“疏肝调神”针法对PTSD睡眠障碍大鼠海马神经编码与功能重构影响的研究

发布时间：2018-07-20 16:15

【摘要】：目的通过与盐酸帕罗西汀比较,观察“疏肝调神”针法对创伤后应激障碍(PTSD)睡眠障碍模型大鼠异常睡眠脑电的调节作用,以及对海马CA1、CA3区异常神经信息编码时空模式与受损神经元超微结构的影响。本研究旨在验证“疏肝调神”针法干预PTSD睡眠障碍的有效性,并从修复海马神经元结构以及重构神经网络动作电位发放功能角度,揭示“疏肝调神”针法影响PTSD睡眠障碍的神经生物学机制,为“疏肝调神”针法治疗PTSD睡眠障碍的有效性提供科学实验依据。方法选取120只SD大鼠,适应性饲养后随机分为两组,第一组70只,按实验要求随机分为空白组、手术组、模型组、抓取组、针刺组和西药组,先后用于实验一、三；第二组50只,按实验要求随机分为空白组、模型组、抓取组、针刺组和西药组,用于实验二。各实验均以复合应激法复制PTSD睡眠障碍大鼠模型,并在造模开始同时对西药组大鼠以盐酸帕罗西汀灌胃治疗,对针刺组选取百会、内关、神门、太冲四穴以“疏肝调神”针法针刺治疗,并对抓取组采用与各治疗组相同的抓取法固定,而第一组大鼠还需在造模前行脑电电极埋置并术后恢复。上述干预结束后,按以下方法进行采集、分析。卖验一对大鼠连接电缆进行8：00～20：00脑电记录,通过脑电区分,获得各组大鼠昼间12h睡眠潜伏期、觉醒—睡眠周期和睡眠时相特征。实验二以在体多通道技术记录海马CA1、CA3区动作电位,以软件计算动作电位发放量、放电频率、波形幅值、峰—峰间期(ISI)和功率谱密度(PSD),并绘制相应图谱。实验三对大鼠进行心脏灌注,取海马CA1、CA3区制备组织切片,并以透谢电镜观察神经元数量、细胞核、细胞器及突触超微结构。对上述资料通过组间比较,获得实验结果,阐明研究结论。结果1.睡眠脑电特征模型组与手术组相比,非快速动眼睡眠(NREMs)和快速动眼睡眠(REMs)潜伏期、觉醒期延长,总睡眠期、NREMs和REMs缩短,8：00～9：00、15：00～18：00觉醒期(总睡眠期)延长(缩短),NREMs在8：00～9：00、15：00～16：00和REMs.在9：00～10：00、11：00～18：00缩短,均有显著性差异(P0.05,P0.01)；其余均未见明显差异(P0.05)。抓取组与模型组相比,均未见明显差异(P0.05)。西药组与抓取组相比,NREMs潜伏期、总睡眠期、NREMs和REM8延长,觉醒期缩短,8：00～10：00、14：00～16：00觉醒期(总睡眠期)缩短(延长),NREMs在8：00～10：00、15：00～17：00和REMs在9：00～1 0：00、11：00～13：00、14：00～15：00延长,均有显著性差异(P0.05,P0.01)；其余均未见明显差异(P0.05)。针刺组与抓取组相比,NREMs潜伏期、总睡眠期、NREMs和REMs延长,觉醒期缩短,8：00～9：00、12：00～13：00、15：00～18：00觉醒期(总睡眠期)缩短(延长),NREMs在8：00～9：00、12：00～13：00、15：00～17：00和REMs在12：00～13：00、15：00～17：.00延长,均有显著性差异(P0.05,P0.01)；其余均未见明显差异(P0.05)。针刺组与西药组相比,觉醒期(总睡眠期)在9：00～10：00较长(较短),在16：00～17：00较短(较长),NREMs在9：00～1 0：00和REMs在11：00～12：00、14：00～15：00较短,均有显著性差异(P0.05)；其余均未见明显差异(P0.05)。2.海马神经编码时空模式模型组与空白组相比,CA1、CA3区动作电位发放量减少,放电频率缩短,集中分布频带降低,由连续性转为阵发性；放电波稀疏、凌乱,波形变窄,波形幅值均减小,放电间隔序列(ISI)延长；PSD下降,谱功率集中分布区域下移,均有显著性差异(P0.05,P0.01)。抓取组与模型组相比,均未见明显差异(P0.05)。西药组与抓取组相比,CA1、CA3区动作电位发放量增多,放电频率延长,集中分布频带升高,由阵发性转为断续性；放电波规则、整齐,波形幅值增大,放电间隔序列(ISI)缩短；PSD上升,谱功率集中分布区域上移,均有显著性差异(P0.05,P0.01)。CA1区波形变宽,但CA3区波形宽度无变化。针刺组与抓取组相比,CAl、CA3区动作电位发放量增多,放电频率延长,集中分布频带升高,由阵发性状态转为断续性；放电波规则、整齐,波形变宽,波形幅值增大,放电间隔序列(ISI)缩短；PSD上升,谱功率集中分布区域上移,均有显著性差异(P0.05,P0.01)。针刺组与西药组相比,CA1、CA3区动作电位发放量、放电频率、放电频率集中分布频带及状态,放电波形态,以及CA1区波形幅值宽度,放电间隔序列(ISI)、PSD和谱功率集中分布区域相近,均未见明显差异(P0.05),但针刺组大鼠CA3区波形幅值较宽,平均波形幅值较高,具有显著性差异(P0.05)。3.海马神经元超微结构手术组与空白组相比,除CA3区神经元数量减少,细胞核略不规则,突触间隙不够明显外,其余均未见明显差异。模型组与手术组相比,细胞肿胀,电子密度低,细胞核不规则,核内染色质结构松散；细胞质内结构空旷或松散,线粒体肿胀,部分线粒体膜及嵴结构消失,粗面内质网扩张；突触不清晰,结构空旷,突触小泡减少。抓取组与模型组相比,均未见明显差异。西药组与抓取组相比,神经元数量增多,细胞核呈圆形,常染色质丰富；CA1区粗面内质网、核糖体增多；突触清晰,突触结构趋于正常,突触小泡增多可见。针刺组与抓取组相比,神经元数量增多,结构清晰,核内染色质均匀且常染色质丰富；除CA3区仍有部分线粒体轻度肿胀外,细胞器增多,线粒体结构清晰,粗面内置网条索样分布,核糖体丰富,高尔基体常见；突触清晰,突触结构趋于正常,突触小泡增多且丰富。针刺组与西药组相比,CA1区神经元数量较多,体积较大,线粒体圆形或杆状,结构清晰,粗面内质网条索样分布,高尔基复合体常见；CA3区神经元结构清晰,电子密度较高,细胞核形状尚不规则,核内染色质分布基本均匀；细胞器丰富,粗面内质网条索样分布,核糖体丰富,高尔基复合体常见,突触小泡更丰富。其余未见明显差异。结论第一,复合应激法可使大鼠睡眠脑电发生异常变化,引起PTSD睡眠障碍; PTSD睡眠障碍模型大鼠海马CA1、CA3区神经信息编码时空模式异常改变可能是PTSD睡眠障碍发生的重要中枢机制,而这种改变可能与该脑区神经元形态结构损伤有关。第二,“疏肝调神”针法可明显改变PTSD睡眠障碍大鼠异常睡眠脑电,促使睡眠好转；“疏肝调神”针法还能恢复动作电位发放特征,调节海马CA1、CA3区异常的神经信息编码时空模式,这可能是其治疗PTSD睡眠障碍的重要中枢机制,而相应脑区神经元修复可能是促进海马功能重构的主要原因。第三,与盐酸帕罗西汀治疗相比,应用“疏肝调神”针法治疗,对异常睡眠脑电、海马神经编码时空模式的影响和神经元修复的调节作用整体较好,且不会引起嗜睡等副反应,进一步说明“疏肝调神”针法干预PTSD睡眠障碍可发挥更好的治疗效应。综上,本研究认为,“疏肝调神”针法可能通过修复海马神经元结构促进神经网络功能重构,从而发挥对PTSD睡眠障碍的治疗效应。
