Mechanism Study Of Magnetic Stimulation Inducing Neuron Synapse Growth And Migration

Background and Objective: A number of animal experiment studyhave shown that rTMS can promote the damaged nerve cell axonregeneration, improve the remodeling of axons, promote the migration ofendogenous neural cells and ultimately improve functional recovery afterischemic brain injury, in recent years. But the exact mechanism is not clear.In this study, the primamy neurons were cultured, the injury model wasestablished by oxygen-glucose deprivation in vitro, to simulate cerebralischemia and hypoxia. Hypoxia-ischemia nerve cell survival was determinedby MTT technique, Its damaged degree was determined by the release oflactate dehydrogenase(LDH). The changes of Robo2 and RhoA expressionwas determined by immunocytochemical and RT-PCR method. To study theeffects of rTMS on axon regeneration, cell activity and RhoA signal pathwayin hypoxia-ischemia rat nerve cells, for exploring its possible mechanism; Inthis study, the primamy neurons were cultured, tanswell chamber wasapplied to simulate the migration of endogenous neurons in vitro andinvestigate the effect of magnetic stimulation on nerve cell migration.Methods(Part I): 1. Primary cortical neurons were prepared from brains 15-18 day SDfetal rat.2. The cortical neurons at 6 days in vitro were identified by immuno-cytochemical method.3. Primary cortical neurons were inoculated in culture paltes,weredivided into five groups: (1)Control group; (2)Oxygen/Glucose Deprivationgroup; (3)Sham simulation group; (4)Forty percent of maximum stimulusintensity to stimulate the OGD group; (5)Sixty percent of maximum stimulusintensity to stimulate the OGD group.4. The cortical neurons survival was determined by MTT technique at 6day, Its damaged degree was determined by the release of lactatedehydrogenase (LDH). The change of Robo2 and RhoA expression wasdetermined by immunocytochemical and RT-PCR method.5. The cortical neurons were stimulated from the next day to the sixth day,at a fixed time every day.Stimulation parameters:3 string/day, 100 times/string, each string interval 1 min.Methods(Part II):1. Primary cortical neurons were prepared from brains 15-18 day SDfetal rat.2. The cortical neurons at 6 days in vitro were identified by immuno- cytochemical method.3. Primary cortical neurons were inoculated in transwell chamber anddivided into four group: Control group; Sham simulation group; Fortypercent of maximum stimulus intensity group; Sixty percent of maximumstimulus intensity group. Crystal violet dye migrated out of the cell at 6 day.4. The cortical neurons were stimulated from the next day to the sixth day,at a fixed time every day.Stimulation parameters:3 string/day, 100times/string, each string interval 1 min.Results(Part I):1. The cortical neurons at 6 day in vitro were clearly visible, attached to agood wall, were round and multipolar shape, were strong refraction, whichneuritis contacted each other to form a network. The positive cells accountedfor about(93.7±5.5)% by immunocytochemical staining with NeuN.2. Microscopic observation of the cells of the ischemic group: Nerve cellmorphology and normal group showed no significant change, OGD for 30min. The part of cells of the ischemic group shrinkaged and were weakrefraction. Part of cells membrane collapsed,OGD for 60 min. The release oflactate dehydrogenase (LDH) significantly increased(p<0.05), comparedwith normal group.3. Primary cortical neurons induced by oxygen-glucose deprivation and reperfusion injury, the nerve cell survival of OGD group compared with thecontrol group were significantly lower(p<0.05), Robo2 and RhoA expressionand cell LDH release than the control group significantly increased (p<0.05).Sham stimulation group, the cell survival, LDH release, Robo2 and RhoAexpression compared with the OGD group, no significant change(p> 0.05).The cell survival, LDH release, Robo2 and RhoA expression compared withthe OGD group, no significant change(p>0.05). The cell survival, Robo2expression of 40% or 60% maximum stimulation intensity groupssignificantly increased(p<0.05), compared with the OGD group. The LDHrelease, RhoA expression of 40% or 60% maximum stimulation intensitygroups significantly declined(p<0.05), compared with the OGD group.Results (Part II)：The cell boby and nucleus showed a purple by crystal violet staining.The number of cells migrated out of was no significant difference(p>0.05),which compared with sham stimulation group. The cell number of 40% or60% maximum stimulation intensity groups significantly increased(p<0.05),compared with normal group and the sham stimulation group.Conclusion:(1) Magnetic stimulation can improve the survival rate of OGD neurons,reduce the OGD neurons in the release of LDH, have a direct neuroprotective effect on OGD neurons. (2)High-strength low-frequencymagnetic stimulation can promote damaged neurons axonal regeneration bypromoting Robo2 expression regulation. (3)High-strength low-frequencymagnetic stimulation can inhibit the expression of damaged neurons RhoA,which may be one of the mechanisms of magnetic stimulation to promoteaxonal regeneration. (4)High-strength low-frequency magnetic stimulationpromote neuronal migration in vitro.