| As one of the most important brain areas of the central nervous system,hippocampus have a close relationship with learning, memory, and otherphysiological function, many neurodegenerative diseases, such as Alzheimer'sdisease and epilepsy, also accompanied by the lesion of it. Repetitivetranscranial magnetic stimulation (rTMS) is a new neural electrophysiologicaltechnique developed on the basis of transcranial magnetic stimulation and itdelivered magnetic stimulation (MS) to the brain in the repetitive impulseform, through the pulse magnetic field generating induction current, activatingthe neurons and playing a therapeutic role by temporarily changing theexcitability of the stimulated tissue. Studies have shown that rTMS has beenused successfully in the treatment of the diseases such as Alzheimer's disease,epilepsy, and depression. Early studies suggested that aging related cognitivefunction decline may be related to the loss of neurons, but more and moreevidences showed that, in the normal aging process, the number of neuronshad no significant change, but the specialized structure of informationtransmission between neurons--the number and the integrity of synapses hadchanged, with a different degree of decline. Synaptic plasticity refers to themodification of synaptic connectivity in the structure and function which isclosely related to the nervous system development, injury repair, learning andmemory, and the other important brain functions, has become a researchhotspot recently.Studies had showed that the constant magnetic field could contribute tothe growth of cultured neurons and affect the direction of newborn axons. Theneurite outgrowth and the secretion of DA all increased significantly after theapplication of MS intervention on PC12cells in the logarithmic phase. Additionally, animal experiments also confirmed that MS could increase thesynaptophysin expression in hippocampal CA1area of the vascular dementiarat and in striatum of MPTP-induced the Parkinson rat, which indicated thatMS could promote the ability of synaptic plasticity. In recent years the studiesabout MS mostly concentrated on synaptic remodeling and regeneration in thepathological conditions, while the research on normal brain development andneural network construction was scarce, the regulation function of MS onsynaptic plasticity involved in many intracellular molecular pathways andcomplex mechanism. The primary cultured hippocampal neurons were morerepresentative of the normal developmental status of nerve cells in vivo thanthose cell lines after several passages in vitro, were the ideal model for thestudy of neurons' growth and development. Therefore, we selected primarycultured hippocampal neurons, to impose different intensity stimulationparameters, by observing the effect of MS on hippocampal neuronal synapticultrastructure, the expression changes of synaptic plasticity related protein,intracytoplasmic calcium concentration, NR2B, pCaMKII and pCREB afterMS, to explore the mechanism of MS on primary cultured hippocampalneurons neurite outgrowth and synaptic plasticity. And throughthe interventionstudy on the calcium signal transduction pathways, we can clear the precisemechanisms of MS playing the role and to provide a theoretical basis forfurther clinical studies on normal brain plasticity.1Research of repetitive magnetic stimulation on neurite growth and c-fosactivation in primary cultured hippocampal neuronsObjective: to investigate whether magnetic stimulation can cause c-fosexpression, promote neurite growth of primary cultured hippocampal neurons,observe the effects of MS on hippocampal neuron survival.Methods: The primary cultured hippocampal neurons were divided intofour groups, control group (remained in the incubator for7days), sham group(exposed to the magnetic stimulation apparatus for the identical length of time,but did not receive stimulation),30%maximum stimulus intensity group(30%intensity,1.1T),40%maximum stimulus intensity group (40%intensity, 1.48T), the neurons were stimulated after24h, at a rate of1Hz, and themaximum output intensity of magnetic field was3.7Tesla. Continuousstimulation5d, the stimulation coil was held paralleled1cm above the dish.Cellular immunofluorescence staining was executed on day7after plating,tocount the multiple neurites neurons and the cell neurite length of MAP2positive neurons, the c-fos expression in each group was detected byimmunofluorescence staining and Western blot. The effect of magneticstimulation on hippocampal neuron survival was detected by MTT.Results: The purity of MAP2positive neurons in primary culturedhippocampal neurons was more