[Abstract]:Objective To observe the effect of "Shugan Tiao Shen Shen" Acupuncture on abnormal sleep EEG in rats with post traumatic stress disorder (PTSD) sleep disorder model and the influence of the spatio-temporal pattern of abnormal nerve information coding in hippocampus CA1 and CA3 area and the ultrastructure of damaged neurons in the hippocampus of the rat model of posttraumatic stress disorder. The effect of acupuncture intervention on PTSD sleep disorder, and from the repair of the structure of hippocampal neurons and the function of reconstructing the neural network action potential, reveal the neurobiological mechanism of "Shugan Tiao Shen" acupuncture method affecting the PTSD sleep disorder, and provide scientific experimental basis for the efficacy of "Shugan Tiao Shen" needle therapy for the treatment of PTSD sleep disorder. 120 SD rats were randomly divided into two groups, the first group and 70 rats were randomly divided into the blank group, the operation group, the model group, the grab group, the acupuncture group and the western medicine group, which were used in the experiment one, the three and the second groups, and were randomly divided into the empty white group, the model group, the grabbing group, the acupuncture group and the western medicine group, which were used in the experiment two. The PTSD sleep disorder rat model was replicated by the compound stress method, and the rats in the western medicine group were treated with paroxetine hydrochloride in the western medicine group at the beginning of the model. The acupuncture group was treated with the acupuncture treatment of Baihui, Neiguan, Shenen, Tai Chung four acupoints with "Shugan Tiao Shen" needle method, and the grasping group was fixed with the same grasping method in the treatment group. In the first group, the brain electroencephalogram (EEG) electrode was required to be buried and recovered after the model. After the intervention, the rats were collected and analyzed according to the following methods. A pair of rats was sold at 8:00 to 20:00 EEG, and the diurnal 12h sleep latency, the awakening sleep cycle and the sleep phase were obtained through the EEG differentiation. In experiment two, the action potential was recorded in the hippocampal CA1 and CA3 region by body multichannel technique. The action potential distribution, discharge frequency, wave amplitude, peak to peak interval (ISI) and power spectral density (PSD) were calculated by software, and the corresponding atlas was plotted. Experimental three rats were perfused in the heart to prepare the tissue section of the hippocampus CA1, CA3 region, and observe the God with the transmission electron microscope. The results of the above data were compared, and the results of the 1. sleep EEG model group were compared with the operation group. Compared with the operation group, the latency of non rapid eye sleep (NREMs) and rapid eye sleep (REMs), the prolongation of the awakening period, the total sleep period, the shortened NREMs and REMs, at 8:00. 9:00,15:00 ~ 18:00 awakening period (total sleep period) prolonged (shortened), NREMs was significantly different (P0.05, P0.01) at 8:00 ~ 9:00,15:00 ~ 9:00,15:00 ~ (P0.05, P0.01), and no significant difference was found in the rest (P0.05). There was no significant difference (P0.05) between the grabbing group and the model group (P0.05). The NREMs incubation period, the total sleep period, the total sleep period, the prolongation of the NREMs and REM8, the awakening period, the period of the awakening from 8:00 to 16:00 to 16:00 (the total sleep period) shortened (the extension), and the NREMs was significantly different (P0.05, P0.01) at 1 0:00,11:00 to 13:00,14:00 to 13:00,14:00. There was no significant difference in the rest (P0.05). Compared with the gripping group, the NREMs latency, total sleep period, NREMs and REMs prolonged, the awakening period shortened, and the awakening period from 8:00 to 13:00,15:00 to 18:00 (the total sleep period) shortened (the total sleep period), and