The Mechanisms Of High Frequent RTMS To Improve The Hippocampal Synaptic Plasticity And Potential Metabolites In Mice With Age-related Cognition Impairment

Brain aging is a most significant physiological change for aging and acts as risk factor for many neurodegenerative diseases such as Alzheimer`s Disease(AD). Cognitive decline is an important symbol of brain aging. Study on how to improve cognitive function, delay the aging of brain and prevent aged-related diseases has great importance and medical and social significance.Some domestic research found that strategy training improved word memory of the old to achieve even better than untrained young people, indicating that the old people’s cognitive ability has some certain plasticity. Other studies have confirmed that synaptic plasticity in the normal aging process still reserve in hippocampus and temporal lobe cortex.Repetitive transcranial magnetic stimulation(rTMS) is a safe,inexpensive, noninvasive, and extracranial stimulation method, by adjusting the stimulation parameters such as frequency, intensity of stimulation, and can affect the neuron activity, improve brain function, so as to play different roles for neural modulation. RTMS has been used for clinical treatment mainly for neurological and psychological diseases at present and can improve the symptoms of the diseases, such as improve the degree of depression, reduce the motor symptoms associated with Parkinson’s disease, regulating the cognitive function of Alzheimer’s patients. Studies have shown that long-term high frequency rTMS treatment can inhibit the impairment of normal cognition in the process of aging. The improvement of individual cognitive function by rTMS was getting through the network and several pathways to improve brain structure and function. Studies have shown that rTMS can improve neuronal plasticity related gene and protein expression and the metabolites to protect neurons, thus improve cognitive function. Generally,low frequency stimulation is considered to inhibit the action of the neural excitability and high-frequency stimulation evokes the excitability. However,the specific mechanism of high frequency rTMS in improving the neural excitability is still not very clear. Some research show that rTMS is state-dependent. We are considering if it can play the same role under this special background in the aging brain and its mechanism of action remains to be discussed. Nowadays in post-genome era, it is emphasized that the research of the whole function of the organism is needed. Metabonomics application for biological body fluids and tissues, combining of non-target method of multivariate statistical analysis, could discover the biochemical changes and biomarkers for disease prevention and treatment. Studies by nuclear magnetic resonance(NMR) method combined with multivariate statistics found that normal aging brain metabolism spectrum compared to adult control has changed significantly. Gas chromatography- time of flight mass spectrometry has high detection sensitivity and repeatability and has been one of the best tool for the metabonomics research.Rodents as the experimental animal model have many in common with brain aging and the aging associated cognitive impairment. The hippocampus and prefrontal cortex are fragile with aging, accompanied by changes in the structure and function of synapse. Aged Kunming mice go through the cognitive impairment and are suitable for the study of normal brain aging as an animal model. Novel object recognition(NOR) and morris water maze(MWM) experiment are classic in assessing cognitive function in mice among lots of behavioral experiments.Elderly Kunming mice were used in this study and were given two different frequency high frequent rTMS(HF-r TMS) treatment. Behaviors associated with cognitive performance were detected with behavior experiment. The change of the synaptic ultrastructure of hippocampus was observed by transmission electron microscopy(TEM). Hippocampal synaptic plasticity was observed by PCR array and western blotting technology by observing the level changes and their interaction of related gene and protein.Thus, to discuss whether synaptic structure and function of older brain are influenced by HF-rTMS. GC- TOF- MS combined with principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) methods were used to detect mice brain metabolites. The cognitive related metabolites might provide possible cognition related metabolic markers. It is possible that rTMS could