| [Background]Due to the high prevalence, mortality and morbidity, cerebrovascular disease has become a kind of important disease that threat to human health, and brings heavy burden to the society and family life. In the traditional view, it is considered neurological forever missing after central nervous system injury, the damage can not be restored. But a lot of research suggests that cell necrosis and apoptosis in adjacent and remote areas after ischemia anoxic injury, and there is brain late-onset reduced and blood flow; can also activate a small amount of endogenous neural stem cells. NSCs are found in the embryonic and adult brains of both rodents and primates and have the potential to differentiate into neurons, astrocytes, and oligodendrocytes. In this time window, the multiplication, the migration, processes and differentiation of NSCs were regulated, It may be an effective way to repair the damaged nerve cells. More and more evidence suggests that the central nervous system has the potential ability to repair itself, mainly because of neural stem cells exists in the subventricular zone, subgranular layer zone, the cerebral cortex, the fourth ventricle and the central canal of the spinal cord and other parts of brain. Most of NSCs are in the resting state under normal physiological conditions, with slow ongoing mitosis for self-renewal. However, under certain conditions, such as ischaemic damage, NSCs cells can be activated, and a large number of proliferated cells migrate to the damaged area and integrate with remaining healthy neurons. This proliferation and migration of NSCs is trigged by injury-induced release of growth factors, cytokines, adhesion molecules and extracellular matrix molecules. Lesions or damage on the activation of neural stem cells in different regions is different. Hippocampus is not only the main areas NSCs existence, but also sensitive to nerve injury. Although not directly effect on the hippocampus, focal cerebral ischemia and brain trauma can cause pathological changes of the hippocampus, and promote the hippocampus nerve regeneration. Nestin staining is widely used for identification of pluripotent neural precursor cells following activation of NSCs because nestin expression is generally low, both prior to activation and following differentiation. Nestin mainly expressed in undifferentiated cells and cells having the ability to divide, its expression and maturation process of stem cells is consistent.5-Bromodeoxyuridine is a substance similar to thymine nucleotides, which embedded in the nuclear DNA in S phase of the cell cycle to mark newborn cells. We treat Brdu positive cells as the newborn cell.Repetitive transcranial magnetic stimulation (rTMS), in which alternating magnetic fields are used to modulate electric currents in the cortical tissue of brain regions, can induce specific changes in cortical activity and excitability depending on the stimulation frequencies Clinical trials in which TMS was applied have yielded promising results relating to improved recovery of sensorimotor and cognitive functions. In combination with conventional therapeutic approaches, rTMS has the potential to become a complementary strategy for enhancing stroke recovery by way of modulating the excitability of targeted brain areas. Different frequencies and intensities of electromagnetic fields have different biological effects. High frequency (> 1 hz) stimulation can make local cortical excitability and increased blood flow, on the contrary, the low frequency (≤1Hz) can reduce the cortical excitability and blood flow. rTMS for multi-pulse magnetic field while the ultra-low-frequency magnetic stimulation is a continuous magnetic with frequency<0.2Hz. ULF-TMS can affect brain function through interven the active of neurotransmitters that have special biophysical characteristics and clinical application. However, despite the fact that understanding the mechanism of rTMS in nerve genesis will be critical for developing better therapies for cerebral ischemia patients, many studies have focused on the effects of rTMS on adult NSCs in central nervous disease. Such as rTMS can promote the expression of BDNF and 5-HT in hippocampus while BDNF and 5-HT are positive regulator of NSCs rTMS works through the induced current that have a moderating effect of neurogenesis; rTMS can cause changes in the brain micro-environment which is the important factors influencing the NSCs. ULF-TMS continuous magnetic field can produce ultra-low frequency currents in brain regions and then simulate the slow synaptic potentials in a variety of neurotransmitters. Applied magnetic field at different frequencies respectively affect excitatory and inhibitory neurotransmitter neurotransmitters. 