The Study Of Repetitive Transcranial Magnetic Stimulation On Neurogenesis And Functional Recovery In Rats With Focal Cerebral Ischemia

Objective:Repetitive transcranial magnetic stimulation has increasingly been studied over the past decade to determine whether it has a therapeutic benefit on focal cerebral ischemia. Here we investigate the effects of rTMS on neurological functions, neural stem cells proliferation in subventricular zone and explored miRNAs that were affected by rTMS after transient focal cerebral ischemia in the adult rats, so as to explore the neuroprotection and possible mechanism of rTMS therapy.Methods:45 male Sprague-Dawley adult rats were randomly distributed into sham-operated group, model group and rTMS group. Each group had 15 animals. Right MCAO for 80 min model was used. rTMS group received therapy once a day for a 7-day period since 24 h after surgery, except sham-operated group and model group. NSSs assessment was performed in all rats on the first and the 7th day after surgery. BrdU was intraperitoneally administered to all groups (n= 5 for each group) three times for 12 h following the last rTMS treatment, and rats were sacrificed within 4 h after the last administration. Ipsilateral samples (n=10 for each group) of cerebral infarction including striatum were dissected for qRT-PCR and Western blotting. NSSs test was used to examine whether rTMS could improve the neurological function and Fluorescent double-label immunobistochemistry was used to exam the proliferation of adult NSCs in ipsilateral SVZ. The expressions of miR-25 were determined by qRT-PCR and its target genes were elvaluated by western blotting.Statistical comparisons of results were performed by one-way ANOVA.Results:1. Animals subjected to MCAO showed severe behavior deficits 1 day after ischemia and the there was no significant difference in average scores among ischemic rats (p>0.05). There was a progressive improvement in NSSs in both model group and rTMS group 7 days after surgery (p<0.005). However, rats in rTMS group had a significantly lower NSSs at the 7th day after surgery, compared with the model group (p=0.019).2. Adult NSCs proliferation in the ipsilateral SVZ was observed by utilizing Brdu+/Nestin+ positive cells. Results showed that an increase in double positive cells in the model group relative to the sham-operated group (p=0.044). Moreover, double positive cells in the rTMS group were significantly enhanced than in other two groups (p<0.005).3. Results of qRT-PCR revealed that the expression of miR-25 increased in model group compared with sham group (p<0.001). And rTMS group had significantly higher expressions of miR-25 than other two groups (p<0.001).4. No significant difference was found in expressions of p57 and PTEN between sham-operated group and model group (p>0.05). The p57 expression in rTMS group significantly decreased than that in other two groups (p<0.01) and the PTEN expression in rTMS group obviously increased than that in other two groups (p<0.01).Conclusion:10Hz rTMS has potential in the rehabilitation of motor function after focal cerebral ischemia.10Hz rTMS can promote the proliferation of adult NSCs in the ipsilateral SVZ and played a regulatory role in the expression of miR-25, which might be associated with the neuroprotection effect of rTMS.Objective:To determine whether miR-25/p57 play a pivotal role in rTMS's promotion of adult NSC proliferation after focal cerebral ischemia, rats were injected with antagomir-25 just prior to MCAO, followed by 7 days of rTMS treatment.Methods:Male Sprague-Dawley adult rats were randomly distributed into rTMS group, rTMS+antagomir-25 group and rTMS+scrambled control group. Right MCAO for 80 min model was used. MCAO model was established after injection of different doses of antagomir-25 or scramble into the lateral ventricle in the rTMS+antagomir-25 group and rTMS+scrambled control group respectively. All rats received rTMS treatment once a day for a 7-day period since 24 h after MCAO. To verify the efficacy of antagomir-25, ipsilateral samples of cerebral infarction including striatum were dissected on the 7th day to detect the expressions of all miR-106b-25 cluster members by qRT-PCR. After determined proper dose of antagomir-25,we first examined p57 and p21 by western blotting (n=5 for each group), which also further verified antagomir-25 specificities. Then other rats (n=5 for each group) were intraperitoneally administered BrdU and the proliferation of adult NSCs in ipsilateral SVZ was observed as previous method. Statistical comparisons of results were performed by one-way ANOVA.Results:1. The results showed that antagomir could induce silence of miRNA in vivo and had some relevance to its concentration. The levels of miR-25 were maintained at lower levels for 7 days after the injection of 2.5 nmol Ant-25, although miR-25 was not totally inhibited.2. Results of qRT-PCR showed that a dose of 2.5 nmol antagomir-25 significantly reduced miR-25(p<0.001) and didn't alter the expression of other members of miR-106b-25 cluster.3. Western blotting revealed that after miR-25 suppressed, the protein level of p57 was remarkably up-regulated in the ipsilateral tissue (p<0.05), confirming