provide an early treatment procedure for age associated cognitive impairment.Part Ⅰ HF-rTMS improved age-related cognitive impairment of Kunming miceObjects: To observe the effect of high frequent repetitive transcranial magnetic stimulation(HF-rTMS) on the cognitive behavior of aged Kunming mice.Methods: 3-month-old and 15-month-old Kunming mice were housed with conditions of temperature(20-25 °C) to adapt the laboratory environment1 day before NOR test. Aged Kunming mice were divided into three groups depending on the discrimination index(DI) to guarantee equivalent cognitive ability between aged groups.Two sessions of rTMS comprising of 1000 pulses in 10 trains were delivered every day for 14 days:(1) Adult sham group: 30 mice(3-month-old) were treated by the reverse side of the coil for a sham purpose.(2) Aged sham group: 30 mice(15-months-old) were treated by the reverse side of the coil for a sham purpose.(3) 5Hz rTMS group: 30 mice(15-months-old) were treated with 5Hz rTMS.(4) 25 Hz rTMS group: 30 mice(15-months-old) were treated with 25 Hz rTMS.Spontaneous activity,NOR and MWM tests were carried out after rTMS procedure.Result:1 No effect on spontaneous activity was shown after rTMS on aging miceSpontaneous activities decreased in aged sham group compared to adult sham group. Compared to aged sham group, spontaneous activities in both rTMS group had no significant difference, indicating that the rTMS procedure in this study is safe.2 Non-spatial memory was significantly improve by rTMS2.1 Non-spatial memory was decreased in aged miceCompared to adult sham group, the DI decreased significantly in aged sham group(P<0.05) and no significant difference was found among aged groups( aged sham, 5Hz and 25 Hz rTMS) at 1h after training. Compared to adult sham group, the DI decreased significantly in aged sham group(P<0.05)and no significant difference was found among aged groups( aged sham, 5Hz and 25 Hz rTMS) at 24 h after training.2.2 Non-spatial memory was significantly improved by rTMS in aging miceCompared to aged sham group, the DI increased significantly in 5Hz and25 Hz rTMS group(P<0.05) at 1h after training. At 24 h after training, the DI increased significantly in 5Hz rTMS group only(P<0.05). No significant difference was found between 5Hz and 25 Hz rTMS group at both time points.3 Spatial learning and memory were significantly improved by rTMSNavigation:The mean escape latency in aged groups( aged sham, 5Hz and 25 Hz rTMS) was significantly increased compared to adult sham group(P<0.05). Compared to aged sham group, the mean escape latency decreased significantly in 5Hz(P<0.01) and 25 Hz rTMS group(P<0.05). Space probe:Compared to the adult sham group, the distance ratio of aged sham and 25 Hz group were significantly increased(P<0.05) and the time of platform crossing was decreased in aged sham group(P<0.05). Compared to the aged sham group, the distance ratio were significantly increased in 5Hz rTMS groups(P<0.05) and the time of platform crossing was significantly increased in 5Hz and 25 Hz rTMS groups(P <0.05).In summary, the spatial learning and memory was impaied in aged mice compared to adult mice. HF-rTMS improved the spatial learning and memory ability in aged mice and 5Hz showed more significant difference than 25 Hz.Part Ⅱ HF-rTMS improved the synaptic structural remodeling in hippocampus of aged miceObjects: To observe the neural structure remodeling as a possible mechanisms of HF-rTMS improved cognitive fuction in aged Kunming mice.Methods: The 15-month-old Kunming mice were divided into three groups after the behavior test in Part one. Totally 18 mice were included in each group.Two sessions of rTMS comprising of 1000 pulses in 10 trains were delivered every day for 14 days.(1) sham group: 18 mice(15-months-old) were treated by the reverse side of the coil for a sham purpose.(2) 5Hz rTMS group: 18 mice(15-months-old) were treated with 5Hz rTMS.