期、稳定的中脑脑片培养体系;鱼藤酮对整个脑片以及脑片上DA能神经元的毒性作用具有浓度依赖性;功能性损伤早于形态学变化,突起的变性是形态学变化的早期特征。

Objective To investigate the early neurotoxicity of rotenone on dopaminergic neurons and explore an ideal tissue model. Methods A long-term midbrain slice cuhure system of SD pup was established according to the interface tissue culture method. After rotenone was added for some time, its toxic effects on the whole slices and the dopaminergic neurons were identified through the measurements of lactate dehydrogenase （LDH） released into the medium from the slices and dopamine （DA） content from the cultured tissue, as well as the observations of immunohistochemistry for tyrosine hydroxylase （TH）. Results In those cultures exposed to rotenone for 24 h, the level of DA in tissue dramatically decreased with the concentrations rising. The processes of TH-positive neurons in slices demonstrated some morphological changes, such as appearance of string of beads, reduce of numbers and even disappearance. The content of dopamine in tissue was dominantly decreased with 5 nmol/L rotenone for 14 days, although its cellular morphology was not seen to change. Conclusion Long-time stable midbrain slice culture system has been set up successfully. The neurotoxicity of rotenone on the whole slices and dopaminergic neurons shows a dose-dependent manner. The functional damages on the neurons may be earlier than their morphological changes, of which the injury in the processes of neurons seems to be an early characteristic.