| ObjectiveTo observe the regulation effect of "Dispersing Liver and Regulating Spirit" Acupuncture Method on the irregular EEG, the damaged neural ultrastructure, and the abnormal spatiotemporal pattern of neural information coding of hippocampal CA1?CA3 regions of PTSD sleep disorder model rats by comparing with paroxetine hydrochloride. To confirm the effectiveness of "Dispersing Liver and Regulating Spirit" Acupuncture Method on PTSD sleep disorder and to reveal its neurobiological mechanism from the perspective of AP function restructure of the neural network and repair of the hippocampal neural structure; moreover, to provide its scientific experimental basis for the effectiveness on PTSD sleep disorder.MethodsAfter adaptive breeding,120 SD rats was randomly divided into 2 groups:70 for the group 1, which was randomly divided into the blank group, the surgery group, the model group, the grasp group, the acupuncture group and the paroxetine hydrochloride group, all of them were used respectively in the experiment 1 and the experiment 3; 50 for the group 2, which was randomly divided into the blank group, the surgery group, the model group, the grasp group, the acupuncture group and the paroxetine hydrochloride group, all of them were used in the experiment 2.In all the experiments, all the PTSD rat models were created by composite stress method, and the paroxetine hydrochloride group began to get the gastric perfusion of paroxetine hydrochloride since the creation of model started; meanwhile the acupuncture group began to get acupuncture treatment by "Dispersing Liver and Regulating Spirit" Method (on the points including GV20?PC6?HT7?LR3); the grasp group was grasped as the same way as all the groups which got treatment. For the first group, all the rats got brain electrode embedment and postoperative recovery before the model creation. After interventions were finished as above, all the records were collected and analyzed as follows.In the experiment 1, EEG from 8:00 to 20:00 was recorded by cable connection on rats to obtain the diurnal 12h Sleep Latency, the sleep-wake cycle and the phasic characteristics of each group. In the experiment 2, the action potential of hippocampal CA1?CA3 regions of rats was recorded by in vivo multi-channel recording methods, and the spike quantity, the discharge frequency, the wave amplitude, the inter-spike interval and PSD were analyzed by software and relative spectrums were drawn. In the experiment 3, the cardiac perfusion, the sampling, the section and preparation of tissues were applied, and then neuronal image of hippocampal CA1?CA3 regions were collected by transmission electron microscope to observe the ultrastructure of nucleus?organelle and synapsis. All the data was compared among groups to obtain the results and clarify the research conclusion.Results1. The character of sleep EEGCompared to the surgery group, the model group showed the lengthening of NREMs and latency of REMs?awakening stage, and the shortening in NREMs, REMs, TST,8:00?9:00 and 15:00?18:00 awakening stage (TST) lengthened (shortened), NREMs shortened in 8:00-9:00 and 15:00?16:00, and REMs shortened in 9:00?10:00 and 11:00-18:00 shortened, which all showed significant difference (P<0.05, P<0.01); the others showed no significant difference (P>0.05). There was no significant difference between the grasp group and the model group (P>0.05). Compared with the grasp group, the paroxetine hydrochloride group showed the lengthening of latency of REMs?TST?NREMs ? REMs, and the shortening in awakening stage, awakening stage (TST) shorted (lengthened) in 8:00?10:00 and 14:00?16:00, NREMs lengthened in 8:00?10:00 and 15:00?17:00 and REMs lengthened in 9:00?10:00,11:00?13:00 and 14:00?15:00, which all showed significant difference (P<0.05, P<0.01); the others showed no significant difference (P>0.05). Compared with the grasp group, the acupuncture group showed the lengthening in latency of NREMs?TST?NREMs and REMs and the shortening in awakening stage, awakening stag (TST) shortened (lengthened) in 8:00?9:00,12:00?13:00,15:00 ? 18:00, NREMs lengthened in 8:00?9:00,12:00?13:00,15:00?17:00 and REMs lengthened in 12:00? 13:00 and 15:00?17:00, which all showed significant difference (P<0.05, P<0.01); the others showed no significant difference (P> 0.05). Compared with the paroxetine hydrochloride group, the acupuncture group showed longer (shorter) awakening stage (TST) in 9:00?10:00, shorter (longer) in 16:00?17:00, shorter NREMs (9:00?10:00) and REMs (11:00?12:00 and 14:00?15:00), which all showed significant difference (P<0.05); the others showed no significant difference (P>0.05).2. The spatiotemporal pattern of hippocampal neural information encodingCompared to the blank group, the model group showed decrease of spike quantity of action potential, shortening of discharge frequency, and the dropping of concentrated band, which changed from continuous to paroxysmal; the discharge wave was sparse and messy, the waveform was narrowing and its amplitude was reducing, and ISI was prolonged; PSD was decreased, concentrated distribution area moved downwards, which all showed significant difference (P<0.05, P<0.01). There was no significant difference between the grasp group and the model group. Compared to the grasp group, the paroxetine hydrochloride group showed increase in pike quantity of action potential, and