Neurotoxic Effects Of Zinc On Dopaminergic Neurons In The Substantia Nigra Of Rats

Parkinson's disease(PD) is a progressive neurodegenerative disorder associated with the loss of dopaminergic neurons originating in the substantia nigra pars compacta(SNpc) and terminating in the striatum(Str).This disorder is characterized symptomatically by bradykinesia,resting tremor and rigidity.The exact pathogenesis of PD has not been revealed yet.However,lots of researches suggest that multiple factors might be involved, such as heredity,environment,oxidative stress,excitotoxin,autoimmunity,iron accumulation and cell apoptosis.Recent studies have begun to pay attention to the role of zinc that played in neurodegenerative diseases.Zinc is an important trace element in biology.It is involved in synthesis of several enzymes and transmitters in brain,and has extensive biological effect.The scarce or excessive zinc will induce disorders of corresponding biochemistry process to lead to diseases.The studies in neurochemistry, clinical medicine and epidemiology found zinc might be involved in the onset of PD.For example:Morbidity of PD was increased in the crowd with long time exposure to zinc; Zinc level was increased by 50-54%in the substantia nigra(SN) in Parkinsonian patients compared to age-matched control brain tissues.But the concrete mechanism about the damage effect of zinc on dopaminergic neurons in the SN is not clear yet,and still need to be investigated further.In order to observe whether zinc could induce the damage of dopaminergic neurons in the SN of rats and to clarify the possible mechanisms of zinc toxicity,we used adult male Wistar rats by the administration of zinc sulfate heptahydrate(ZnSO₄·7H₂O) intraperitoneally once daily for 14 days.Using ICP-2(Inductively Coupled Plasma Detector),we detected the content of zinc in the SN.Using high performance liquid chromatography-electrochemical detection(HPLC-ECD),tyrosine hydroxylase(TH) immunohistochemistry,Nissl staining and fast cyclic voltammetry(FCV),we measured the damage of dopaminergic neurons in the SN of rats.Using biochemistry methods,we investigated the changes of oxidative stress in the midbrain and caspase-3 activity in the SN of rats.Using western blots,we detected the location of cytochrome c in cytosol and mitochondria of the SN.Using double immunofluorescent staining,we obversed the apoptosis of dopaminergic neurons in the SN of rats.The results were as follows:1.In the 12.5,25 mg/kg group,there was no significant change in zinc contents compared with that of control group in the SN.However,in the 50 mg/kg group,zinc contents were found to be significantly higher than that of control and the other two groups(P＜0.01).2.In the 12.5,25 mg/kg groups,there was no significant difference in the DA and its metabolites levels compared with that of control group in the Str.However,in 50 mg/kg group,the DA and its metabolites HVA levels decreased in the Str compared with that of control and the two groups(P＜0.01).But no significant difference can be observed in the content of DOPAC.3.In the 25,50 mg/kg groups,there was no significant change in the DA release compared with that of control group.4.After Nissl staining,in the 12.5,25 mg/kg groups,there was no significant difference in the cell number compared with that of control group in the SN.But,in the 50 mg/kg group,a significant reduction in cell number in the SN was observed compared with that of control and the two groups(P＜0.01).5.In the 12.5,25 mg/kg groups,there was no significant difference in the TH-immunoreactive (TH-ir) cell number compared with that of control group in the SN. However,in the 50 mg/kg group,the cell number markedly decreased compared with that of control and the other two groups(P＜0.01).6.In the 12.5,25 mg/kg groups,there was no significant difference in the content of malondialdehyde(MDA) compared with that of control group in the midbrain. However,the content of MDA was higher in the 50 mg/kg group than that of control and the other two groups(P＜0.01).7.In the 12.5,25 mg/kg groups,there was no significant difference in the activity of superoxide dismutase(SOD) compared with that of control group in the midbrain. However,in the 50 mg/kg group,the activity of SOD was decreased compared with that of control and the other two groups(P＜0.05).8.In the 50 mg/kg group,neuronal apoptosis was found in the SN of rats by TH immunofluorescent staining and Hoechst 33258 staining.9.In the 12.5,25 mg/kg groups,there was no significant difference in the activity of caspase-3 compared with that of control group in the SN.However,in the 50 mg/kg group,the activity of caspase-3 was increased in the SN compared with that of control and the other two groups(P＜0.01).10.Low dose of zinc(12.5,25 mg/kg) did not significantly alter the level of cytosolic cytochrome c,while zinc evoked abrupt increases in cytosolic cytochrome c in the 50 mg/kg group(P＜0.01).In contrast,in the 50 mg/kg group,cytochrome c level decreased in the mitochondrial fractions(P＜0.05),which indicated that cytochrome c release from mitochondria into cytosol.In conclusion,the content of zinc was increased in the SN by intraperitoneal injection of ZnSO₄·7H₂O.High dose of zinc could induce the decrease of DA and its metabolites in the Str,but no significant change was found in DA release.Zinc also decreased the numbers of TH-ir cell in the SN.These results demonstrated that intraperitoneal injection of ZnSO₄·7H₂O could damage dopaminergic neurons in the nigrostriatal system of rats.The probable mechanism of this effect was as follows:The level of oxidative stress was elevated in the SN of rats by increasing the content of MDA and decreasing the activity of SOD.The mitochondria were damaged and cytochrome c was released into cytocol,which activated caspase-3 and apoptosis of dopaminergic neurons happened.Our findings provide experimental evidence about the possible mechanism that zinc might be involved in the etiology of PD.