Effects Of Infra-low-frequency Transcranial Magnetic Stimulation On The Endogenous Neural Stem Cells Proliferation, Migration And Differentiation In Hippocampus Dentate Gyrus After Transient Focal Cerebral Ischemia-reperfusion In The Adult Rats

[Background]With the development of national economy and the aging of the population,Cerebrovascular disease(CVD) has become a kind of important disease that threat to elderly health and life.Among them,the ischemic stroke accounted for about 75% of the cerebrovascular disease.Most of the stroke patients left behind more or less of limb movement function impaired, aphasia and cognitive disorder,which not only directly affect the living quality and their rehabilitation treatment progress of the patients,but also bring great damage and heavy economic burden to the society and their family life. In the traditional points of view,it is difficult for nerve regeneration once nerve impaired.The loss of neurological function would be permanent and could not be restored. However,some researches in recently years indicated that the endogenous neural stem cells in lateral ventricle and the hippocampus in adult rodent brain could be sustainable activating, proliferating, migrating and differentiating under the condition of ischemia, trauma, stimuli, epilepsy, additional growth factors and other factors.The neurogenesis of endogenous neural stem cells could play an important role in repairing of the damaged neural network and reconstruct nerve function,which provide a new breakthrough for the treatment of cerebrovascular diseases,such as cerebral apoplexy. Although,many studies had revealed that cerebral ischemia could lead to the neurogenesis of endogenous neural stem cells in hippocampal,the number of endogenous neural stem cells were inadequate.Besides,the regulation effects of survival, proliferation migration and differentiation in the process of neurons repairing and the factor of synapses forming were also insufficient.Therefore, it is important for further study to research the methods of using the biochemical and physical factors to expand the neurogenesis of endogenous neural stem cells,promote their proliferation, migration to the ischemic necrosis focal,and differentiating to nerve cells eventually. It is meaningful and could have a great help to repair the neural network,improve the plasticity of the brain,and recover of neural function after stroke.· Transcranial Magnetic Stimulation (TMS) is non-invasive technology based on the fast electromagnetic conversion theory,using the magnetic field effect on central nervous system,induced biological electric current in the cerebral cortex and subcortical nerve cells,which could have effect in the brain metabolism and electrical activity of nerve cells.Thus,causing a series of physiological and biochemical effect.Infra-low frequency transcranial magnetic stimulation is using the stimulating frequency between 0～0.2 Hz. At present, most of the researches on the frequency of TMS were foucs on Hertz level,the study which contrapose lower stimulation frequency was rarely reported.But a large number of clinical and animal experiments did confirmed that ILF-TMS had a significant curative effect not only in the treatment of brain impaired diseases, such as Parkinson’s disease, depression, cerebrovascular disease, etc, but also the mental illness, such as depression, anxiety, insomnia, etc. Clinical trials with TMS were applied to have yielded promising results relating to improved recovery of sensorimotor and cognitive functions, the motor function, language ability.ILF-TMS could affect brain function through intervention the active of neurotransmitters that have special biophysical characteristics and clinical application. With the growing popular application of ILF-TMS, the study aimed at its.treatment mechanism is also increasingly. At present, a lot of research suggested that ILF-TMS could cure central nerve disease by influencing the eNecs. On the one hand, ILF-TMS could promote the expression of BDNF and 5-HT in hippocampus while BDNF and 5-HT have a positive effect in regulating the neurogenesis process of eNSCs; on the other hand, ILF-TMS could impact the changing of micro-environment in the brain which also have accommodation for the eNscs. Accordingly, the continuous magnetic field of ILF-TMS could produce urtra-low frequency current in the cerebral cortex, and imitating slow postsynaptic potential to regulate the releasing of neurotransmitter. Moreover, various frequencies have different effects for excitatory neurotransmitter and inhibitory neurotransmitter, In our prophase experiments,we used the therapy of 11mHz ILF-TMS to intervene the transient focal cerebral ischemia-reperfusion adult rats.After treatment,the immunohistochemical staining was used to detect the expression of the 5-Bromo-2-deoxyUridine (BrdU) and nestin. Compared with the shame stimulation groups, we founded that endogenous neural stem cells in the rats of the ILF-TMS treatment group had continous activation. Furthermore, the rats’ 18 points Nerve dysfunction score assessment in ILF-TMS groups are better than the shame stimulation groups.[Objectgive]This experiment via further observation the effects of infra-low-frequency transcranial magnetic stimulation on the endogenous neural stem cells proliferation, migration and differentiation in hippocampus dentate gyrus after transient focal cerebral ischemia-reperfusion in the adult rats. Besides, the the mNSS nerve dysfunction rating methods was used to assess the neurologic evaluation of the rats in each groups.By that, we aim to explore the mechanism of ILF-TMS in in the process of nerve repairing of the hippocampal dentate gyrus after ischemic cerebral stroke,and try to provide more adequate theoretical basis of the ILF-TMS applied in clinical treatment of patients with cerebral apoplexy.