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

发布时间：2018-09-08 17:14

【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（P0.05）。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（P0.05）；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（P0.05）。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（P0.05）。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（P0.05），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（P0.05），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（P0.05）。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（P0.05）。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（P0.05）。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（P0.05）。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（P0.05）。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（P0.05）。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（P0.05）。而应用rTMS后，可显著降低老年组的后超级化幅度（P0.05）。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（P0.05）。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VIP1，91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（P0.05）。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（P0.05），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（P0.05）。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（P0.05），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。
[Abstract]:Repeated transcranial magnetic stimulation (rTMS) is a non-invasive and non-invasive physical therapy that has attracted increasing attention. It has been reported that rTMS can improve cognitive impairment in normal aging and Alzheimer's disease (AD) patients. The effects of rTMS on neuronal excitability and synaptic plasticity were reported. The effects of rTMS on synaptic plasticity were manifested as long-term potentiation (LTP). Animal experiments showed that LTP in hippocampal slices was significantly enhanced while rTMS was used to improve cognitive function. Studies on gene, protein and metabolite levels have shown that rTMS can protect neurons by regulating the expression of plasticity-related genes, protein and metabolite homeostasis. However, the specific mechanism of high-frequency rTMS in improving cognitive function and the substances related to rTMS in improving cognitive function are discussed. The foundation is not clear at the moment.
The hippocampus and frontal cortex are brain regions closely related to cognitive function, and are also the most vulnerable areas in the aging process and some neurodegenerative diseases. Studies have shown that the excitability of hippocampal neurons decreases during normal aging, and shows an increase in the amplitude of post-hyperpolarization and the hyperpolarization of resting potential. Transmission codes are action potentials, so if action potentials are slowed down, the rate of information transmission is affected, and thus the efficiency of neural network information transmission is reduced. It plays a key role in the process. The specific mechanism of neuronal excitability reduction, the material basis, is how to change. How this change in pathophysiological stimuli, gene changes and other ways of action to show dynamic, multi-system changes. Biological fluids and tissues on the body's pathological mechanisms for the detection of metabolic substances, combined with non- Target multivariate statistical analysis is widely used in many fields to analyze the basis of material changes in the course of disease. The advantage of this method is that biochemical markers can be found in the process of disease prevention and treatment. Methods The cerebrospinal fluid of AD patients was analyzed. It was found that the metabolism of AD patients was significantly different from that of normal controls.
Brain aging is a change in the structure and function of brain tissues with age.As individuals age, their learning and memory abilities decrease during brain aging.Rodents as experimental models have many advantages in studying cognitive impairment related to brain aging and aging: the hippocampus of rodents. The prefrontal cortex is very vulnerable to damage and accompanied by structural and functional changes in aging. Kunming mice exhibit cognitive impairment during aging and are ideal animal models for studying brain aging. And the new object recognition experiment is a classic cognitive function test method.
In this study, we observed how rTMS improves cognitive impairment in aging Kunming mice, and its intrinsic electrophysiological mechanism and metabolic basis.
1rTMS improves cognitive impairment caused by aging
To investigate whether rTMS can ameliorate cognitive impairment in Kunming mice during aging, passive avoidance response (PAR) test and new object recognition (NER) test were used to test the cognitive impairment in mice.
Methods: Kunming mice aged from 3 to 4 months, Kunming mice aged from 9 to 10 months and Kunming mice aged from 16 to 17 months were raised at 20 to 25 degrees Celsius in the laboratory for one day to adapt to the laboratory environment.
Experimental grouping:
(1) Aged rTMS group: aged Kunming mice aged 16-17 months were treated with 25 Hz magnetic stimulation at 10 groups per day on the top of the skull. The total number of stimulation pulses was 1000, lasting 14 days.
(2) Aged sham group: aged Kunming mice aged 16-17 months were treated with magnetic head reversed on the cranial top of the mice with similar nonfunctional magnetic stimulation as rTMS group.
(3) adult group: 9-10 month old adult Kunming mice were the same as the old sham group mice.
(4) young group: 3-4 month old young Kunming mice were the same as the old sham group mice.
Each group was fed in the same environment, and then the learning and memory abilities of each group were tested by passive escape response test and new object recognition test.