1mHzULF-TMS affect the activity inhibitory neurotransmitter γ-GABA and 11mHzULF-TMS corresponds excitatory neurotransmitter dopamine (DA). This study compare 1mHz and 11mHz ULF-TMS with lOHz rTMS to investigate effects of different frequencies magnetic stimulation on recovery of neurological function and activation, proliferation of endogenous neural stem cells(NSCs) in hippocampus of cerebral ischemia-reperfusion(I-R) rats in different time.[Objective]To investigate 1mHz and 11mHz ULF-TMS with 10Hz rTMS to investigate effects of different frequencies magnetic stimulation on recovery of neurological function and activation, proliferation of endogenous neural stem cells(NSCs) in hippocampus of cerebral ischemia-reperfusion(I-R) rats in different time.[Methord]1. The filament method was used to establish the focal cerebral ischemia-reperfusion rat model. Successful model in rats were divided into shame stimulation group and three different frequencies TMS groups randomly. All groups were randomly assigned to 1 of 3 subgroups according to time after treatment at 7th, 14th and 21th day. The control group was also prepared at the same time.2. I-R TMS groups was delivered at a stimulation frequency of 1mHz, 1mHz, 10Hz and stimulation intensity of 500 Gs for 15 min once per day using magnetic stimulator. Rats in sham-stimulated group was also fixed as I-R TMS groups, but magnetic stimulation was not implemented. The control group was not fixed.3. Rats were intraperitoneally injected 4 times with 50 mg/kg BrdU at 4h intervals on the day before sacrifice.4. A comprehensive 18-point test battery was used to evaluate neurological dysfunction at 7,14 and 21 days.5. Remove the brain in rats after death. Use paraffin embedded the brain that fixed in 4% paraformaldehyde overnight. Take sections were stained with HE. The expression of nestin was detected by immunohistochemical and western blotting at all time points. Immunohistochemical technique was used to detect the expression of BrdU.[Statistical analysis]Film with a scanner input image into computer. Statistical analysis was performed using SPSS 16.0 software. Using the Image Pro Plus software to detect the mean optical density of nestin-positive and BrdU-positive expression. Image-Pro Plus software used to measure the average gray value of nestin and GAPDH bands, the ratio of the relative value on behalf of nestin expression. Descriptive statistic is showed by mean and standard deviation(X±S). Groups were compared using ANOVA. The significance level was set at P<0.05. Two further comparison with LSD method, P< 0.05 for the difference has significant meaning.[Results]1. Rats in the control group exhibited normal neurological function at 7th,14th and 21th day after treatment, and their deficit scores were significantly higher than the others. Neurological deficit scores did not differ significantly between sham-stimulated and I-R TMS groups at 7th day, but did differ at 14th and 21th day. However, the score of lOHz group is higher than 1mHz group and llmHz group. 1mHz group is no significant difference compared with 11mHz group.2. Nestin-positive cells in the hippocampus exhibited tan staining mainly in the cytoplasm. A significant increase in nestin positive cells was observed in the sham-stimulated and I-R TMS groups relative to control group. Compared with sham-stimulated group, I-R TMS groups exhibited significantly higher average optical density at all measurement times. But lOHz group had significant differences with 1mHz and 11mHz group.3. BrdU-positive cells exhibited staining mainly in the nucleus. The control group showed significantly less staining than sham-stimulated and I-R TMS groups at points. 10Hz group compared with 1mHz group, 11mHz group and sham-stimulated group, Brdu positive average optical density increased. There were no significant differences among groups 1mHz, 11mHz and sham-stimulated group at three points.4. Rarely amount of nestin protein produced in the control group. The relative content of nestin protein in I-R TMS groups exhibited significantly more than I-R sham-stimulated group at three time points. The expression of nestin protein in 10 Hz group more than lmHz and 11 mHz group that have significantly differences at different time points.[Consulsion]1. The frequency of 1mHz and 11mHz ULF-TMS and 10Hz transcranial magnetic stimulation can promote recovery of I-R model rat model of nerve motor function. But at different time points, 1mHz, 11mHz ULF-TMS and 10Hz rTMS at different levels to improve neurologic recovery.2. Focal cerebral ischemia-reperfusion injury can cause the activation and proliferation of NSCs in rat hippocampus.3. The frequency of 1mHz,11mHz ULF-TMS and 10Hz transcranial magnetic stimulation facilitated nestin expression. However, magnetic stimulation with different frequencies to promote model rats at different levels of nestin expression in hippocampus.4. Relative to sham-stimulated group, the frequency of 10Hz rTMS can promote the proliferation of NSCs in I-R rat hippocampus, but the effect was not observed in 1mHz and 11mHz ULF-TMS. |
PDF Full Text Request