The Mechanisms Of Transcranial Magnetic Stimulation To Improve The Electrophysiological And Potential Metabolites With Age-related Cognition Impairment

Repetitive transcranial magnetic stimulation (rTMS) is a noninvasivebrain stimulation technique that has recently received increasing interests as atherapeutic neurorehabilitative tool. Studies confirmed that chronic highfrequency rTMS ameliorated cognitive impairment in normally agingindividuals and patients with Alzheimer disease. The function of rTMS tocognition was related to multiple pathways and networks. rTMS can increasethe excitability of the neuron, and promote the neuronal synaptic plasticity.These aftereffects have been proposed to represent long-lasting changes ofsynaptic efficacy and were therefore termed ‘long-term potentiation (LTP)like’ phenomena. In parallel with the increased LTP recorded fromhippocampal slices, there was a significant enhancement of cognitiveperformance in behavioral test in animals exposed to high-frequency rTMS.Several studies have shown that rTMS promoted neuronal plasticity relatedgenes, proteins expression, and metabolites, remodeling of the neurons.However, the mechanisms underlying the improvement in the neuronalexcitability and cognitive performance after high-frequency rTMS have notbeen well understood.Brain aging is associated with structural and functional changes thatinvariably lead to impairment in cognitive functions even in healthyindividuals, as well as to changes that increase the brain’s susceptibility toneurodegenerative disorders. Rodents offer several benefits as models toinvestigate the mechanisms and to identify the potential treatment ofage-related cognitive decline. Such as, similar to humans, information thatrequires prefrontal cortex and hippocampus processing is particularlyvulnerable to ageing. Previous study reported that Kunming mice as an ideal animal model exhibited an age-related cognitive impairment during normalaging. Passive avoidance task and novel object recognition test were theclassic methods to appraise the cognitive ability in rodent.It is well accepted that the hippocampus and prefrontal cortex is a veryimportant region related to learning and memory and also known as the mostvulnerable region affected by the internal and external changes such as aging,stroke or other neurodegenerative diseases. Studies indicated that the neuronalexcitability in hippocampus decreased during normal aging, represented asenhanced after-hyperpolarization (AHP) and hyperpolarized resting membranepotential (RMP). Such neuronal hypoexcitability, when placed within thecontext of an active neuronal network, will likely result in less efficienttransmission of information encoded in the form of action potential (AP),which in turn will reduce the computational efficacy of the network. Severalstudies shown that compared with younger counterparts, the reduced intrinsicneuronal excitability in central nervous system structure in aged individualsplayed a crucial role in the impairment of cognitive processing. The materialbasis of specific mechanisms to reduce neuronal excitability changes in thepathophysiological stimuli with a mode of action of multi-system changes.Metabonomics combined with untargeted multivariate analysis, has beenextensively applied to many fields, such as understanding the diseases of thebiochemical basis in the process of diagnosis and treatment according to themetabolic profiles in biological fluids and tissues. In the study with untargetedmultivariate analysis to analyze cerebrospinal fluid of AD patients found thatcompared with normal individuals, metabolism in AD patients was obviouslychanged.In this study, we observed the application of rTMS to improve theage-related cognition impairment, and the internal mechanisms and materialbasis.1rTMS improved age-related cognitive dysfunctionApplication of passive avoidance response task and novel objectrecognition (NOR) task to test the cognition of mice，we observed that whether rTMS can improve cognitive impairment in aged Kunming mice.Methods:3-4month-old Kunming mice,9-10month-old Kunming mice,and16-17month-old Kunming mice were housed with conditions oftemperature (20-25°C) to adapt the laboratory environment, and then used forthe experiments.Groups:(1) Aged rTMS group: mice (16-17months old) exposed to highfrequency rTMS (25Hz) with the coil placed just above the head of the micefor14consecutive-days,10trains per day,100pulses per train.(2) Aged group: mice (16-17months old) were treated similar to agedrTMS mice by the reverse side of the coil without rTMS effect.(3) Adoult group: mice (9-10months old) were treated same as agedmice by the reverse side of the coil for a sham purpose.