than90%, suitable for the experiment. Thepercentage of multiple neurites neurons(n=2,n≥3)and the cell neurite lengthin both stimulation group were significantly higher than that in control group(P<0.05). Meanwhile it was higher in30%intensity group than that in40%intensity group, there was significant difference in the results (P<0.05). Whilethere was no significant difference between the control and sham stimulationgroup was observed in the growth of neurons (P>0.05). C-fosimmunofluorenscence staining showed that after MS the proportion of c-fospositive neurons increased significantly, and the increase in MS group wasstatistically significant compared with control and sham group (P<0.01), andit had no statistical meaning between control and sham group (P>0.05).Western blot analysis showed that30%intensity MS significantly increasedthe c-fos protein expressin (P<0.05), while the increase in40%intensitygroup had no statistical significance (P>0.05). Cell survival tested by MTTshowed that30%intensity MS had no effect on it, while the survivaldecreased obviously in40%intensity group.Conclusion: Both intensities (1.11T,1.48T) low frequency (1Hz) MSpromoted the neurite outgrowth of primary cultured hippocampus neurons,increased the number and length of neurites, and made the connectionsbetween neurons more closely.30%intensity (1Hz,1.11T) low frequency MSactivated the cell function, increased c-fos protein expression in hippocampalneurons and had no harmful effects on neuron survival, while the cell survival rate decreased obviously in40%intensity group.2The influence of repetitive magnetic stimulation on synapticultrastructure of primary cultured hippocampal neurons and NR2Breceptor expressionObjective: to explore the influence of repetitive MS on synapticultrastructure and NR2B receptor expression of primary cultured hippocampalneurons, make clear the role of MS in the synaptic plasticity regulation ofhippocampal neurons.Methods: Cell transmission electron microscope was used to observe thechanges in the synaptic interface parameters of primary cultured hippocampalneurons. We used immunofluorescence staining, RT-PCR and Western-blotanalysis to explore the expression of NR2B mRNA and protein in differentgroup after MS.Results: After MS, the postsynaptic density thickness and curvature ofthe synaptic interface in30%intensity group had a significant increase(P<0.01) compared to control group, and the increase in presynaptic length ofthe active zoneswas also statistically significant (P<0.05). While40%intensity MS only promoted the postsynaptic density thickness and curvatureof the synaptic interface (P<0.01, P<0.05), the change in presynaptic length ofthe active zones had no statistical significance compared to control group. TheMS had no effect on the synaptic cleft width. Immunofluorescencequantitative analysis showed that NR2B-positive neurons immunoreactivity in30%and40%intensity group was significantly higher than that in controlgroup and sham group after MS, and the difference was statisticallysignificant (P<0.01), RT-PCR result showed that the increase of NR2B mRNAin both MS group was statistically significant compared with control andsham group, and Western blot result also indicated significant difference(P<0.01).Conclusion: Both intensities (1.11T,1.48T) low frequency (1Hz) MScould promote the NR2B mRNA and protein expression of primary culturedhippocampal neurons, had a regulatory role on the synaptic interface parameters, and the adjustment effect of30%intensity (1.11T) MS is betterthan that of40%intensity (1.48T). Upregulation of NR2B receptor mRNAand protein expression in hippocampal neurons may be one of the mechanismsof MS to improve synaptic plasticity.3The influence of repetitive magnetic stimulation on calcium signalingtransduction pathway of primary cultured hippocampal neuronsObjective: To investigate the effect of MS on intracellular Ca2+concentration and the activation of downstream signaling pathways, to explorethe possible mechanism of MS regulating the synaptic plasticityMethods: Flow cytometry analytical technique was used to observe thechanges of the intracellular Ca2+concentration and intervention of MS. Weused Western-blot analysis to explore the effect of MS on the proteinexpression of CaMKII(pCaMKII),CREB(pCREB) in different group. Theinfluence of MS on neurons apoptosis rate was detected by Hoechst33258immunofluorescence staining.Results: Intracellular [Ca2+]i showed a significant increase both in30%and40%intensity group compared to control group (P<0.05, P<0.01), and theincrease in40%intensity group was significantly higher than that in30%intensity