重复经颅磁刺激改善老化相关的认知功能损伤的电生理机制及潜在代谢产物的变化
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摘要
重复经颅磁刺激（rTMS）是一种无损伤、无侵入性的物理治疗方法，目前已逐渐得到人们关注。研究报道rTMS可以提高正常老化和阿尔兹海默病（AD）患者的认知功能损伤。rTMS对认知功能的改善是通过网络化，多通路调节脑组织结构和功能发挥作用的。研究报道，rTMS作用于神经系统可以提高神经元的兴奋性，并增加突触可塑性。rTMS改善突触可塑性的效应可以表现为长时程增强（LTP）效应。动物实验证明，在应用高频rTMS改善动物认知功能的同时，所记录到的海马脑片LTP显著增强了。在基因水平、蛋白质水平、代谢产物水平的研究表明，rTMS可以通过调节改善神经元可塑性相关基因、蛋白的表达和代谢产物平衡保护神经元。然而，高频rTMS改善认知功能时提高神经元兴奋性的具体机制及rTMS改善认知功能相关的物质基础目前还不是十分清楚。
海马和额皮层是与认知功能密切相关的脑区，在老化过程和一些神经系统退行性疾病中也是最易受损伤的区域。研究表明海马神经元兴奋性在正常老化过程中是下降的，并表现为后超极化幅度的增大、静息电位的超极化。神经元的信息传递编码为动作电位进行传递，因此，如果动作电位的形成变慢，信息传递速率会受到影响，这样在神经网络信息传递中，效率就会降低。研究已经证明与青年个体相比，老年个体的神经元兴奋性显著下降低，而神经元兴奋性的下降在认知过程中起到了关键作用。神经元兴奋性降低的具体机制--物质基础，是如何变化的。这一变化在病理生理刺激，基因改变等作用方式下如何表现为动态的、多系统的变化。应用生物体液和组织对机体病理机制进行代谢物质的检测，结合非靶标多元统计分析方法，分析疾病过程中的物质变化基础，目前在许多领域得到了广泛应用。这种物质检测分析方法的优势是在疾病预防和治疗过程中，可以发现机体生化改变的生物标志物。在对AD疾病的研究中，已有应用非靶标多元统计方法对AD病人脑脊液进行了分析，发现AD病人机体的新陈代谢与正常对照人员相比，发生了明显变化。
脑老化是脑组织随着年龄增长发生的组织结构和功能的变化。随着年龄的增长，个体在脑老化过程中可表现为学习记忆能力的降低。啮齿类动物作为实验模型研究脑老化及老化相关的认知功能障碍具有很多优点：啮齿类动物的海马和前额皮层在老化中与人类相似，是非常容易受损的，并伴有结构和功能的变化。昆明小鼠在老化过程中可表现出认知功能的损伤，是研究脑老化的理想动物模型。在对啮齿类动物进行认知功能评估的行为学实验中，被动逃避反应实验和新物体识别实验是经典的认知功能测试方法。
本课题中我们观察了rTMS是如何改善昆明小鼠老化过程中认知功能的损伤，及其内在电生理机制和代谢物质基础。
1rTMS改善老化引起的认知功能障碍
观察rTMS是否可以改善昆明小鼠在年龄增长过程中出现的认知功能损害。为证明rTMS对认知功能的影响，应用被动逃避反应实验和新物体识别实验对小鼠的认知功能进行检测。
方法：3-4月龄（青年）昆明小鼠，9-10月龄（成年）昆明小鼠，16-17月龄（老年）昆明小鼠。在实验室恒温20-25摄氏度适应饲养1天以适应实验室环境，然后用于实验。
实验分组：
（1）老年rTMS组：16-17月龄老年昆明小鼠，磁头于小鼠的颅顶部每天进行10组频率为25赫兹的磁刺激，总共刺激脉冲数为1000个，连续14天。
（2）老年sham组：16-17月龄老年昆明小鼠，以磁头反面于小鼠颅顶部进行与rTMS组相似的无功能的磁刺激。
（3）成年组：9-10月龄成年昆明小鼠，与老年sham组小鼠刺激方式相同。
（4）青年组：3-4月龄青年昆明小鼠，与老年sham组小鼠刺激方式相同。
各组动物在相同环境下饲养，然后通过被动逃避反应实验和新物体识别实验测试各组动物学习记忆能力的差异。
