| Objective: Parkinson's disease (PD) is a major neurodegenerative disease in elderly people, The exact pathogenesis of this disease is still unknown, and therapeutic mechanism is not perfect. Traditional drug therapy and surgery only could improve the symptom partly, but the long-term effect is not very optimistic. Therefore, at present, it has become a hot research in neuroscience. Promotion of eNSCs cells has become a potential treatment which be helpful of the restoration of the nervous system. SVZ area is an important neurogenesis area, the ability of its neurogenesis could keep for life. eNSCs are usually in a resting state, activating in the condition, eNSCs could proliferate in a short period when the nervous system was damaged. In this time window, the multiplication, the migration, processes and differentiation of eNSCs were regulated, It may be an effective way to protect nervous tissue. eNSCs under the pathological state, for example disease or the damage, the number of eNSCs increase suddenly, these proliferating cell tends to repair the nervous tissue injuried in disease state. While it cannot prevent disease from progress or contribute disease to recover. Therefore recently the people are looking for methods of promoting eNSCs proliferation. rTMS is a indolence treatment method by stimulating the cerebral cortex neuron. Neuron's activity can be adjusted partial widely separated by given pair or a sequence pulse. In recent years rTMS has already been a potential therapy to psychology disease such as depression, PD, ataxia and so on. It is demonstrated that high frequency (>1 Hz) and the low frequency(<1 Hz) rTMS anti-depression effect is better, the clinical practice shows that low frequency stimulation is safe, however treatment mechanism of rTMS was still unclear. In addition, some experiments indicated that rTMS may promote the SGZ area eNSCs proliferation and the nerve function recovery in cerebral ischemia rat models. The most experiments indicated that the low frequency electromagnetic field will promote neural cell proliferation in vivo and in vitro. The objective of the research is to observe 1 Hz rTMS whether can induce SVZ area eNSCs proliferation in the acute MPTP-PD mouse model and whether MPTP injection and rTMS for 1 day could impair the emotion.Methods:①Animals: Adult healthy male C57BL/6J mice of closed group and clean grade and neonatal C57BL/6J mice were purchased from Laboratory Animal Center of Hebei Medical University and peace. The treatment of animals in the experimental process was in accordance with the criteria of Animal Ethics.②Methods: 72 male C57BL/6J mice were randomly divided into four experimental groups: normal saline group (NS), PD model group (PD), sham-rTMS group (s-rTMS) and rTMS group (rTMS), 18 mice every group. The mice in PD,s-rTMS and rTMS groups received four MPTP (15 mg/kg, s.c., dissolved in 0.9% saline) injection at 2 h intervals for 1 day to establish acute PD model mice. The mice in NS group were injected the same volume saline instead of MPTP at same time point. And twenty-four hours after the last injection of MPTP, the mice in rTMS group received 5 trains of 1pulses/s for 25 s, at an intensity of 1 Tesla(T) daily for 1,3,7 consecutive days. Sham control mice were exposed to the same noise during the stimulation. No any treatment was performed in the mice of NS and PD group. in the behavioral and EPM testing.③Experiment evaluation: the rTMS intervention ago and intervened latter to carry on the EPM experiment, to examines if MPTP treatment and rTMS result the acute PD model have the mood change. The immunohistochemical straining of coronal paraffin section of SVZ area, examines the nestin-psitive cells.Results: 1,The general performance of mice: Shortly after the first injection of MPTP, mice showed piloerection, bradykinesia, stroub tail reaction , instability of gait, tremor and the toes of the hind feet were widely separated. The symptoms remained for 5 to 6 h. The same symptoms were observed after the subsequent injections,and the symptoms of bradykinesia, stiffness and instability of gait became more obvious. However, all symptoms recovered after 24 h after injection. No abnormality was found in control group. No dysfunction was observed in the stimulated animals during or after rTMS. 2,Elevated plus-maze:①The comparison of EPM testing at the different time points in each group. After intervention and intervention on the 1st day and the 2nd day, There were no statistical significance about percentage of opening arm time acout for total time on various time among NS group, PD group, s-rTMS group and rTMS group (P>0.05).②The comparison of EPM testing among each group. There were no statistical significance about percentage of opening arm time account for total time around the intervention among groups(P>0.05).③Although there was no statistical significance, after intervening the mice about percentage of opening arm time account for total time shows the tendency of decrease. 3,The results of nestin immunohistochemical staining:①The comparion of NS group and PD group: the number of nestin-positive cells of NS Group on 1st, 3rd, 7th day were (24.33±5.57), (22.33±4.50), (23.00±5.40) , the number of nestin-positive cells of PD Group on 1st, 3rd, 7th day were (25.00±3.90), (50.50±5.13), (52.67±7.45). There was no statistical significance in the number of nestin-positive cells between NS group and PD group in 1st day (P>0.05), while there was statistical significance in between on the 3rd day and 7th day (P<0.05).②The comparion of PD group and s-rTMS group: the number of nestin-positive cells of s-rTMS group on 1st, 3rd, 7th day were were (29.00±2.83), (47.83±4.02), (53.00±4.98), There was no statistical significance in the number of nestin-positive cells between PD group and s-rTMS group in 1st , 3rd, 7th day (P>0.05).③The comparion of s-rTMS group and the rTMS group: the number of nestin-positive cells of rTMS group on 1st, 3rd, 7th day were (51.50±9.40), (64.33±9.07), (100.50±13.94), There was statistical significance in the number of nestin-positive cells between PD group and s-rTMS group in 1st , 3rd, 7th day (P<0.01).④The comparion of PD group and rTMS group: There was statistical significance in the number of nestin-positive cells between PD group and rTMS group in 1st , 7th day (P<0.01), while there was no statistical significance in between on the 3rd day (P<0.05).⑤The comparion of PD group, s-rTMS group and rTMS group of nestin positive cells at different time points were also statistically significant (P<0.05). The proliferation of eNSCs was time dependent.⑥eNSCs proliferate along certain way toward the outside migrates gradually, some cells were able to migrate to the cerebral cortex on the 7th day.Conclusion:①MPTP treatment could inducethe eNSCs proliferation in acute PD model mouse.②rTMS can promote the eNSCs proliferation.③The proliferation of eNSCs was time dependent. eNSCs begin to proliferate on the 1st day, proliferation of eNSCs is obvious on the 3rd day, proliferation of eNSCs achieves the peak on the 7th day. eNSCs proliferate along certain way toward the outside migrates gradually, some cells were able to migrate to the cerebral cortex on the 7th day.⑤EPM experiment indicated that MPTP treatment and rTMS result the acute PD model have no the mood change.
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