NREMs in 9:00,12:00 to 13:00,15:00 to REMs and REMs was 13. There were significant differences between 00,15:00 and 17:.00 (P0.05, P0.01), and the rest were not significantly different (P0.05). Compared with the western medicine group, the waking period (total sleep period) was longer (shorter) from 9:00 to 10:00 and shorter (longer) in the acupuncture group than in the western medicine group. There were significant differences (P0.05), and the rest were not significantly different (P0.05). Compared with the blank group, the spatial and temporal model group of.2. hippocampal neural coding was less than that in the blank group, CA1, the discharge of action potential was reduced, the frequency of discharge was shortened, the frequency band of the concentration distribution was reduced, and the frequency of the concentration distribution was reduced, and the radio waves were sparse, messy, and the waveform was narrowed, and the amplitude of the waveform decreased. The discharge interval sequence (ISI) was prolonged, PSD decreased and the concentration distribution of spectral power decreased significantly (P0.05, P0.01). There was no significant difference between the grabbing group and the model group (P0.05). Compared with the model group, the western medicine group and the grabbing group were compared with the grabbing group, CA1, CA3 area action potential distribution increased, the discharge frequency extended, the concentration distribution band increased, from paroxysmal to broken. Continuity; radio wave rules, neatly, wave amplitude increase, discharge interval sequence (ISI) shortening; PSD rise, the spectrum power concentrated distribution area moves up, there are significant differences (P0.05, P0.01).CA1 region width, but the CA3 area waveform width has no change. Compared with the grasping group, the acupuncture group increases the action potential in CAl, CA3 area, and the discharge frequency is prolonged. The concentration distribution band increased from paroxysmal state to intermittent; the radio wave rules, neatly, wave width, the amplitude of the waveform increased, the discharge interval sequence (ISI) shortened; the PSD increased, the spectral power concentrated distribution area moved up, there were significant differences (P0.05, P0.01). Compared with the western medicine group, the acupuncture group and the CA1, CA3 area action potential distribution and discharge frequency were compared. The distribution frequency and state of the discharge frequency, the shape of the discharge wave, the width of the amplitude of the CA1 region, the sequence of the discharge interval (ISI), the distribution of PSD and the spectral power are close, and there is no significant difference (P0.05), but the amplitude of the CA3 region in the CA3 region of the rats of the acupuncture group is wide, the amplitude of the average waveform is higher, and there is a significant difference (P0.05).3. hippocampal neurons. Compared with the blank group, the number of neurons in the microstructural operation group decreased, the nucleus was slightly irregular and the synaptic gap was not obvious. The cells were swollen, the electron density was low, the nucleus was irregular and the chromatin structure in the nucleus was loose, and the internal structure of the cytoplasm was empty or loose and the mitochondria swollen in the model group compared with the operation group. Swelling, partial mitochondrial membrane and ridge structure disappeared, rough endoplasmic reticulum dilated, synapses were not clear, structure