| Parkinson’s disease(PD)is the secondly most common neurodegenerative disease in the elderly Alzheimer’s disease.The main pathological feature is the degeneration of dopaminergic neurons in substantia nigra pars compacta(SNpc)and the formation of Lewy bodies(LBs).The main clinical manifestations are rigidity,resting tremor,postural instability,and bradykinesia.At present,it was generally accepted that oxidative stress,mitochondrial dysfunction,apoptosis,and inflammation might be involved in the pathogenesis of PD,but the exact mechanisms are still not fully understood.Alpha-synuclein,encoded by the SNCA gene,is the major component of the characteristic marker LBs of PD.The results of brain autopsy showed that LBs were deposited in SNpc of PD patients.Abnormal aggregation of α-synuclein was considered to be the core mechanism of PD in sporadic or genetic PD.It was reported that α-synuclein expression in cerebrospinal fluid(CSF)of PD patients decreased at the pre-disease,but gradually increased with the progression of PD.Another study found that in some neurodegenerative diseases with obvious cognitive impairment,the expression of α-synuclein in CSF was significantly increased,and the close relationship between the expression of α-synuclein in CSF and the severity of PD development was observed.The level of α-synuclein in CSF can reflect not only the disease itself,but also the severity of the disease,however,the effects of intracerebroventricular injection of different concentrations of α-synuclein on the nigrostriatal system have rarely been reported.The aim of this study was to investigate the effects of intracerebroventricular injection of different concentrations of α-synuclein on the nigrostriatal system of normal and PD model mice and its possible mechanisms.In this experiment,C57BL/6 mice were used as research objects to research the effects of intracerebroventricular injection of different concentrations of α-synuclein on DA neurons in the substantia nigra and striatum regions by preparing chronic and subacute PD mice models.By behavioral tests,western blots,High Performance Liquid Chromatography and other research methods were applied to explore possible mechanisms.The results are as follows: 1.ELISA was applied to detect the concentration of α-synuclein in CSF.The results showed that α-synuclein in CSF in the control group and MPTP group was undetectable,and did not reach the detection limits in mice with 0.2ng or 2ng α-synuclein injection into the lateral ventricle.After dilution of α-synuclein with freshly extracted CSF,the results showed that the concentration in the 0.2ng group was approximately 110.8 ± 7.612 pg/ml,and the concentration in the 2ng group was approximately 579.0 ± 15.93 pg/ml.The results suggest that α-synuclein concentration in CSF did not directly increase with α-synuclein injection into CSF for 7 consecutive days.2.Injection of 0.2ng or 2ng α-synuclein into the lateral ventricle does not affect motor coordination in normal mice.Rotarod test showed that the movement time on the rotarod did not significantly change in the 0.2ng and 2ng α-synuclein groups.The results of the pole test showed that the time taken to crawl freely from the top of the wood to the bottom did not significantly change in the 0.2ng and 2ng α-synuclein groups.The results of open field test showed that the average rate of spontaneous activity of the 0.2ng and 2ng α-synuclein mice in the open box for a period of time did not significantly change.3.DA contents in the striatum of 0.2ng and 2ng α-synuclein groups decreased by 33.3 % and 54.9 %,respectively,compared to the control group(P<0.01;P<0.001);DOPAC contents in the striatum of the 0.2ng and 2ng α-synuclein groups did not significantly change.HVA contents increased by 57.2% and 57.4%,respectively,compared to the control group(P<0.05;P<0.05);DOPAC/DA ratio of 0.2ng and 2ng α-synuclein mice increased by 85.1% and 149.8%,respectively,compared to the control group(P<0.05;P<0.05);HVA/DA ratio increased by 157.0% and 212.4%,respectively,compared to the control group(P<0.05;P<0.05).4.There was no significant difference in the expression of TH protein in the SN and striatum of mice with saline,0.2ng or 2ng α-synuclein injection.5.Injection of 0.2ng or 2ng α-synuclein into the lateral ventricle does not affect motor coordination in MPTP-induced subacute PD mice.Rotarod test showed that the movement time on the rotarod decreased by 29.8%,32.8% and 34.3%,respectively,compared to the control group,(P<0.05;P<0.05;P<0.01)in the MPTP+ saline,MPTP + 0.2ng and MPTP + 2ng α-synuclein group.The results of the pole test showed that the time taken to crawl freely from the top of the wood to the bottom increased by 94.0%,115.8% and 97.2%,respectively,compared to the control group.