[Methord]1.The way of Longa’ was adopted to establish the model of focal cerebral middle cerebral artery ischemia-reperfusion rats. Successful model in rats were divided into shame stimulation group and Infra-low-frequency transcranial magnetic stimulation groups. All groups were randomly assigned to 1 of 4 subgroups according to time after treatment at 7th day,14th day,21th day and 28th day. The control groups was established at the same time.2.The rats of ILF-TMS groups were received the frequency of 11mHz, field intensity of 500GS,]nfra-low-frequency transcranial magnetic stimulation for 30min per day. Rats in shame stimulated group was also fixed as ILF-TMS groups, but magnetic stimulation was not implemented. The control group was not fixed.3. Each groups of rats were intraperitoneal injection of BrdU 4 hours/time, a total of 3 times of 24 hours before death.12 h after the last injection executed animal.4.By using the method of improved degree of nerve function defect score (mNSS) rated the assess of each neural function in rats at the time of 7th day,14th day,21th day and 28th day.5. Fetch the brain specimens in rats after executed animal. Using paraffin embedded the brain after fixed in 4% paraformaldehyde overnight. The hematoxylin-eosin staining method (HE staining) was used to dye before observing the pathological morphology of ischemic tissue.Besides, the immunofluorescence staining was used to detecte the change number of the BrdU+cells, the BrdU+/PSA NCAM+cells, the BrdU+/NeuN+cells and the BrdU+/GFAP+cells expressing in the hippocampus dentate gyrus subgranular zone(SGZ) and granular cell layer (GCL) after 7 days,14 days,21 days and 28 days ischemia/reperfusion.The Image-Pro Plus 6.0 software was uesd to analysis and calculate the number of positive cells.[statistical analysis]Statistical analysis was performed using SPSS 19.0 software. Descriptive statistic is showed by mean and standard deviatio(-:S). Groups were compared with the methods of ANOVA. The significance level was set at P<0.05.Two further comparison with LSD-t method, P<0.05 for the difference has significant meaning.[Result]1.The rats’ activities of the shame operation groups were normal in each time phase, compared with the rats of the ILF-TMS groups and the shame stimulation groups,the difference was statistically significant (P<.05).Compared with the shame stimulation groups,the assessment of the mNSS in ILF-TMS groups were significant lower(P<0.05).2.Pathological observation HE staining showed that the structure of brain cortex and hippocampus in shame operation group is clear and intact, meanwhile the cells are intact as well. In addition, the structure of focal ischemic cerebral cortex was chaos, cells were degeneration and apoptosis. The cells in hippocampus arranged disorder, parts of the cellular structure is not clear, nucleus pycnosis, nucleoli disappear. The Pathological changes in ILF-TMS groups were less than the shame stimulation groups.3.The changing expression of BrdU positive cells in dentate gyrus of hippocampus:Immunofluorescence staining shows that BrdU positive cells could be detected in each groups of rats. In the 7th day,14th day,21st and 28th day, the number of BrdU positive cells in dentate gyrus of hippocampus in rats after transient focal cerebral ischemia-reperfusion were significantly higher than the sham operation groups (P<0.05). Moreover, the number of BrdU positive cells in dentate gyrus of hippocampus in ILF-TMS groups were significantly higher than the shame stimulation group in the same time phase(P<0.05).4.The changing expression of BrdU+/PSA-NCAM+ cells in dentate gyrus of hippocampus:Immunofluorescence staining shows that there were only a small amount of BrdU+/PSA-NCAM+ cells could be detected in the shame operation groups. The number of BrdU+/PSA-NCAM+ cells in dentate gyrus of hippocampus in rats after transient focal cerebral ischemia-reperfusion were significantly higher than the sham operation groups (P<0.05).After the 14th day,The number of BrdU+/PSA-NCAM+ cells in ILF-TMS groups were increased markedly as compared with shame stimulation group(P<0.05).5.The changing expression of BrdU+/NeuN+ cells in dentate gyrus of hippocampus:Immunofluorescence staining shows that there were only a small amount of BrdU+/NeuN+ cells could be detected in the shame operation groups. After the 7th day,the number of BrdU+/NeuN+cells in dentate gyrus of hippocampus of rat after transient focal cerebral ischemia-reperfusion were significantly higher than the sham operation groups (P<0.05).In subgranular zone, the number of BrdU+/NeuN+ cells in dentate gyrus of hippocampus had no significantly different as compared with the shame stimulation group in the same time phase(P<0.05). However, in the 21st day and the 28th day, the number of BrdU+/NeuN+ cells of ILF-TMS groups were significantly higher than the shame stimulation groups in granular cell layer.(P<0.05).6.The changing expression of BrdU+/GFAP+ cells in dentate gyrus of hippocampus:Immunofluorescence staining shows that there were only a small amount of BrdU+/GFAP+cells could be detected in the shame operation groups. After the 14th day, the number of BrdU+/GFAP+cells in dentate gyrus of hippocampus of rat after transient focal cerebral ischemia-reperfusion were significantly higher than the sham operation groups (P<0.05).In subgranular zone, the number of BrdU+/GFAP+ cells in ILF-TMS groups were significantly lower than the shame stimulation group after 14th day(P<0.05). Furthermore, in the 21st day and the 28th day,as compare with the shame stimulation groups,the number of BrdU+/NeuN+ cells in ILF-TMS groups were also significantly lower (P<0.05).[Consulsion]1.Endogenous neural stem cells in hippocampal dentate gyrus could be sustainable activated after transient focal cerebral ischemia-reperfusion in the adult rats,and the activated endogenous neural stem cells could migrate and differentiate into neurons or glial cells.2.Rats could be incompletely rehabilitated after transient focal cerebral ischemia-reperfusion, especially in ILF-TMS groups.Their assessment of modified Neurological Severity Scores were ameliorated.It maybe contributed to the progress of neurogenesis.3.11mHz infra-low-frequency transcranial magnetic stimulation could promoted the endogenous neural stem cells proliferation, migration and differentiation to neurons in hippocampus dentate gyrus after transient focal cerebral ischemia-reperfusion in the adult rats.