| Dysfunction of central nervous system causes disability of the corresponding part of the body and lower the survival quality of patients, which also significantly add to the social economic burden. How to effectively promote the functional rehabilitation and reconstruction of the central nervous system after injury is the focus of neuroscience. Electrical stimulation has been widely used in clinical practice as one of the important means to promote the functional recovery of central nervous system after injury. Recently, transcranial magnetic stimulation (TMS) has received widespread attention as a new non-invasive treatment. This technique has been used abroad for the treatment of central degenerative diseases, but has not achieved the ideal effects. Moreover, both the magnetic and electrical stimulation have their shortcomings. How to make magnetic and electrical stimulation work effectively on the functional remodeling of central nervous system after injury always attracts many medical researchers.We have developed an apparatus which can output the magnetic and electrical signals simultaneously, based on the researches of the magnetic and electrical treatment. This equipment was used combined with transplantation of neural stem cells on the cerebral motor cortex injured rats, to investigate the effects of them on functional remodeling of central nervous system. The changes in content of BDNF and NCAM in the injured region of the cerebral motor cortex were also analyzed to reveal the underlying mechanisms of the remodeling action of the combined stimulation. The results are as follows.1. Development of the magnetic and electric stimulating apparatus. According to the principle of magnetic and electric stimulating therapy, the tolerable stimulating parameters to animals is optimized and integrated, thus we developed the magnetic and electric stimulating apparatus. This apparatus can produce magnetic and electrical signals simultaneously, which can also arrive at the cerebral cortex synchronously. The magnetic stimulation is to the left gastrocnemius muscle (peripheral stimulation), and the magnetic stimulation is directly to the cerebral cortex. Both stimulations can reach the cerebral cortex synchronously after signal processing.In animal experiments, the adult Sprague-Dawley (SD) rats were divided into three groups, simple electrical stimulation group (ESG, n=40), simple magnetic stimulation group (or transcranial magnetic stimulation group, TMSG, n=10), combined stimulation group (or transcranial magnetic and electrical stimulation group, TMESG, n=70). The ESG is constitute of 4 subgroups (n=10/subgroup), the TMESG is constitute of 7 subgroups (n=10/subgroup). Detecting the latency and amplitude of the action potential of the gastrocnemius muscle induced by electric stimulation to the contralateral cerebral cortex.In TMESG, three subgroups were used to observe the latency and amplitude of the action potential of the gastrocnemius muscle and vital signs of animals under different magnetic stimulating intensity (300, 660, 800mT). And the other four subgroups were used to observe effects of different electric stimulating intensity (15, 20, 30, 50mV) on the indexes above under the same magnetic stimulating intensity (660mT). Thus the optimized combined stimulation program was formulated, which is, 660mT magnetic simulations for 0.2ms, 20mV electric stimulations with a 200μs pulse width, the two stimulations were in synchronism for 10ms. The experiments of the third and fourth part are carried out based on the program above, the frequency of combined stimulation is once per 5s, for 30min, with an interval of 2h, and this process is repeated for 3 times.This apparatus facilitated the researches restricted to the tolerance threshold of the rats, and achieved the miniaturization of researching equipment, which filled the domestic blank of this area.2. Establishment of experiments with methods of molecular biology. Rat embryonic neural stem cells (NSCs) were isolated, cultured, identified, cryopreservated and resuscitated. The culturing process is simple and effective, which could ensure us to obtain a lot of NSCs in a short period. And the morphosis integrity of the NSCs could be kept well. This part had established the groundwork for the third and fourth part.3. Promotion of combined stimulation to the functional recovery of paralyzed limbs of cerebral motor cortex injured rats. Experiments were carried out on 50 SD rats, which were randomly divided into 5 groups (n=10/group), normal group (NG), simple destroy group (SDG), transplanted NSCs group (TNG), destroy and transcranial magnetic and electrical stimulation group (SDSG) and transplanted NSCs and TMES group (TNSG).Three days after injuring operation to cerebral motor cortex, all the neural functional loss was first or second order. Five days after injuring operation, comparing with the TNG and SDG, the paralyzed limbs of the TNSG was bettered significantly (P<0.05) (motor function of one rat recovered totally). These results demonstrated that combined stimulation had apparent promotion to the functional recovery of paralysis limbs of cerebral motor cortex injured rats.4. Effect of combined stimulation on BDNF and NCAM of the injured area and its vicinity. Experiments were carried out on 78 SD rats, which were randomly divided into 5 groups, NG (n=6), SDG (n=18), TNG (n=18), SDSG (n-18), TNSG (n=18). Every experimental group is divided into three subgroups (n=6) according to the time after injury (1d, 3d, 5d).Comparing to the NG, the expression of BDNF of SDG increased after injury, reaching top at the 1d (P<0.05), back to the normal level at the 5d (P>0.05). Changes of BDNF of TNG, SDSG and TNSG were the same with SDG, but still higher than NG. Comparing to SDG, expression of BDNF of TNG increased more significantly, the expression of 1d was significantly different from the 3d (P<0.05). Expression of SDSG did not change significantly (P>0.05). Expression of TNSG increased significantly at the Id and 3d (P<0.05), while did not change at the 5d (P>0.05). Comparing to the TNG, BDNF of TNSG had no change at all the time points (P>0.05). Comparing to the SDSG, expression of BDNF increased significantly at the 1d and 3d (P<0.05), while did not change at the 5d (P>0.05).Comparing to the NG, the expression of NCAM of SDG increased after injury at the 1d, back to the normal level at the 3d and 5d (P>0.05). TNG, SDSG and TNSG showed the same change with SDG, at the top in 1d, and recovered gradually. Comparing to SDG, NCAM of TNG increased more significantly, and kept at a high level in 3d (P<0.01), recovering at 5d (P>0.05). Comparing to SDG, NCAM of SDSG had no significant change in every time point (P>0.05), but NCAM of TNSG was significantly raised (P<0.05). NCAM of TNSG was significantly higher than TNG in 1d and 3d (P<0.05) and was significantly higher than TNSG in 1d, 3d and 5d (P<0.05).These results indicate that 1) the magnetic and electric stimulating apparatus met our experimental requirements, which was very convenient, reliable and safe. This apparatus is an ideal equipment to carry out researches on magnetic and electric stimulation. 2) combination of magnetic and electric stimulation could promote the functional recovery of the central nervous system after injury, shorten the recovering time of the paralyzed limbs after injury of cerebral motor cortex. 3) combination of magnetic and electric stimulation could change the expression of the BDNF and NCAM of the injured area and the vicinity, which suggested that BDNF and NCAM played a role in the promotion of combined stimulation to the functional recovery of central nervous system.Our research first demonstrated that combination of magnetic and electric stimulation was an effective therapy to injury of central nervous system. We have provided the experimental evidence for clinical application of this therapy on functional recovery of cerebral cortex after injury, expecting to benefit the human beings.
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