(4) Young group: mice (3-4months old) were treated same as aged miceby the reverse side of the coil for a sham purpose.Each group of animals housed in the same environment, and then testedwith passive avoidance response task and novel object recognition task.Result: the performance of NOR: The total time of the mice touching thetwo objects was no significant difference between young, matured and agedmice during the NOR test at1h or24h point. It was found that compared withyoung and matured mice, the cognitive index decreased significantly in agedmice in the NOR test at either1h or24h point (P<0.05). The total time of themice touching the two objects was not significantly different between rTMSand sham mice at1h or24h point. The aged mice in the sham group devotedmore time exploring familiar object than novel object, while the aged mice inrTMS group spent more time in exploring the novel object than familiar object.Thus, the cognitive index in rTMS group tested at either1h or24h increased,compared with those in the sham group (P<0.05).The latency of passive avoidance test was not significantly differentamong young mice, matured mice, aged mice and aged rTMS mice inadaptation trial indicated that aging and rTMS did not affect the native tendency of rodent step-down off a small, elevated platform to a corner. Timesof electric shock in aged mice were significantly increased, compared withyoung mice in the acquisition sessions (P<0.05), and no difference betweenyoung group and matured mice, and application of rTMS in aged mice coulddecrease the times of electric shock significantly (P<0.05). It was found thatcompared with the young mice, passive avoidance latency in matured micewas no significant difference, and passive avoidance latency in aged micesignificantly decreased (P<0.05). The latency in aged mice delivered14successive-days rTMS increased significantly (P<0.05), compared with agedmice without rTMS effect.These data showed that, cognition was no difference between younggroup and adult group, and age-dependent learning and memory deficits canbe detected at16months of age in Kunming mice. The deficits of cognition inthe aged mice were significantly improved by chronic high-frequency rTMS.2The electrophysiological mechanisms of rTMS to improve theage-related cognition imparimentWe observed that whether rTMS improved cognitive dysfunction in agedmice by modulating neuronal excitability by regulating Voltage-dependentCa2+current (VDCC).Methods:3-4month-old Kunming mice,9-10month-old Kunming mice,and16-17month-old Kunming mice were housed with conditions oftemperature (20-25°C), and then used for the experiments.Groups:(1) Aged rTMS group: mice (16-17months old) exposed to highfrequency rTMS (25Hz) with the coil placed just above the head of the micefor14consecutive-days,10trains per day,100pulses per train.(2) Aged group: mice (16-17months old) were treated similar to agedrTMS mice by the reverse side of the coil without rTMS effect.(3) Young group: mice (3-4months old) were treated same as aged miceby the reverse side of the coil for a sham purpose.Each group of animals housed in the same environment, and then recorded the electrophysiological indexes, after the behavioral task.Result: Whole-cell current clamp recordings revealed that the RMP was morehyperpolarized in CA1pyramidal neurons (CA1-PNs) in slices prepared fromaged mice compared with young mice (P<0.05). Application of rTMSsignificantly elevated the RMP in CA1-PNs prepared from aged micecompared with the aged mice without rTMS (P<0.05). The number of APs wassignificantly reduced in CA1-PNs of aged mice compared with those of youngmice (P<0.05). High-frequency rTMS significantly increased the number ofAPs elicited by the current injection in CA1-PNs of aged mice compared withthose without rTMS (P<0.05). The amplitude of AHP after a series of APs wassignificantly increased in aged mice compared with that in young mice(P<0.05). Application of rTMS significantly decreased the AHP amplitude inthe aged mice compared with the aged mice without rTMS (P<0.05). Thethreshold potential of the first AP fired in response to a300-pA currentinjection revealed no significant differences among matured, aged and agedrTMS mice.VDCC of the CA1-PNs was recorded with whole-cell patch clamp involtage-clamped at-50mV and depolarized from-40mV to20mVincremental10mV voltage steps with150-ms duration. The full currentdensity-voltage curves indicated that Ca2+currents recorded from aged rTMSneurons were decreased compared with those from aged sham neurons.Analysis indicated that the Ca2+current density was significantly smaller fromaged rTMS