group (P<0.01). Compared to control group, the change of [Ca2+]ihad no significant difference in sham group (P>0.05). Western blot analysisshowed that, the total protein level of CaMKII was not different in each group(P>0.05), while the expression of pCaMKII was significantly up-regulated by28.9%and17.7%in30%and40%intensity group respectively. The proteinexpression of pCaMKII had no significant difference in control and shamgroup (P>0.05). The pCREB protein expression also increased by59.1%and13.6%in30%and40%intensity group respectively and MS had nosignificant effect on the total protein level of CREB (P>0.05). Hoechst33258fluorescence staining showed that the neuron apoptosis rate had no obviouschange in30%intensity group compared to control group, but increasedsignificantly in40%intensity group.Conclusion:30%intensity MS elevated intracellular Ca2+concentration, activated calcium signal transduction pathway, improved pCaMKII andpCREB protein expression, had no harmful effect on hippocampal neurons.There was no significant changein apoptosis rate was found in30%intensitygroup compared to control group. While in40%intensity group, MSsignificantly promoted the intracellular Ca2+concentration, althoughimproved the protein expression of pCaMKII and pCREB, calcium overloadcaused an obvious increase in apoptosis rate of the hippocampal neurons.4CaMKII-pCREB pathways involved in the regulatory role of magneticstimulation promoting the synaptic plasticity-related proteins expressionObjective: To research the influence of repetitive MS on the expressionof synaptic plasticity-related proteins, and whether CaMKII inhibitor KN93administration can antagonize the regulatory role, further to make clear therole and mechanism of MS on synaptic plasticity in hippocampal neurons.Methods: The primary cultured hippocampal neurons were divided intosix groups, control group, sham group,30%maximum stimulus intensitygroup, K1group (pretreated with0.5umol/L KN93for3h, and then began toaccept the30%intensity of MS),40%maximum stimulus intensity group, K2group (pretreated with0.5umol/L KN93for3h, and then began to accept the40%intensity of MS). MS intervention condition and time was the same asbefore. Immunofluorescence staining, RT-PCR and Western-blot analysis wereused to detect the expression of synaptophysin (SYN), Activity regulatedcytoskeletal protein (Arc) and microtubule-associated protein2(MAP2).Results: In primary cultured hippocampal neurons, SYNimmunofluorescence-positive material showed beaded distribution along theneuronal processes, granular or point-like, and the cellularimmunofluorescence intensity in30%intensity group was significantly higherthan that in control group. Arc protein expression was mainly located inneuronal cell bodies, processes and dendritic spines, some positive productsshowed punctate distribution. A marked increase of the punctate positivematerial was observed in30%intensity group. MAP2was mainly expressedin the body and dendrites of the neurons and the connection among the neurons in30%intensity group was more closely than that in other groups.Fluorescence quantitative analysis showed that the SYN,Arc and MAP2immunofluorescence intensity in K1group decreased by13.5%,29.6%and13.9%respectively compared to control group. RT-PCR and Western blotanalysis showed that in30%intensity group, SYN,Arc and MAP2mRNAexpression levelswere significantly increased by77.8%,58.7%and46.4%thanthat in control group, and protein levels increased respectively41.8%,89.1%and43.7%. While in K1group, SYN, Arc and MAP2mRNA and proteinexpression showed a significant decrease compared to30%intensity group,mRNA levels were decreased by35.4%,27.4%and22.7%, and protein levelswere decreased by21.7%,41.1%and18.4%respectively. SYN, Arc andMAP2mRNA and protein levels had no significant change in K2groupcompared with control group (P>0.05). These suggested that KN93administration could partly antagonize the regulatory role of MS, reduced thesynaptic plasticity-related protein expression. We further to make clear therole of MS on neurite outgrowthandplasticity regulation in hippocampalneurons.Conclusion: By Ca2+-CaM/CaMKII-pCREB pathways, MS improvedthe expression of synaptic plasticity-related protein SYN,Arc and MAP2,strengthened the regulatory role of MS on synaptic plasticity, KN93pretreatment could partially inhibit the effect of MS. But different stimulatingparameters results in different stimulus effects. The regulation function of30%intensity MS (1.11T) on hippocampal neuronal synaptic plasticity wasbetter than that of the40%intensity (1.48T). |
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