(3) 25 Hz rTMS group: 18 mice(15-months-old) were treated with 25 Hz rTMS.Hippocampus of mice was collected at 1 day and 7 day after rTMS treatment. Synaptic ultrastructure was observed by TEM. The marker of neuron and synapse were detected by Western blotting(WB).Results:1 Ultrastructure of neurons and synaptic parameters were significantly improved by rTMS1.1 Ultrastructure of neuronsNeurons exhibited poor cellular health presenting mitochondria ridges,slight membrane deficiencies and fusion of mitochondrial cristae, dense agglomerate lipofuscin accumulations and autophagosomes in the cytoplasm,decreased cytoplasmic organelles, distention of the rough endoplasmic reticulum. Compared to the sham group, the level of lipofuscin deposition in rTMS groups was lower, and the micrographs of mitochondria were healther which was observed in both 1day and 7day time point.1.2 Synaptic parameters changeThickness of postsynaptic density(PSD)(nm), the width of synaptic cleft(nm),synaptic curvature and the number of perforated synapses was counted in typical Gray I synapse. At 1 day after rTMS: Compared to the sham group,the percentage of perforated synapses and synaptic curvature were increased and the width of synaptic cleft was decreased significantly in 5Hz rTMS group.The thickness of PSD was increased(P <0.05). At 7 day after rTMS:Compared to the sham group, the thickness of PSD was increased and the width of synaptic cleft was decreased significantly in 5Hz rTMS group(P<0.05). No significant difference of all the parameters was found in 25 Hz rTMS group. Compared to the, synaptic curvature increased at 1 day point and the width of synaptic cleft increased at 7 day in 25 Hz rTMS group(P <0.05),indicating a change over time.2 The marker of neuron and synapse was improved by rTMSAt 1 day after rTMS: Compared to the sham group, DCX was increased significantly in 5Hz rTMS group(P <0.05) but not in 25 Hz group observed by Western blotting. At 7 day after rTMS: No significant difference was found between the three groups. Compared to the sham group, SYN and PSD95 were both increased at 1 and 7 day after rTMS. Compared to 5Hz rTMS group,no significant difference was found for SYN or PSD95 in 25 Hz group.Part Ⅲ HF-r TMS modified the genes and proteins related to synaptic functional plastisity in hippocampus of aged miceObjects: To observe the synaptic functional plastisity as a possible mechanism of HF-rTMS improved cognitive fuction in aged Kunming mice.Methods: The experimental group and rTMS treatment were same as Part Ⅱ. At 7 day after rTMS, i.e. after MWM test, hippocampus of mice was collected. Differential expression of synaptic plasticity associated genes after rTMS was analyzed with the Mouse Synaptic Plasticity RT2 Profiler PCR Array. Synaptic plasticity associated proteins were observed by immunohistochemical(IHC) and Western blotting test.Results:1 Synaptic plasticity associated genes were modified by rTMSTotally 84 genes were classified into ten groups associated with synapticplasticity, which consist of Immediate-early response genes(IEGs), late response genes, LTP, LTD, cell adhesion, CREB cofactors, Extracellular matrix & proteolytic processing(EMPP), neuronal receptors(NR),postsynaptic density(PSD) and other genes important for synapse remodeling.Different gene expression pattern was shown after different rTMS procedure. More than 1.5 fold change for up-regulated or down-regulated was considered as differential expression and totally 58 genes were detected in three groups. Compared to sham group, twelve genes were significantly up-regulated more than 1.5 fold in 5Hz rTMS group and 4 of them more than 2 fold: Bdnf、Adcy1(AC1)、Dlg4(Psd95)、Camk IIg. The maximum up-regulated was BDNF, i.e. 4.22 fold. PP1 was the only significant down-regulated gene, i.e. 2.8 fold. As for 25 Hz rTMS group v.s. sham group,three genes was found to be significant changed and all were down-regulated,i.e. Timp1 、 Tnf- α 、 Nt-4/5, with a maximum of 2.64 fold for Timp1.Compared to 5Hz rTMS group, twenty-one genes were significantly up-regulated more than 1.5 fold in 25 Hz rTMS group and 10 of them more than 2 fold. The maximum up-regulated was BDNF, i.e. 3.27 fold. The differential expression genes mainly belong to CREB cofactors, LTP and IEGs according to the classification. Compare with the KEGG pathway database,neurotrophin signaling pathway and cAMP signaling pathway were matched most for 5Hz rTMS group v.s. sham group,no significant matching for 25 Hz rTMS group v.s. sham group. Glutamatergic synapse signaling pathway was matched most for 25 Hz rTMS group v.s. 5Hz rTMS group.2 Synaptic plasticity associated pwere modified by rTMS2.1 The protein level of BDNF and pCREB were promoted in aged hippocampus by rTMS.Compared to the sham group, BDNF and pCREB was increased significantly in 5Hz rTMS group(P <0.05), but not in 25 Hz group observed by Western blotting.2.2 BDNF and pCREB were promoted in DG region in aged hippocampus by rTMSThe reaction positive cells were brown stained and dispersed distributed in the subregions of hippocampus. Compared to the sham group, BDNF-ir and