结果：新物体识别实验成绩显示，在1h时间点，小鼠触碰两物体的总时间在青年组、成年组、老年组之间没有明显差异，在24h小鼠触碰两物体的总时间在三组之间同样也没有发现显著差异。在1h时间点，青年组和成年组之间的认知指数没有明显差异；与青年组和成年组相比，老年组的认知指数显著降低（P<0.05）。同样在24h时间点，与青年和成年组相比，老年组小鼠的认知指数显著降低（P<0.05）；而青年组和成年组之间的认知指数没有显著差异。在1h时间点，小鼠触碰两物体的总时间在老年组和老年rTMS组没有明显差异，同样在24h也没有发现显著差异。在1h时间点，与老年组相比，老年rTMS组的认知指数显著提高（P<0.05）。同样在24h时间点，与老年组相比，老年rTMS组小鼠的认知指数显著提高（P<0.05）。
被动逃避反应实验成绩显示，适应阶段的潜伏期在青年组、成年组和老年组没有显著差异。与青年组和成年组相比，学习阶段的电击次数在老年昆明鼠明显增多（P<0.05），而青年组和成年组之间无显著差异。与青年鼠和成年鼠相比，记忆阶段的被动逃避潜伏期在老年鼠出现了显著缩短（P<0.05），同样青年组和成年组之间没有显著差异。被动逃避反应实验适应阶段的潜伏期在老年组和老年rTMS组没有显著差异。与老年组相比，学习阶段的电击次数在老年rTMS昆明鼠明显减少（P<0.05）。同样与老年鼠相比，记忆阶段的被动逃避潜伏期在老年rTMS鼠出现了显著增长（P<0.05）。
以上结果提示，昆明小鼠的认知功能在青年组和成年组没有出现明显差异，而在16月龄时开始出现下降，而应用rTMS可以改善老化引起的认知功能损伤。2rTMS改善老化相关的神经元电生理变化
观察rTMS是否可以通过改善电压依赖性钙通道（VDCC）来调节神经元兴奋性，进而改善老年小鼠的认知功能障碍。
方法：3-4月龄昆明小鼠，16-17月龄昆明小鼠。在相同实验室条件饲养，实验室恒温20-25摄氏度，然后用于实验。
实验分组：
（1）老年rTMS组：16-17月龄老年小鼠，磁头于小鼠的颅顶部每天进行10组频率为25赫兹的磁刺激，总共刺激脉冲数为1000个，连续14天。
（2）老年sham组：16-17月龄老年小鼠，以磁头反面于小鼠颅顶部进行与rTMS组相似的无功能的磁刺激。
（3）青年组：3-4月龄青年小鼠，与老年sham组小鼠刺激方式相同。
各组动物在相同环境下饲养，然后通过行为学实验测试后，而后进行神经元兴奋性，VDCC等电生理指标的记录。
结果：全细胞电流钳记录结果显示，rTMS可以改善老化引起的神经元兴奋性变化。与青年鼠相比，在老年鼠海马CA1区的神经元静息电位发生了显著超级化（P<0.05）。在应用rTMS后发现，与老年鼠相比，在老年磁刺激组小鼠海马CA1区的神经元静息电位显著去极化（P<0.05）。与青年组相比，老年组海马CA1区神经元的动作电位产生频率显著下降（P<0.05）。而高频经颅磁刺激可以显著提高老年神经元的动作电位的产生频率（P<0.05）。同样与青年组相比，在老年组海马CA1区神经元的后超极化幅度显著增大（P<0.05）。而应用rTMS后，可显著降低老年组的后超级化幅度（P<0.05）。而动作电位的阈值记录结果显示，在青年组、老年组和老年磁刺激组之间，没有发现显著差异。
全细胞电流钳记录结果显示，rTMS可以显著降低老年海马CA1区神经元的VDCC。与老年对照组神经元相比，在测试电压分别为-20mV、-10mV和0mV时，在老年rTMS组神经元所记录的电流强度均显著降低（P<0.05）。
以上结果表明，rTMS可以通过改善VDCC来调节神经元兴奋性，进而改善老年小鼠的认知功能障碍。3rTMS改善老化相关的脑组织代谢产物变化
观察应用气质联用分析方法对小鼠脑组织代谢产物进行检测，应用偏最小二乘法判别分析（PLS-DA）和主成分分析（PCA）方法对结果进行分析，进一步应用one-wayANOVA统计方法深入分析差异代谢物。
方法：3-4月龄昆明小鼠，9-10月龄昆明小鼠，16-17月龄昆明小鼠。在相同实验室条件饲养，实验室恒温20-25摄氏度，然后用于实验。
