【摘要】：目的通過與鹽酸帕羅西汀比較,觀察“疏肝調(diào)神”針法對(duì)創(chuàng)傷后應(yīng)激障礙(PTSD)睡眠障礙模型大鼠異常睡眠腦電的調(diào)節(jié)作用,以及對(duì)海馬CA1、CA3區(qū)異常神經(jīng)信息編碼時(shí)空模式與受損神經(jīng)元超微結(jié)構(gòu)的影響。本研究旨在驗(yàn)證“疏肝調(diào)神”針法干預(yù)PTSD睡眠障礙的有效性,并從修復(fù)海馬神經(jīng)元結(jié)構(gòu)以及重構(gòu)神經(jīng)網(wǎng)絡(luò)動(dòng)作電位發(fā)放功能角度,揭示“疏肝調(diào)神”針法影響PTSD睡眠障礙的神經(jīng)生物學(xué)機(jī)制,為“疏肝調(diào)神”針法治療PTSD睡眠障礙的有效性提供科學(xué)實(shí)驗(yàn)依據(jù)。方法選取120只SD大鼠,適應(yīng)性飼養(yǎng)后隨機(jī)分為兩組,第一組70只,按實(shí)驗(yàn)要求隨機(jī)分為空白組、手術(shù)組、模型組、抓取組、針刺組和西藥組,先后用于實(shí)驗(yàn)一、三；第二組50只,按實(shí)驗(yàn)要求隨機(jī)分為空白組、模型組、抓取組、針刺組和西藥組,用于實(shí)驗(yàn)二。各實(shí)驗(yàn)均以復(fù)合應(yīng)激法復(fù)制PTSD睡眠障礙大鼠模型,并在造模開始同時(shí)對(duì)西藥組大鼠以鹽酸帕羅西汀灌胃治療,對(duì)針刺組選取百會(huì)、內(nèi)關(guān)、神門、太沖四穴以“疏肝調(diào)神”針法針刺治療,并對(duì)抓取組采用與各治療組相同的抓取法固定,而第一組大鼠還需在造模前行腦電電極埋置并術(shù)后恢復(fù)。上述干預(yù)結(jié)束后,按以下方法進(jìn)行采集、分析。賣驗(yàn)一對(duì)大鼠連接電纜進(jìn)行8：00～20：00腦電記錄,通過腦電區(qū)分,獲得各組大鼠晝間12h睡眠潛伏期、覺醒—睡眠周期和睡眠時(shí)相特征。實(shí)驗(yàn)二以在體多通道技術(shù)記錄海馬CA1、CA3區(qū)動(dòng)作電位,以軟件計(jì)算動(dòng)作電位發(fā)放量、放電頻率、波形幅值、峰—峰間期(ISI)和功率譜密度(PSD),并繪制相應(yīng)圖譜。實(shí)驗(yàn)三對(duì)大鼠進(jìn)行心臟灌注,取海馬CA1、CA3區(qū)制備組織切片,并以透謝電鏡觀察神經(jīng)元數(shù)量、細(xì)胞核、細(xì)胞器及突觸超微結(jié)構(gòu)。對(duì)上述資料通過組間比較,獲得實(shí)驗(yàn)結(jié)果,闡明研究結(jié)論。結(jié)果1.睡眠腦電特征模型組與手術(shù)組相比,非快速動(dòng)眼睡眠(NREMs)和快速動(dòng)眼睡眠(REMs)潛伏期、覺醒期延長,總睡眠期、NREMs和REMs縮短,8：00～9：00、15：00～18：00覺醒期(總睡眠期)延長(縮短),NREMs在8：00～9：00、15：00～16：00和REMs.在9：00～10：00、11：00～18：00縮短,均有顯著性差異(P0.05,P0.01)；其余均未見明顯差異(P0.05)。抓取組與模型組相比,均未見明顯差異(P0.05)。西藥組與抓取組相比,NREMs潛伏期、總睡眠期、NREMs和REM8延長,覺醒期縮短,8：00～10：00、14：00～16：00覺醒期(總睡眠期)縮短(延長),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延長,均有顯著性差異(P0.05,P0.01)；其余均未見明顯差異(P0.05)。