neurons when compared with aged neurons at test potential of-20mV,-10mV and0mV (P<0.05).These results suggest that, rTMS can modulate neuronal excitability byregulating VDCC, to improve age-related cognitive dysfunction.3rTMS impoved cognition impairment by regulating the age-relatedmetabolites disordersThe metabolites of prefrontal cortex in mice were tested with gaschromatography mass spectrometry (GC-MS), and analyzed with partial leastsquares analysis (PLS-DA) and principal component analysis (PCA)，then analsized the different metabolites with one-way ANOVAMethods:3-4month-old Kunming mice,9-10month-old Kunming mice,and16-17month-old Kunming mice were housed with conditions oftemperature (20-25°C), and then tested for the experiments.Groups:(1) Aged rTMS group: mice (16-17months old) exposed to highfrequency rTMS (25Hz) with the coil placed just above the head of the micefor14consecutive-days,10trains per day, and100pulses per train.(2) Aged group: mice (16-17months old) were treated similar to agedrTMS mice by the reverse side of the coil without rTMS effect.(3) Young group: mice (3-4months old) were treated same as aged miceby the reverse side of the coil for a sham purpose.Each group of animals housed in the same environment, and thenrecorded the metabolites in prefrontal cortex, after the behavioral task.Result: The score plot of the PLS-DA model showed separation ofsamples in different groups. The model generated with two components had acumulative R2Y of0.84and a cumulative Q2of0.69. According to the valueof VIP>1and after merging the variables from the same metabolites,23identified variables were collected.To accurately evaluate the changes of metabolites level, one-way ANOVA andpost hoc analysis was employed to these metabolites ratios, and significantdifferences were found in the23variables from young group, aged group andaged rTMS group, which were considered as the potential different biomarkers.Plot data of above indicated that compared with young group,16metaboliteswere altered significantly in aged mice， metabolites of Alanine (Ala),Phosphoric acid (Pho), Serine (Ser), Threonine (Thr), Malic acid (Mal), Lacticacid (Lac), Urea and Myo-Inositol (M-In) decreased significantly (P<0.05).Metabolites of Gamma-Aminobutyric acid (GABA), Citric acid (Cit), Oleicacid (Ole),5,8,11,14,17-Eicosapentaenoic acid (Eic), Monostearin (M-Ste),Trans-9-Octadecenoic acid (Oct), Ascorbic acid (Asc) and Cholesterol (Cho)were significantly increased during aging (P<0.05). Compared with aged group,21metabolites were altered in aged rTMS mice. It could be found thatcompared with aged mice metabolites of Pho, Fumaric acid (Fum), Thr, Mal,Cit, Ala, Urea, GABA, Ser, Pyrophosphate (P-Pho), M-In, Lac, Pyroglutamicacid (P-Glu), Aspartic acid (Asp), Creatinine (Cre), Asc and Cho decreasedsignificantly (P<0.05), while metabolites of Ole, Eic, N Acetyl aspartic (NAA),Phosphaglyceride (P-Gly) were increased significantly (P<0.05).Based on selected23difference metabolites from young mice, aged mice,and aged rTMS mice, PCA analysis was used as the verify classificationmethod for modeling the discrimination. The score plots of the first twoprincipal components allowed visualization of the data and comparing thethree-group samples. The R2X and Q2were0.60and0.45. The PCA scoreplot showed the samples from different groups were scattered into threedifferent regions.Cholesterol, GABA, ascorbic acid and citric acid are as the screenedmetabolites of brain tissue by the way of rTMS reversed metabolic profile inthe aging process. The correlation between the performance of passiveavoidance task and the levels of metabolites of cholesterol, GABA, Asc, andCit in the tested mice were assessed. The Pearson Correlation of cholesterolwith passive avoidance latency is-0.413(P<0.05), and metabolites of GABA,Asc, and Cit with the avoidance latency is-0.25,-0.080, and-0.273withoutsignificant difference.The above results show that, rTMS can improve cognition-relatedmetabolites disorders during aging.Conclution(1) These data showed that under the conditions used here, cognitionindex was no significant difference between young group and adult group, andage-dependent learning and memory deficits can be detected at16months inKunming mice. The deficits of cognition in the aged mice were significantlyimproved by chronic high-frequency rTMS. (2) These results suggest that, rTMS can modulate neuronal excitabilityby regulating VDCC, to improve age-related cognitive dysfunction.(3) The above results show that, rTMS can improve cognition-relatedmetabolites disorders during aging, and cholesterol may be one of the mostimportant metabolites correlated with cognition.