实验分组：
（1）老年rTMS组：16-17月龄老年小鼠，磁头于小鼠的颅顶部每天进行10组频率为25赫兹的磁刺激，总共刺激脉冲数为1000个，连续14天。
（2）老年sham组：16-17月龄老年小鼠，以磁头反面于小鼠颅顶部进行与rTMS组相似的无功能的磁刺激。
（3）青年组：3-4月龄青年小鼠，与老年sham组小鼠刺激方式相同。
各组动物在相同环境下饲养，然后通过行为学实验测试后，应用气质联用方法进行脑组织代谢产物的测试。
结果：脑组织代谢物轮廓在老化过程中和rTMS后的变化。PLS-DA得分图显示，三组样本被分布到了不同的区域。前两个主成分的累积R2Y是0.84，Q2是0.69。根据VIP>1，91种变量被选出来作为差异变量，其中一些变量被确定来自同一中代谢物，合并这些来自同一代谢产物的变量后，共得到23种差异代谢产物。
进一步应用one-wayANOVA对这些差异代谢物进行进一步统计，发现这23种代谢物在青年组、老年组和老年磁刺激组具有显著性差异。与青年组相比，16种代谢产物在老年组发生了显著变化，其中丙氨酸、磷酸、丝氨酸、苏氨酸、苹果酸、乳酸、尿素和肌醇的含量显著下降，GABA、柠檬酸、油酸、5,8,11,14,17-二十碳五烯酸、单硬脂酸甘油脂、反式-9-十八碳烯酸、抗坏血酸和胆固醇含量显著增多（P<0.05）。与老年对照组小鼠相比，在老年rTMS组小鼠，21种代谢产物发生了变化，其中磷酸、富马酸、苏氨酸、苹果酸、柠檬酸、丙氨酸、尿素、GABA、丝氨酸、焦磷酸、乳酸、焦谷氨酸、天冬氨酸、肌酐、天冬氨酸和胆固醇含量显著减少（P<0.05），油酸、5,8,11,14,17-二十碳五烯酸、N乙酰天冬氨酸、甘油磷酸含量显著增多（P<0.05）。
根据所筛选出的23种代谢差异物对青年组、老年组和老年rTMS组进行验证性主成分分析。得分图显示，根据前两个主成分，青年组、老年组和老年磁刺激组被显著区分开来，R2X和Q2分别是0.60和0.45。
对所有差异代谢产物进行变化趋势分析，胆固醇、GABA、抗坏血酸和柠檬酸为所筛选出的rTMS逆转脑组织老化过程中的代谢产物。对被动逃避反应实验的成绩和胆固醇、GABA、抗坏血酸和柠檬酸的水平进行相关性分析。发现，胆固醇和被动逃避潜伏期的Pearson Correlation是-0.413（P<0.05），GABA、抗坏血酸和柠檬酸和被动逃避潜伏期的PearsonCorrelation是-0.25、-0.080和-0.273。
上述结果表明，rTMS可以改善老化过程中认知功能相关的代谢产物紊乱，在这些差异代谢产物中，胆固醇可能是一个较为特殊、重要的认知功能相关的代谢标志物。
结论
（1）昆明小鼠的认知功能在青年组和成年组没有出现明显差异，而在16月龄时开始出现下降，应用rTMS可以改善老化引起的认知功能损伤。
（2）rTMS可以通过改善VDCC来调节神经元兴奋性，进而改善老年小鼠的认知功能障碍。
（3）rTMS可影响脑内代谢产物的变化，改善老化过程中认知功能相关的代谢产物紊乱，在这些差异代谢产物中，胆固醇可能是一个较为特殊、重要的认知功能相关的代谢标志物。
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 dysfunction
Application 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 impariment
We 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 ANOVA
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 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.