the direct suppression of p57 by miR-25. The level of p21 was also examined to confirm specificity, and no difference was detected among groups (p>0.05),4. After miR-25 suppressed, no meaningful difference was found in Brdu+/Nestin+ positive cells between rTMS group and scrambled control group, while an significantly decrease in double positive cells in antagomir-25 group compared to other two groups (p<0.005).Conclusion:10 Hz rTMS can promote the proliferation of adult NSCs in the SVZ after focal cerebral ischemia by regulating the miR-25/p57 pathway.Objective:To investigate the effect of 10 Hz rTMS on cognitive dysfunction following focal cer-ebral ischemia and to confirm whether rTMS-induced neuroprotective is associated with corresponding changes in hippocampal neurogenesis and apoptosis.Methods:Male Sprague-Dawley adult rats were randomly divided into the 7 days groups and 14 days groups and further divided into sham-operated group, model group and rTMS group. Right MCAO for 80 min model was used. rTMS groups received therapy once a day since 24 h after surgery, except sham-operated groups and model groups, until the ends of each time point. MRI scan was performed in all rats on the first, the 7th and the 14th day after surgery. DWI were obtained and ADC maps were calculated from the DWI data. For 7 days groups, BrdU was intraperitoneally administered three times for 12 h following the last rTMS treatment and rats were sacrificed within 4 h after the last administration. For 14 days groups (n=5 for each group), BrdU was intraperitoneally administered once daily over the initial 7 consecutive days starting 24 h after surgery. Other rats in 14 days groups were assessed in the Morris water maze since the 12th day after surgery and ipsilateral hippocampus were dissected for Tunel staining (n=5 for each group) and Western blotting (n=5 for each group). Lesion volumes (LV) were evaluated from ADC images as a ratio of the ischemic region to the contralateral hemisphere. Learning and memory changes of rats were observed with Morris water maze. Fluorescent double-label immunohistochemistry was used to exam the proliferation and differentiation of adult NSCs in ipsilateral hippocampus. Apoptotic neuronal death in ipsilateral hippocampus was evaluated using TUNEL staining. Besides, apoptosis-related proteins Bcl-2 and Bax in ipsilateral hippocampus were detected through western blotting to confirm cell survival.Statistical comparisons of results were performed by one-way ANOVA.Results:1. In the sham group, ADC revealed similar signals in both cerebral hemispheres. While both groups started with very similar LV at 1d post-occlusion for the MCAO and rTMS group (p=0.878). Despite that the difference between the LV values of the two groups at day 7(p=0.246) and 14(p=0.132) post-MCAO was not statistically significant, a trend of improvement in the tissue condition was observed for the rTMS group compared with the model group. Besides,7d and 14d post-occlusion, the LV of the model and the rTMS rats both decreased compared to the first day (p<0.001). But statistical differences were not observed in the LV between 7 days and 14 days in model (p=0.716) and rTMS group (p=0.264).2. Rats demonstrated prolonged latency in model group compared with the sham group (p=0.042). However, the latency was shorter after rTMS treatment than that of the model group (p=0.006). Besides, the frequency of swimming across the platform in 60 s in the model group decreased compared with that of the sham group (p=0.001). While in the rTMS group, the frequency increased compared with that of the model group (p=0.045).3. Results showed that the number of BrdU+/Nestin+ cells in the SGZ was increased in the model group and rTMS group relative to the sham group (p=0.042 and p<0.001 respectively). Moreover, Brdu+/Nestin+ cells in the rTMS group were prominently more numerous than in the model group (p<0.001), implying that focal cerebral ischemia could induce adult NSCs proliferation in SGZ, and rTMS treatment facilitated it.4. An increase in the total number of BrdU/NeuN double-labeled cells was observed in the SGZ of rats exposed to rTMS compared with other groups (p=0.006 vs. sham group and p=0.021 vs. model group), indicating a promotion in cell differentiation. Only few positive cells were expressed 14 days after MCAO in the ipsilateral SGZ and no statistical significance was found.5. The percentage of Tunel-positive cells was 0.083±0.042 in the sham group, 0.738±0.091 in the model group and 0.474±0.095 in the rTMS group. p<0.001 for model group versus sham group and p<0.001 for rTMS group versus model group.6. A significant decrease in Bcl-2 expression and increase in Bax expression in model group compared with the sham group (p<0.01) were found. However, rTMS enhanced the expression of Bcl-2 and reduced that of Bax, compared with model group (p<0.005). The present results demonstrated that neuron apoptosis was enhanced by the induction of isch-emia, while rTMS treatment significantly reduced it.Conclusion:10 Hz rTMS could improve cognitive impairment, stimulate neurogenesis in the ischemic hippocampus and inhibit apoptosis by regulation of related proteins after focal cerebral ischemia.