(P<0.001;P<0.001;P<0.001)The results of open field test showed that the average rate of spontaneous activity in the open box for a period of time did not significantly change.There was no significant difference in the above behavioral tests in MPTP-induced subacute PD mice when compared with the MPTP group,suggesting that the injection with 0.2ng or 2ng α-synuclein does not cause further decline in autonomic motor coordination in MPTP-induced subacute PD mice.6.Injection of 0.2ng or 2ng α-synuclein into the lateral ventricle may affect motor coordination in MPTP-induced chronic PD mice.Rotarod test showed that the movement time on the rotarod decreased by 35.8%,65.7% and 84.8%,respectively,compared to the control group(P<0.05;P<0.001;P<0.001)in the MPTP + saline group,MPTP + 0.2ng group and MPTP + 2ng α-synuclein group.The results of the pole test showed that the time taken to crawl freely from the top of the wood to the bottom increased by 135.76%,307.05% and 372.85% in the MPTP group,the MPTP + 0.2ng group and the MPTP + 2ng α-synuclein group.(P<0.01;P<0.001;P<0.001)The results of open field test showed that the average rate of spontaneous activity of the mice in the open box for a period of time decreased by 38.8% and 69.7% in the MPTP + saline,MPTP + 0.2ng and MPTP + 2ng α-synuclein mice group.(P<0.05;P<0.001)Rotarod test showed that the movement time on the rotarod decreased by 76.3%,compared with the MPTP group(P<0.05)in the MPTP + 2ng α-synuclein group.The results of the pole test showed that the time taken to crawl freely from the top of the wood to the bottom increased by 72.7% and 100.56%,respectively,compared with the MPTP group(P<0.01;P<0.001)in the MPTP + 0.2ng and the MPTP + 2ng α-synuclein groups.The results of open field test showed that the average rate of spontaneous activity of the mice in the open box for a period of time decreased by 36.1% and 68.4% in the MPTP + 0.2ng and MPTP + 2ng α-synuclein groups.(P<0.05;P<0.001)These results suggest that injection of 0.2ng α-synuclein and 2ng α-synuclein into the lateral ventricle resulted in the further decline of the coordination in MPTP-induced chronic PD mice.7.DA contents in the striatum of MPTP + saline,MPTP + 0.2ng and MPTP + 2ng α-synuclein groups decreased by 75.1%,84.6% and 89.8%,respectively,compared to the control group(P<0.001;P<0.001;P<0.001);DOPAC contents in the striatum of decreased by 81.2%,86.4% and 89.4%,respectively,compared to the control group(P<0.001;P<0.001;P<0.001);HVA contents decreased by 56.2%,59.8% and 68.7% respectively,compared to the control group(P<0.01;P<0.05;P<0.01)DOPAC/DA ratio and HVA/DA ratio in the striatum did not significantly change.HVA/DA ratio increased by,respectively,compared to the control group(P<0.05;P<0.05)There was no significant difference in the contents of DA and its metabolites DOPAC and HVA,however,HVA/DA ratios in the MPTP + 0.2ng and the MPTP + 2ng α-synuclein groups have significant increased trends.8.TH level in the mice striatum was significantly lower in MPTP group,MPTP + 0.2ng group and MPTP + 2ng α-synuclein group when compared to the control group(P<0.001;P<0.001;P<0.001),however,the level of TH in the striatum in the MPTP + 0.2ng and MPTP + 2ng α-synuclein groups was not significantly different from the MPTP+ saline group.9.There was no significant difference in the expression of 14-3-3 protein in the striatum of the normal mice and MPTP-induced PD mice with saline,0.2ng or 2ng α-synuclein injection.10.There was no significant difference in the expression of XBP-1s,p-eIF2α and GRP78 protein in the striatum of mice with saline,0.2ng or 2ng α-synuclein injection.The above results indicate that intracerebroventricular injection of α-synuclein monomer might affect DA metabolism in normal and MPTP-induced mice,and 14-3-3-mediated transfer and propagation of pathogenic α-synuclein and ER stress were not involved in the process.The effects of intracerebroventricular injection of different concentrations of α-synuclein on the nigrostriatal system and underlying mechanisms in this study is to further explore the α-synuclein in CSF during the pathogenesis and progression of PD,as well as new ideas and experimental evidence for PD therapeutics and interventions. |
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