海马和额皮层是与认知功能密切相关的脑区，在老化过程和一些神经系统退行性疾病中也是最易受损伤的区域。研究表明海马神经元兴奋性在正常老化过程中是下降的，并表现为后超极化幅度的增大、静息电位的超极化。神经元的信息传递编码为动作电位进行传递，因此，如果动作电位的形成变慢，信息传递速率会受到影响，这样在神经网络信息传递中，效率就会降低。研究已经证明与青年个体相比，老年个体的神经元兴奋性显著下降低，而神经元兴奋性的下降在认知过程中起到了关键作用。神经元兴奋性降低的具体机制--物质基础，是如何变化的。这一变化在病理生理刺激，基因改变等作用方式下如何表现为动态的、多系统的变化。应用生物体液和组织对机体病理机制进行代谢物质的检测，结合非靶标多元统计分析方法，分析疾病过程中的物质变化基础，目前在许多领域得到了广泛应用。这种物质检测分析方法的优势是在疾病预防和治疗过程中，可以发现机体生化改变的生物标志物。在对AD疾病的研究中，已有应用非靶标多元统计方法对AD病人脑脊液进行了分析，发现AD病人机体的新陈代谢与正常对照人员相比，发生了明显变化。
脑老化是脑组织随着年龄增长发生的组织结构和功能的变化。随着年龄的增长，个体在脑老化过程中可表现为学习记忆能力的降低。啮齿类动物作为实验模型研究脑老化及老化相关的认知功能障碍具有很多优点：啮齿类动物的海马和前额皮层在老化中与人类相似，是非常容易受损的，并伴有结构和功能的变化。昆明小鼠在老化过程中可表现出认知功能的损伤，是研究脑老化的理想动物模型。在对啮齿类动物进行认知功能评估的行为学实验中，被动逃避反应实验和新物体识别实验是经典的认知功能测试方法。
本课题中我们观察了rTMS是如何改善昆明小鼠老化过程中认知功能的损伤，及其内在电生理机制和代谢物质基础。
1rTMS改善老化引起的认知功能障碍
观察rTMS是否可以改善昆明小鼠在年龄增长过程中出现的认知功能损害。为证明rTMS对认知功能的影响，应用被动逃避反应实验和新物体识别实验对小鼠的认知功能进行检测。
方法：3-4月龄（青年）昆明小鼠，9-10月龄（成年）昆明小鼠，16-17月龄（老年）昆明小鼠。在实验室恒温20-25摄氏度适应饲养1天以适应实验室环境，然后用于实验。
实验分组：
（1）老年rTMS组：16-17月龄老年昆明小鼠，磁头于小鼠的颅顶部每天进行10组频率为25赫兹的磁刺激，总共刺激脉冲数为1000个，连续14天。
（2）老年sham组：16-17月龄老年昆明小鼠，以磁头反面于小鼠颅顶部进行与rTMS组相似的无功能的磁刺激。
（3）成年组：9-10月龄成年昆明小鼠，与老年sham组小鼠刺激方式相同。
（4）青年组：3-4月龄青年昆明小鼠，与老年sham组小鼠刺激方式相同。
各组动物在相同环境下饲养，然后通过被动逃避反应实验和新物体识别实验测试各组动物学习记忆能力的差异。
结果：新物体识别实验成绩显示，在1h时间点，小鼠触碰两物体的总时间在青年组、成年组、老年组之间没有明显差异，在24h小鼠触碰两物体的总时间在三组之间同样也没有发现显著差异。在1h时间点，青年组和成年组之间的认知指数没有明显差异；与青年组和成年组相比，老年组的认知指数显著降低（P<0.05）。同样在24h时间点，与青年和成年组相比，老年组小鼠的认知指数显著降低（P<0.05）；而青年组和成年组之间的认知指数没有显著差异。在1h时间点，小鼠触碰两物体的总时间在老年组和老年rTMS组没有明显差异，同样在24h也没有发现显著差异。在1h时间点，与老年组相比，老年rTMS组的认知指数显著提高（P<0.05）。同样在24h时间点，与老年组相比，老年rTMS组小鼠的认知指数显著提高（P<0.05）。
