| Objective:Parkinson's disease(PD) is a relatively common extrapyramidal diseases. The main symptom is bradykinesia, muscle rigidity, tremor and abnormal posture. With population aging, the incidence of PD is increasing.It seriously affects the life quality of patient, and is a heavy burden for the family and society. Currently, the mainly clinical treatment of PD is drug therapy and surgical treatment, however, they could not protect nerve degeneration and remodel nerve function, are not an ideal means of treating PD. In recent years, transcranial magnetic stimulation (TMS) technology become a hot research at home and abroad. It is a biological technology which with the advantages of painless, non-invasive, easy operation ,safety and so on. The first clinical reports appeared in 1985, after 20 years of development, TMS clinical application of research has been focused on the psychiatric mental illness and neurodegenerative diseases. TMS is a powerful electromagnetic field under the effect of deep brain resulting in the induction of biological currents, thus affecting the electrical activity of neuron and the corresponding partial function as a non-invasive physical methods. It has different effects on the central nervous system function by regulating the frequency, intensity, intermittent stimulation and stimulation time, resulting in therapeutic effects on the central and peripheral nervous system diseases. The previous research of laboratory confirmed that TMS can increase the expression of GAP-43 and SYN of striatum on MPTP-induced PD mice [1], which suggesting that TMS can promote nerve regeneration and synaptic remodeling .The number of cell processes and content of DA are increased when PC12 cells intervented by the low-frequency magnetic stimulation [2].Application of magnetic stimulation (MS) on primary cultured neurons, the impact of MS on the growth of neurons processes and morphology of synaptic was observed. The mechanism was observedMethods: The routine methods were used to extract hippocampus and midbrain neurons from Kunming mouse 1 day after birth. The hippocampus and midbrain neuron were planted in culture dish of 35mm and culture medium was DMEM contained 10% fetal bovine serum. The culture medium was changed to Neurobasal medium added B27 when the cells were cultured for 24h and 72h. Cells were cultured for 6 days. There are four groups: 1. control group (C). 2. sham stimulation group (S). 3. 40% of maximum intensity of stimulation (M1). 4. 60% of maximum intensity of stimulation (M2). The cells was stimulated at the same time each day. Stimulus is divided into three series .Each series has 20 sequences. Interval of each sequence is 1second. Interval of each series is 1minute,continuous stimulation 5days. Stimulation frequency of 1 Hz, stimulus intensity reference to experimental groups. Cellular immunofluorescence staining of MAP-2 was executed in the fifth day after stimulation. The growth state of cells were observed by fluorescence microscope and counted. Expression of Growth-associated protein-43 and Synapsin I mRNA were detected by using RT-RCR technology. Statistics analyses were performed by SPSS 13.0 software. Results were expressed as mean±standard deviation. Single-factor analysis of variance was used in this study.Result:1 The impact of magnetic stimulation on primary neuronal processes:1.1 The impact of magnetic stimulation on hippocampus primary neurons:There are uniformly distributed cells in the dish and the small number of cells can be seen protruding short after 24h of magnetic stimulation. There was no significant difference among the groups. After Cells grew well and most of the cells show small processes growth after 48h, there was no significant difference among the groups. Cells grew in good condition after 72h.Compared with C , S group, contact between processes and processes in M1, M2 group were increased. Contact between processes and processes were significantly increased in each group after 96 h. Some contact formed like net. Connections is obviously after 120h.There are very hard to make a clear difference among the groups by microscopic observation.1.2 The impact of magnetic stimulation on midbrain primary neurons: There are uniformly distributed cells in the dish and the small number of cells can be seen short processes after 24 hours of magnetic stimulation. There was no significant difference among the groups Cells grew well and a small processes was seen in part of cells after 48h . Cells were in good condition after 72h. Compared with C , S group, the length of cell processes became long in M1, M2 group. Contact between processes and processes was increased after 96h in each group and some contact like net in M1, M2 group . There are very hard to make a clear difference among the groups by microscopic observation after 120h.2 The results of cell processes counting and statistic analyses:2.1 The processes of hippocampus primary neuronal were counted in each group: The percentage of two processes neurons: The percentage of two processes of neurons in M1 and M2 groups (M1 group:45.61%±14.92%;M2 group:45.18%±15.64%) were more than that in C and S groups(C group:33.42%±9.49%;S group:34.09%±8.44%; P<0.05). The percentage of three and above of processes of neurons: The percentage of three and above of processes of neurons in M1 and M2 groups (M1 group:30.25%±10.80%;M2 group:29.32%±11.49%) were significantly more than that in C and S groups(C group:22.81%±10.36%;S group:24.05%±9.12%; P<0.01).2.2 The processes of midbrain primary neuronal were counted in each group: The percentage of two processes of neurons: The percentage of two processes of neurons in M1 and M2 groups (M1 group: 29.76%±11.85%;M2 group: 32.45%±12.65%) significantly more than that in C and S groups(C group:20.19%±13.92%;S group:23.02%±10.90%; P<0.01). There was no significant difference between M1 and M2 group (P> 0.05). The percentage of three and above of processes of neurons in M1 and M2 groups (M1 group:18.53%±13.69%;M2 group:23.53%±12.56%) were significantly more than that in C and S groups(C group:13.10%±8.14%;S group:11.95%±8.32%; P<0.01). And there was some difference between M1 and M2 group.2.3 The length of processes in midbrain primary neuronal measurement: The length of processes in M1 and M2 groups were significantly longer than that in C and S groups (P<0.01).3 SYN-I and GAP-43 analysis:3.1 Expression of SYN-I mRNA in hippocampus primary neurons: Compared with C and S groups, the relative optical densities(ROD) of SYN-I bands in M2 group became higher (P<0.05) .There was no statistically significant difference between M1 and other groups(P>0.05).3.2 Expression of SYN-I mRNA in midbrain primary neurons:Compared with C, S and M1groups, ROD of SYN-I bands in M2 group became higher (P<0.05) , But there was no statistically significant difference between M1 and other groups(P>0.05).3.3 Expression of GAP-43 mRNA in hippocampus and midbrain primary neurons : There was no significant difference among the four groups. Conclusion: In this study, application intensity of 0.76T and 1.14T, a frequency of 1Hz magnetic field on the primary cultured hippocampus and midbrain primary neurons, and immunofluorescence staining method and RT-PCR technology was used.Preliminary study on the magnetic stimulation to promote cell differentiation mechanisms Study confirmed that:1.Field strength of 0.76T and 1.14T, the pulse frequency of 1Hz magnetic field can promote processes growth in primary neuronal. 2. SYN-I may play a role in the promotion.
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