open, and synaptic vesicles decreased. Compared with the model group, the grab group had no significant difference. Compared with the grabbing group, the number of neurons increased, the nucleus was round, and the chromatome was abundant; the CA1 area rough endoplasmic reticulum, ribosome increased; Synapses; Synapses The synaptic structure tended to be normal and the synaptic vesicles increased. Compared with the grabbing group, the number of neurons in the acupuncture group was increased, the structure was clear, the chromatin in the nucleus was uniform and the chromatin was abundant. In addition to the CA3 region, some mitochondria were slightly swelling, the organelle increased, the mitochondria structure was clear, the rough surface of the net stripe like distribution, the ribosome rich and the high levels were high. The synapses were common, the synapses were clear, the synapse structure tended to be normal, the synaptic vesicles increased and abundant. Compared with the western medicine group, the number of neurons in the CA1 area was more, the volume was larger, the mitochondria were round or rod-shaped, the structure was clear, the rough endoplasmic reticulum like distribution, the Golgi complex were common, the structure of the CA3 area was clear, the electron density was high, and the density was fine. The chromatin distribution in the nucleus is not regular, the chromatin distribution in the nucleus is basically uniform, the organelle is rich, the rough surface of the endoplasmic reticulum, the ribosome is rich, the Golgi complex is common, the synaptic vesicles are more abundant. The rest no obvious difference is found. Conclusion first, the compound stress method can cause abnormal changes of sleep electroencephalogram in rats, cause PTSD sleep disorder, and PTSD sleep. The abnormal changes in the spatio-temporal pattern of neural information coding in the hippocampal CA1 and CA3 region of the rat hippocampus may be an important central mechanism for the occurrence of PTSD sleep disorders. This change may be related to the morphological structure damage of the neurons in the brain. Second, the "Shugan Tiao Shen" needle method can obviously change the abnormal sleep brain electricity of the PTSD sleep disorder rats and promote sleep. The "Shugan Tiao Shen" needle method can restore the characteristics of the action potential distribution and regulate the spatio-temporal pattern of neural information coding in the CA1 and CA3 regions of the hippocampus, which may be an important central mechanism for the treatment of PTSD sleep disorders, and the corresponding neuron repair in the corresponding brain region may be the main cause of the reconstruction of the hippocampal power. Third, and Pa Rossi Dean hydrochloric acid. Compared with the treatment, the effect of "Shugan Tiao Shen" acupuncture therapy on abnormal sleep EEG, the influence of the spatio-temporal pattern of hippocampal neural coding and the regulation of neuron repair is better, and it will not cause somnolence and other side effects. It further illustrates that the intervention of "Shugan Tiao Shen Shen" needling can play a better therapeutic effect on PTSD sleep and sleep disorders. It is believed that "Shugan Tiao Shen" acupuncture method may promote the functional reconstruction of neural network by repairing hippocampal neuron structure, thereby playing a therapeutic effect on PTSD sleep disorders.
【学位授予单位】：成都中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R245

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