the prolonged discharge frequency, and raising of concentrated band, which changed from paroxysmal to continuous; the discharge wave was orderly and regulated, the waveform amplitude was amplified, and ISI was shortened; PSD was increased and concentrated distribution area raised, which all showed significant difference (P<0.05. P<0.01). The waveform showed broadening in CA1 region but no change in CA3 region. Compared to the grasp group, the acupuncture group showed increase in the spike quantity of action potential, and the prolonged discharge frequency, and raising of concentrated band, which changed from paroxysmal to continuous; the discharge wave was orderly and regulated, the waveform amplitude was amplified, and ISI was shortened; PSD was increased and its concentrated distribution area raised, which all showed significant difference (P<0.05, P<0.01). Compared to the paroxetine hydrochloride group, the acupuncture group showed no significant difference in the spike quantity of action potential, the discharge frequency, the concentrated band, the shape and amplitude of waveform, ISI, PSD and its concentrated distribution area (P> 0.05). However, the acupuncture group showed significantly broadening in the waveform CA3 region and higher average amplitude of waveform (P<0.05).3. The ultrastructure of hippocampal neuronCompared to the blank group, the acupuncture group showed decrease in the amount CA3 region neuron, and slightly irregular cell nucleus and obscure synaptic cleft; the others showed no significant difference. Compared to the surgery model, the model group showed more swollen cells, lower electron density, the more irregular cell nucleus and looser chromatin structure; the structure of cytoplasm was looser, and the chondriosome was more swollen with the disappearance of some mitochondrial membrane and ridge structure, and rough endoplasmic reticulum was expanding; the synaptic cleft was obscure, its structure of cytoplasm was looser, and the synaptic vesicle was decreased. There was no significant difference between the grasp group and the model group. Compared with the grasp group, the paroxetine hydrochloride group showed increased in the amount CA3 region neuron, and cell nucleus round with abundant euchromatin; the rough endoplasmic reticulum and ribosome were increased; the synapsis was clear in the structure and tended to be normal, and the synaptic vesicle was increased. Compared with the grasp group, the acupuncture group showed increased in the amount of neuron with clear structure, abundant and well-distributed euchromatin; excepted some slightly swollen chondriosome in CA3 region, the organelle was increased, and the chondriosome was clear in the structurem and rough endoplasmic reticulum distributed like cores, ribosome was abundant, golgi apparatus was easy to be observed; the synapsis was clear in the structure and tended to be normal, and the synaptic vesicle was increased. Compared with the paroxetine hydrochloride group, the acupuncture group showed more amount of CA1 region neuron which larger in size, and mitochondria was round or rod-shaped with clear structure, rough endoplasmic reticulum distributed like bar. golgi apparatus was easy to be observed; CA3 region neuron showed clear structure and higher electron density, the shaped of cell nucleus still irregular with basically well-distributed chromatin; the organelle was rich, the rough endoplasmic reticulum distributed like cores, ribosome was abundant, golgi apparatus was easy to be observed, and more abundant synaptic vesicle; the others showed no significant difference.ConelusionFirstly, the stimulation from composite stress method could cause the abnormal change of sleep EEG and lead to PTSD sleep disorder; the abnormal changes of sspatiotemporal pattern of hippocampal CA1?CA3 regions could be the important central mechanism of PTSD sleep disorder, which could be related to the morphological and structural damage of neuron in these regions.Secondly, "Dispersing Liver and Regulating Spirit"Acupuncture Method could significantly promote the recovery of abnormalities of sleep EEG in rats to improve sleep; it could also help in the recovery of the action potential's character, and regulate the abnormal changes of spatiotemporal pattern of neural information encoding of hippocampal CA1?CA3 regions, which could be the important central mechanism of acupuncture in treating PTSD sleep disorder. Also, the functional reconfiguration in relative brain regions could be the main reason of the recovery of spatiotemporal pattern of neural information encoding.Thirdly, "Dispersing Liver and Regulating Spirit"Acupuncture Method showed better effect in treating abnormal sleep EEG, spatiotemporal pattern of hippocampal neural information encoding, and regulating morphological recovery of neuron than paroxetine hydrochloride without causing side effects like somnolence, which furtherly suggested "Dispersing Liver and Regulating Spirit"Acupuncture Method has the better therapeutic effect on PTSD sleep disorder.In summary, this study suggested that "Dispersing Liver and Regulating Spirit"Acupuncture Method could promote the functional reconfiguration of neural network by repairing the hippocampal neural structure, thereby its therapeutic effect can showed in treating PDST sleep disorder. |
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