被动逃避反应实验成绩显示，适应阶段的潜伏期在青年组、成年组和老年组没有显著差异。与青年组和成年组相比，学习阶段的电击次数在老年昆明鼠明显增多（P<0.05），而青年组和成年组之间无显著差异。与青年鼠和成年鼠相比，记忆阶段的被动逃避潜伏期在老年鼠出现了显著缩短（P<0.05），同样青年组和成年组之间没有显著差异。被动逃避反应实验适应阶段的潜伏期在老年组和老年rTMS组没有显著差异。与老年组相比，学习阶段的电击次数在老年rTMS昆明鼠明显减少（P<0.05）。同样与老年鼠相比，记忆阶段的被动逃避潜伏期在老年rTMS鼠出现了显著增长（P<0.05）。
以上结果提示，昆明小鼠的认知功能在青年组和成年组没有出现明显差异，而在16月龄时开始出现下降，而应用rTMS可以改善老化引起的认知功能损伤。2rTMS改善老化相关的神经元电生理变化
观察rTMS是否可以通过改善电压依赖性钙通道（VDCC）来调节神经元兴奋性，进而改善老年小鼠的认知功能障碍。
方法：3-4月龄昆明小鼠，16-17月龄昆明小鼠。在相同实验室条件饲养，实验室恒温20-25摄氏度，然后用于实验。
实验分组：
（1）老年rTMS组：16-17月龄老年小鼠，磁头于小鼠的颅顶部每天进行10组频率为25赫兹的磁刺激，总共刺激脉冲数为1000个，连续14天。
（2）老年sham组：16-17月龄老年小鼠，以磁头反面于小鼠颅顶部进行与rTMS组相似的无功能的磁刺激。
（3）青年组：3-4月龄青年小鼠，与老年sham组小鼠刺激方式相同。
各组动物在相同环境下饲养，然后通过行为学实验测试后，而后进行神经元兴奋性，VDCC等电生理指标的记录。
结果：全细胞电流钳记录结果显示，rTMS可以改善老化引起的神经元兴奋性变化。与青年鼠相比，在老年鼠海马CA1区的神经元静息电位发生了显著超级化（P<0.05）。在应用rTMS后发现，与老年鼠相比，在老年磁刺激组小鼠海马CA1区的神经元静息电位显著去极化（P<0.05）。与青年组相比，老年组海马CA1区神经元的动作电位产生频率显著下降（P<0.05）。而高频经颅磁刺激可以显著提高老年神经元的动作电位的产生频率（P<0.05）。同样与青年组相比，在老年组海马CA1区神经元的后超极化幅度显著增大（P<0.05）。而应用rTMS后，可显著降低老年组的后超级化幅度（P<0.05）。而动作电位的阈值记录结果显示，在青年组、老年组和老年磁刺激组之间，没有发现显著差异。
全细胞电流钳记录结果显示，rTMS可以显著降低老年海马CA1区神经元的VDCC。与老年对照组神经元相比，在测试电压分别为-20mV、-10mV和0mV时，在老年rTMS组神经元所记录的电流强度均显著降低（P<0.05）。
以上结果表明，rTMS可以通过改善VDCC来调节神经元兴奋性，进而改善老年小鼠的认知功能障碍。3rTMS改善老化相关的脑组织代谢产物变化
观察应用气质联用分析方法对小鼠脑组织代谢产物进行检测，应用偏最小二乘法判别分析（PLS-DA）和主成分分析（PCA）方法对结果进行分析，进一步应用one-wayANOVA统计方法深入分析差异代谢物。
方法：3-4月龄昆明小鼠，9-10月龄昆明小鼠，16-17月龄昆明小鼠。在相同实验室条件饲养，实验室恒温20-25摄氏度，然后用于实验。
实验分组：
（1）老年rTMS组：16-17月龄老年小鼠，磁头于小鼠的颅顶部每天进行10组频率为25赫兹的磁刺激，总共刺激脉冲数为1000个，连续14天。
（2）老年sham组：16-17月龄老年小鼠，以磁头反面于小鼠颅顶部进行与rTMS组相似的无功能的磁刺激。
（3）青年组：3-4月龄青年小鼠，与老年sham组小鼠刺激方式相同。