針刺組與抓取組相比,NREMs潛伏期、總睡眠期、NREMs和REMs延長,覺醒期縮短,8：00～9：00、12：00～13：00、15：00～18：00覺醒期(總睡眠期)縮短(延長),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延長,均有顯著性差異(P0.05,P0.01)；其余均未見明顯差異(P0.05)。針刺組與西藥組相比,覺醒期(總睡眠期)在9：00～10：00較長(較短),在16：00～17：00較短(較長),NREMs在9：00～1 0：00和REMs在11：00～12：00、14：00～15：00較短,均有顯著性差異(P0.05)；其余均未見明顯差異(P0.05)。2.海馬神經(jīng)編碼時(shí)空模式模型組與空白組相比,CA1、CA3區(qū)動(dòng)作電位發(fā)放量減少,放電頻率縮短,集中分布頻帶降低,由連續(xù)性轉(zhuǎn)為陣發(fā)性；放電波稀疏、凌亂,波形變窄,波形幅值均減小,放電間隔序列(ISI)延長；PSD下降,譜功率集中分布區(qū)域下移,均有顯著性差異(P0.05,P0.01)。抓取組與模型組相比,均未見明顯差異(P0.05)。西藥組與抓取組相比,CA1、CA3區(qū)動(dòng)作電位發(fā)放量增多,放電頻率延長,集中分布頻帶升高,由陣發(fā)性轉(zhuǎn)為斷續(xù)性；放電波規(guī)則、整齊,波形幅值增大,放電間隔序列(ISI)縮短；PSD上升,譜功率集中分布區(qū)域上移,均有顯著性差異(P0.05,P0.01)。CA1區(qū)波形變寬,但CA3區(qū)波形寬度無變化。針刺組與抓取組相比,CAl、CA3區(qū)動(dòng)作電位發(fā)放量增多,放電頻率延長,集中分布頻帶升高,由陣發(fā)性狀態(tài)轉(zhuǎn)為斷續(xù)性；放電波規(guī)則、整齊,波形變寬,波形幅值增大,放電間隔序列(ISI)縮短；PSD上升,譜功率集中分布區(qū)域上移,均有顯著性差異(P0.05,P0.01)。針刺組與西藥組相比,CA1、CA3區(qū)動(dòng)作電位發(fā)放量、放電頻率、放電頻率集中分布頻帶及狀態(tài),放電波形態(tài),以及CA1區(qū)波形幅值寬度,放電間隔序列(ISI)、PSD和譜功率集中分布區(qū)域相近,均未見明顯差異(P0.05),但針刺組大鼠CA3區(qū)波形幅值較寬,平均波形幅值較高,具有顯著性差異(P0.05)。3.海馬神經(jīng)元超微結(jié)構(gòu)手術(shù)組與空白組相比,除CA3區(qū)神經(jīng)元數(shù)量減少,細(xì)胞核略不規(guī)則,突觸間隙不夠明顯外,其余均未見明顯差異。模型組與手術(shù)組相比,細(xì)胞腫脹,電子密度低,細(xì)胞核不規(guī)則,核內(nèi)染色質(zhì)結(jié)構(gòu)松散；細(xì)胞質(zhì)內(nèi)結(jié)構(gòu)空曠或松散,線粒體腫脹,部分線粒體膜及嵴結(jié)構(gòu)消失,粗面內(nèi)質(zhì)網(wǎng)擴(kuò)張；突觸不清晰,結(jié)構(gòu)空曠,突觸小泡減少。抓取組與模型組相比,均未見明顯差異。西藥組與抓取組相比,神經(jīng)元數(shù)量增多,細(xì)胞核呈圓形,常染色質(zhì)豐富；CA1區(qū)粗面內(nèi)質(zhì)網(wǎng)、核糖體增多；突觸清晰,突觸結(jié)構(gòu)趨于正常,突觸小泡增多可見。