p CREB-ir positive cells are increased in DG region in 5Hz rTMS group. No significant difference of total CREB-ir positive cells was found between subregions and no significant difference of protein level was found between rTMS groups.Part Ⅳ HF-rTMS improved cognition impairment by regulating thehippocampal related metabolites in aged miceObjects: To observe the profile of differential metabolites after rTMS treatment and explore possible metabolic markers for aged mice after rTMS treatment.Methods: The experimental group and rTMS treatment were same as Part Ⅱ. At 1 day after rTMS, i.e. after NOR test, hippocampus of mice was collected. The metabolites of hippocampus were tested with GC-TOF-MS, and analyzed with principal component analysis(PCA) and orthogonal partial least squares analysis(OPLS-DA) to explore the differential metabolites.Results:1 Hippocampal metabolite profile changed after rTMS interventionFirst of all, 499 peaks could be left through interquartile range denoising method for further analysis. PCA analysis with SIMCA-P software showed a fairly clear separation between three groups, shown as different regions in score plots. In order to obtain a higher level of group separation and get a better understanding of variables responsible for classification, supervised orthogonal projections to latent structures-discriminate analysis(OPLS-DA)were applied. The R2 Y value of in OPLS-DA analysis were 0.983, 0.994 and 0.990, and the Q2 Y value were 0.423,0.732 and 0.678, which suggested that the model were stable and good to fitness and prediction. Furthermore,7-fold cross validation and 200 permutation tests were used to estimate the robustness and the predictive ability of our mode, the low values of Q2 intercept further validate the robustness of the models, and thus show a low risk of over fitting and reliable.2 Profile of differential metabolites after rTMSSPSS13.0 was used to further analysis.To refine this analysis, the first principal component of variable importance projection(VIP) was obtained.The VIP values>1.0 were first selected as changed metabolites, the remaining variables were then assessed by Student’s T test(P<0.05). For sham v.s. 5Hz rTMS, 28 metabolites were identified as significant differential. For sham v.s.25 Hz rTMS, 13 metabolites was identified as significant differential. For5 Hz rTMS v.s.25 Hz rTMS, Cholesterol(Cho) was the only significant differential metabolites, similarity:722(reliable),VIP=1.9959,P=0.0400,Fold change:up-regulated 0.92 fold.Compared to sham group, elaidic acid was the only decreased metabolites as in 5Hz rTMS group, while the levels of oleic acid,lauric acid, ketovaleric acid,linoleic acid,zymosterol,aminomalonic acid and gluconic lactone were significantly increased(P<0.05). As for 25 Hz rTMS, the levels of acid,lauric acid, linoleic acid, zymosterol and gluconic lactone were significantly increased, however, sophorose decreased significantly(P<0.05). Oleic acid and lauric acid were both identified as reliable and the fold change >20, the other metabolites were identified as putative.3 The correlation between the performance of NOR and the levels of metabolitesThe correlation between the performance of NOR with aged mice and the levels of oleic acid and lauric acid was selected by the |Pearson correlation|>0.5 and P<0.05. Oleic acid, but not lauric acid, was closely correlated with the NOR and considered as a possible cognitive-related metabolites.Conclusions:1 Aging associated cognitive impairment occurred in 15-month-old Kunming mice and could be an ideal model for normal brain aging. HF-r TMS improved cognitive function impaired with normal aging.The procedures for5 Hz and 25 Hz HF-r TMS in this study are safe to aged mice.2 HF-r TMS improved the structural neuronal plasticity of the aging brain,both the presynaptic and postsynaptic can be affected for a certain time.HF-r TMS also improved the functional synaptic plasticity of the aged hippocampus. Multiple signaling pathways, paticularly c AMP/PKA/CREB and BDNF-TrkB/CREB play the role of improving cognitive function in aging mice after 5Hz rTMS. Hippocampal dentate gyrus could be main subregion for rTMS to play a role.3 The metabolic profile of aged hippocampus was changed by HF-r TMS.Oleic acid is one of the differential metabolites and could be a special potential metabolic markers associated with cognitive function.4 In general, 5Hz rTMS showed a greater influence on the cognitive function of aging brain and related mechanism than 25 Hz rTMS.