各组动物在相同环境下饲养，然后通过行为学实验测试后，应用气质联用方法进行脑组织代谢产物的测试。
结果：脑组织代谢物轮廓在老化过程中和rTMS后的变化。PLS-DA得分图显示，三组样本被分布到了不同的区域。前两个主成分的累积R2Y是0.84，Q2是0.69。根据VIP>1，91种变量被选出来作为差异变量，其中一些变量被确定来自同一中代谢物，合并这些来自同一代谢产物的变量后，共得到23种差异代谢产物。
进一步应用one-wayANOVA对这些差异代谢物进行进一步统计，发现这23种代谢物在青年组、老年组和老年磁刺激组具有显著性差异。与青年组相比，16种代谢产物在老年组发生了显著变化，其中丙氨酸、磷酸、丝氨酸、苏氨酸、苹果酸、乳酸、尿素和肌醇的含量显著下降，GABA、柠檬酸、油酸、5,8,11,14,17-二十碳五烯酸、单硬脂酸甘油脂、反式-9-十八碳烯酸、抗坏血酸和胆固醇含量显著增多（P<0.05）。与老年对照组小鼠相比，在老年rTMS组小鼠，21种代谢产物发生了变化，其中磷酸、富马酸、苏氨酸、苹果酸、柠檬酸、丙氨酸、尿素、GABA、丝氨酸、焦磷酸、乳酸、焦谷氨酸、天冬氨酸、肌酐、天冬氨酸和胆固醇含量显著减少（P<0.05），油酸、5,8,11,14,17-二十碳五烯酸、N乙酰天冬氨酸、甘油磷酸含量显著增多（P<0.05）。
根据所筛选出的23种代谢差异物对青年组、老年组和老年rTMS组进行验证性主成分分析。得分图显示，根据前两个主成分，青年组、老年组和老年磁刺激组被显著区分开来，R2X和Q2分别是0.60和0.45。
对所有差异代谢产物进行变化趋势分析，胆固醇、GABA、抗坏血酸和柠檬酸为所筛选出的rTMS逆转脑组织老化过程中的代谢产物。对被动逃避反应实验的成绩和胆固醇、GABA、抗坏血酸和柠檬酸的水平进行相关性分析。发现，胆固醇和被动逃避潜伏期的Pearson Correlation是-0.413（P<0.05），GABA、抗坏血酸和柠檬酸和被动逃避潜伏期的PearsonCorrelation是-0.25、-0.080和-0.273。
上述结果表明，rTMS可以改善老化过程中认知功能相关的代谢产物紊乱，在这些差异代谢产物中，胆固醇可能是一个较为特殊、重要的认知功能相关的代谢标志物。
结论
（1）昆明小鼠的认知功能在青年组和成年组没有出现明显差异，而在16月龄时开始出现下降，应用rTMS可以改善老化引起的认知功能损伤。
（2）rTMS可以通过改善VDCC来调节神经元兴奋性，进而改善老年小鼠的认知功能障碍。
（3）rTMS可影响脑内代谢产物的变化，改善老化过程中认知功能相关的代谢产物紊乱，在这些差异代谢产物中，胆固醇可能是一个较为特殊、重要的认知功能相关的代谢标志物。
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 dysfunction
Application 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 impariment
We 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 ANOVA
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 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.
引文
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