針刺組與抓取組相比,神經(jīng)元數(shù)量增多,結(jié)構(gòu)清晰,核內(nèi)染色質(zhì)均勻且常染色質(zhì)豐富；除CA3區(qū)仍有部分線粒體輕度腫脹外,細(xì)胞器增多,線粒體結(jié)構(gòu)清晰,粗面內(nèi)置網(wǎng)條索樣分布,核糖體豐富,高爾基體常見；突觸清晰,突觸結(jié)構(gòu)趨于正常,突觸小泡增多且豐富。針刺組與西藥組相比,CA1區(qū)神經(jīng)元數(shù)量較多,體積較大,線粒體圓形或桿狀,結(jié)構(gòu)清晰,粗面內(nèi)質(zhì)網(wǎng)條索樣分布,高爾基復(fù)合體常見；CA3區(qū)神經(jīng)元結(jié)構(gòu)清晰,電子密度較高,細(xì)胞核形狀尚不規(guī)則,核內(nèi)染色質(zhì)分布基本均勻；細(xì)胞器豐富,粗面內(nèi)質(zhì)網(wǎng)條索樣分布,核糖體豐富,高爾基復(fù)合體常見,突觸小泡更豐富。其余未見明顯差異。結(jié)論第一,復(fù)合應(yīng)激法可使大鼠睡眠腦電發(fā)生異常變化,引起PTSD睡眠障礙; PTSD睡眠障礙模型大鼠海馬CA1、CA3區(qū)神經(jīng)信息編碼時(shí)空模式異常改變可能是PTSD睡眠障礙發(fā)生的重要中樞機(jī)制,而這種改變可能與該腦區(qū)神經(jīng)元形態(tài)結(jié)構(gòu)損傷有關(guān)。第二,“疏肝調(diào)神”針法可明顯改變PTSD睡眠障礙大鼠異常睡眠腦電,促使睡眠好轉(zhuǎn)；“疏肝調(diào)神”針法還能恢復(fù)動(dòng)作電位發(fā)放特征,調(diào)節(jié)海馬CA1、CA3區(qū)異常的神經(jīng)信息編碼時(shí)空模式,這可能是其治療PTSD睡眠障礙的重要中樞機(jī)制,而相應(yīng)腦區(qū)神經(jīng)元修復(fù)可能是促進(jìn)海馬功能重構(gòu)的主要原因。第三,與鹽酸帕羅西汀治療相比,應(yīng)用“疏肝調(diào)神”針法治療,對(duì)異常睡眠腦電、海馬神經(jīng)編碼時(shí)空模式的影響和神經(jīng)元修復(fù)的調(diào)節(jié)作用整體較好,且不會(huì)引起嗜睡等副反應(yīng),進(jìn)一步說明“疏肝調(diào)神”針法干預(yù)PTSD睡眠障礙可發(fā)揮更好的治療效應(yīng)。綜上,本研究認(rèn)為,“疏肝調(diào)神”針法可能通過修復(fù)海馬神經(jīng)元結(jié)構(gòu)促進(jìn)神經(jīng)網(wǎng)絡(luò)功能重構(gòu),從而發(fā)揮對(duì)PTSD睡眠障礙的治療效應(yīng)。
[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.
【學(xué)位授予單位】：成都中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R245

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8 賀弋;韓珍;陶虹;;中西醫(yī)結(jié)合臨床睡眠障礙學(xué)教學(xué)再實(shí)踐與探討[A];2008年中國睡眠研究會(huì)第五屆學(xué)術(shù)年會(huì)論文摘要匯編[C];2008年

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5 中國消費(fèi)者報(bào) 裴立英;睡眠障礙會(huì)導(dǎo)致兒童發(fā)育遲緩[N];中國消費(fèi)者報(bào);2005年

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