氟哌啶醇对斑马鱼胚胎的神经毒性作用

Neurotoxicity of haloperidol on zebrafish embryos

目的探讨用斑马鱼制备帕金森病(PD)动物模型的可能性。方法每组500枚1 hpf受精卵(培养1 h的受精卵)分别加入10 ml氟哌啶醇(Hal)0.27,0.54,1.08,2.16和4.32μmo.lL-1,于3,12,24,48,72,96和120 h显微镜下观察胚胎发育,统计胚胎的孵化率、畸变率和死亡率;免疫组织化学染色法观察96 hpf幼斑马鱼脑部多巴胺(DA)能神经元面积。孵育5 d后,分别将Hal 0.54和1.08μmo.lL-1组的一半幼鱼转移至左旋多巴20 mmo.lL-1中孵育2 d,记录5 min幼鱼游泳速度。结果与正常对照组相比,Hal 0.54,1.08,2.16和4.32μmol.L-1孵育120 h,斑马鱼胚胎的孵化率明显降低〔(81±4)%,(56±5)%,(31±4)%和(8±3)%vs(94±2)%〕(P<0.01),胚胎发育迟缓。孵育96 h时斑马鱼胚胎LC50为1.31μmo.lL-1。免疫组织化学染色结果显示,与正常对照组相比,Hal 0.54,1.08和2.16μmol.L-1组幼鱼DA能神经元面积分别从(10 460±734)μm2减少至7237±1054,5044±389和(3342±320)μm2(P<0.01);Hal 0.54,1.08和2.16μmol.L-1组7 dpf幼鱼游泳速度从(1.94±0.21)mm.s-1下降至0.96±0.17,0.64±0.16和(0.38±0.15)mm.s-1(P<0.01),并出现僵直和侧翻等PD症状,转移至左旋多巴20 mmol.L-1中2 d后,Hal 0.54和1.08μmol.L-1组游泳速度恢复至1.56±0.31和(1.23±0.29)mm.s-1(P<0.01)。结论 Hal使斑马鱼产生类似哺乳动物的PD行为表现,左旋多巴能够改善PD样行为表现,说明斑马鱼有可能用来制备PD实验模型。

OBJECTIVE To investigate the possibility of generating a Parkinson disease（PD） model using zebrafish.METHODS After 10 ml haloperidol（Hal） 0.27,0.54,1.08,2.16 and 4.32 μmol·L-1 was respectively added to the solution that contained 500 1 hpf zebrafish embryos in each group,the morphologic changes of embryonic development of zebrafish in 3,12,24,48,72,96 and 120 hpf were observed under a microscope.The incubation rate,aberration rate and death rate were also calculated;meanwhile,the changes in the dopaminergic neuron area in 96 hpf larval zebrafish′s brain was observed using immunostaining method.Five days later,half of the larval zerbrafish in Hal of 0.54 and 1.08 μmol·L-1 groups were transferred respectively into L-dopa 20 mm·L-1 solution for 2 d,and their locomotor activity was recorded for 5 min.RESULTS Compared with normal control,after incubation in Hal of 0.54,1.08,2.16 and 4.32 μmol·L-1 for 120 h,the zerbafish showed developmental delay,and the incubation rate was significantly decreased（（94±2）%,（81±4）%,（56±5）%,（31±4）%,（8±3）%）（P0.01）;the 96 h LC50 of zebrafish embryos was 1.31 μmol·L-1;results of immunostaining showed that after treatment with Hal of 0.54,1.08 and 2.16 μmol·L-1,the dopaminergic neuron area in the zerbrafish＇s brain was reduced to 7237±1054,5043±389 and（3342±320）μm2（P0.01）,respectively.The swimming speed of larval zebrafish in Hal of 0.54,1.08 and 2.16 μmol·L-1 groups dropped to 0.96±0.17,0.64±0.16 and（0.38±0.15）mm·s-1（P0.01）,respectively.The larval zebrafish showed rigidity and inclination symptoms that were similar to those of PD;but after they were transferred to L-dopa 20 mmol·L-1 for 2 d,the swimming speed of larval zebrafish in Hal of 0.54 and 1.08 μmol·L-1 groups rose to 1.56±0.31 and（1.23±0.29）mm·s-1（P0.01）.CONCLUSIONThe behavior of zebrafish irritated by Hal is similar to that of PD disease,but can be improved by L-dopa.Zebrafish can be used to establish a PD model.