Results: The results of the new object recognition experiment showed that there was no significant difference in the total time of touching two objects between the young group, the adult group and the old group at 1 hour, and there was no significant difference in the total time of touching two objects between the three groups at 24 hours. The cognitive index of the aged group was significantly lower than that of the young group and the adult group (P 0.05). At the same time, the cognitive index of the aged group was significantly lower than that of the young group and the adult group (P 0.05). There was no significant difference in the cognitive index between the young group and the adult group. There was no significant difference in time between the elderly group and the elderly rTMS group, and no significant difference was found at 24 hours. At 1 hour, the cognitive index of the elderly rTMS group was significantly higher than that of the elderly group (P 0.05). At the same time, the cognitive index of the aged rTMS group was significantly higher than that of the elderly group (P 0.05).
The results of passive evasion test showed that there was no significant difference in the latency of adaptation stage between the young group, the adult group and the old group. There was no significant difference in the latency of passive escape reaction between the aged group and the adult group (P 0.05). There was no significant difference in the latency of passive escape reaction between the aged group and the aged rTMS group. The passive escape latency of the memory stage in the aged rTMS mice increased significantly (P 0.05).
These results suggest that there is no significant difference in cognitive function between young and adult Kunming mice, but it begins to decline at the age of 16 months. rTMS can improve cognitive impairment induced by aging. 2rTMS can improve neuronal electrophysiological changes associated with aging.
To observe whether rTMS can regulate neuronal excitability by improving voltage-dependent calcium channel (VDCC) and improve cognitive dysfunction in aged mice.
METHODS: Kunming mice aged 3-4 months and Kunming mice aged 16-17 months were fed in the same laboratory conditions at a constant temperature of 20-25 degrees Celsius in the laboratory, and then used in the experiment.
Experimental grouping:
(1) Aged rTMS group: aged 16-17 months old mice, magnetic head in the top of the mouse cranium every day for 10 groups of 25 Hz magnetic stimulation, a total of 1000 stimulation pulses, 14 days.
(2) Aged sham group: aged 16-17 months old mice were treated with magnetic head on the head of the head of the mice, which was similar to that of rTMS group.
(3) young group: 3-4 month old young mice were the same as the old sham group mice.
The animals in each group were fed in the same environment, then were tested by behavioral experiments, and then the neuronal excitability, VDCC and other electrophysiological indicators were recorded.
Results: Whole cell current clamp recording showed that rTMS could ameliorate neuronal excitability induced by aging. Compared with young rats, resting potentials of neurons in hippocampal CA1 region of aged rats were significantly increased (P 0.05). The frequency of action potential production in hippocampal CA1 neurons in the aged group was significantly lower than that in the young group (P 0.05). High frequency transcranial magnetic stimulation could significantly increase the frequency of action potential production in the aged neurons (P 0.05). The amplitude of posthyperpolarization in hippocampal CA1 neurons in the aged group was also higher than that in the young group. After rTMS was used, the post-hyper amplitude (P 0.05) of the aged group was significantly decreased, while the threshold of action potential was recorded. There was no significant difference between the young group, the aged group and the aged magnetic stimulation group.
Whole-cell current clamp recording showed that rTMS could significantly reduce the VDCC of neurons in hippocampal CA1 region in the aged. Compared with the aged control group, the recorded current intensity of neurons in the aged rTMS group decreased significantly at - 20 mV, - 10 mV and 0 mV respectively (P 0.05).
These results suggest that rTMS can regulate neuronal excitability by improving VDCC, thereby improving cognitive impairment in aged mice. 3rTMS can improve metabolites of aging-related brain tissues.
The metabolites in brain tissue of mice were detected by GC-MS, the results were analyzed by PLS-DA and PCA, and one-way ANOVA was used to analyze the metabolites.
Methods: Kunming mice aged from 3 to 4 months, Kunming mice aged from 9 to 10 months and Kunming mice aged from 16 to 17 months were fed under the same laboratory conditions at a constant temperature of 20 to 25 degrees Celsius in the laboratory.
Experimental grouping:
(1) Aged rTMS group: aged 16-17 months old mice, magnetic head in the top of the mouse cranium every day for 10 groups of 25 Hz magnetic stimulation, a total of 1000 stimulation pulses, 14 days.
(2) Aged sham group: aged 16-17 months old mice were treated with magnetic head on the head of the head of the mice, which was similar to that of rTMS group.
(3) young group: 3-4 month old young mice were the same as the old sham group mice.
All groups of animals were fed in the same environment, and then the metabolites of brain tissue were measured by GC-MS after behavioral tests.
Results: Changes in metabolite profiles of brain tissues during aging and after rTMS. PLS-DA scores showed that three groups of samples were distributed in different regions. The cumulative R2Y of the first two principal components was 0.84 and Q2 was 0.69. A total of 23 differential metabolites were obtained from the variables of the same metabolite.
Furthermore, one-way ANOVA was used to analyze these metabolites. The results showed that the 23 metabolites were significantly different in the youth group, the elderly group and the elderly magnetic stimulation group. Compared with the youth group, 16 metabolites had significant changes in the elderly group, including alanine, phosphoric acid, serine, threonine, malic acid, lactic acid, urea. The contents of GABA, citric acid, oleic acid, 5,8,11,14,17-eicosapentaenoic acid, glycerin monostearate, trans-9-octadecanoic acid, ascorbic acid and cholesterol increased significantly (P 0.05). Compared with the aged control group, 21 metabolites in the aged rTMS group were changed, including phosphoric acid, fumaric acid, threonine. Contents of acid, malic acid, citric acid, alanine, urea, GABA, serine, pyrophosphate, lactic acid, pyroglutamic acid, aspartic acid, creatinine, aspartate and cholesterol decreased significantly (P 0.05), oleic acid, 5, 8, 11, 14, 17-eicosapentaene.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R741.044

【共引文献】