Wei Yu-ting (魏玉婷), Yan Xing-ke (嚴(yán)興科), Han Ya-di (韓雅迪), Zhang Yan-feng (張彥峰)

Gansu University of Chinese Medicine, Lanzhou 730000, China

Abstract

Keywords: Acupuncture Therapy; Point, Baihui (GV 20); Point, Shenmen (HT 7); Point, Neiguan (PC 6); Point, Taichong (LR 3);Stress Disorders, Post-traumatic; Electroencephalography; Rats

Post-traumatic stress disorder (PTSD) is a long-lasting mental disorder that can develop after a person is exposed to a sudden, threatening and/or catastrophic event[1].It is characterized by three major symptoms:intrusive, recurrent recollections, dissociative episodes of reliving the trauma (known as ‘flashbacks’), and an increase in the fight-or-flight response[2].Epidemiological studies found that the incidence of PTSD in Wenchuan and Yushu earthquake areas in China was 85% and 60% respectively[3-4]; the average prevalence of PTSD in the United States is 8%[5].PTSD seriously affects the physical and mental health, as well as the social function of patients, and it is difficult to be cured[6].At present, treatment of this disease mainly relies on medication and psychological interventions.The long-term application of medication can cause drug addiction; the psychological intervention requires compliance and is time-consuming.Therefore, it has become an inevitable trend in the future to explore a relatively safe and effective therapeutic method with fewer adverse reactions for this disease.

In recent years, acupuncture has been gradually applied to the clinical treatment of PTSD[7], but there are relatively few reports on the mechanism of acupuncture in the prevention and treatment of PTSD.In this experiment, based on the effective treatment of PTSD by liver-soothing and mind-regulating acupuncture method, rat behaviors were observed and the changes in resting-state electroencephalography(EEG) in the rat models were detected, thus to reveal and investigate the regulation of neurological function by liver-soothing and mind-regulating acupuncture method in PTSD rat models, and to provide evidence for the mechanism study and clinical application of acupuncture on PTSD.

1 Materials and Methods

1.1 Laboratory animals and grouping

Sixty male adult Sprague-Dawley (SD) rats, 6-week old, weighing (180±20) g[8], were provided by the SPF Experimental Animal Center of the Research Center of Gansu University of Chinese Medicine.The sixty rats with normal memory were selected by screening with Morris water maze test.After adaptive feeding for 7 d in the rat breeding room, the rats were randomly divided into a blank control group, a model group, a grasping group, a paroxetine group and an acupuncture group,with 12 rats in each group.The disposal of animals during the experiment was in line with of theGuiding Opinions on the Treatment of Experimental Animalsissued by the Ministry of Science and Technology of the People's Republic of China in 2006.

1.2 Main instruments and reagents

Multichannel EEG acquisition and analysis system(Blackrock Microsystem Inc., USA); WMT-100 Morris water maze video acquisition and analysis system(Chengdu Taimeng Software Technology Co., Ltd.,China); self-made model preparation box for incarceration plus electric shock (output voltage: 60 V;electric current: 8 mA); paroxetine hydrochloride tablets(production batch number: 150104, specification: 20 mg/tablet, Zhejiang Jianfeng Pharmaceutical Co., Ltd.,China); chloral hydrate (production batch number:20150715, Shanghai Zhanyun Chemical Co., Ltd., China);disposable sterile acupuncture needle (specification:0.25 mm in diameter, 25 mm in length, Beijing Keyuanda Medical Products Factory, China).

1.3 Modeling methods

Except for rats in the blank control group, rats in the other groups were subjected to preparing the PTSD models using ‘incarceration plus electric shock’method[9].The self-made model preparation box was placed above the subplate and connected to alternating current (AC) (60 V, 8 mA) power through the electrical fence.A rat was put into a model preparation box at one time, and covered with a cover plate and then pressed by a heavy object to fix the cover for creating a dark and closable environment, as well as preventing animals from escape.The power was turned on to produce an inescapable electrical stimulation to the sole of rats’ paws.The power was turned off after 5 s of stimulation, and the stimulation interval was randomized for a total of 30 times.Rats were then taken out from the model preparation box, and put back into cages for breeding.The modeling time was recorded.The modeling preparation was performed once each morning and evening (30 min/time), at an interval of 4-8 h between two times of modeling preparation.Modeling preparation was continued for 7 d.Rats in each group received designated intervention from the first modeling preparation day.

1.4 Intervention methods

1.4.1 Acupuncture group

Acupoints: Baihui (GV 20), Neiguan (PC 6), Shenmen(HT 7) and Taichong (LR 3).

Methods: Acupoint location was referred to theExperimental Acupuncture Science[10]andSectional Anatomical Atlas of Sprague-Dawley Rat[11].The acupoints were routinely sterilized after the rats were grasped and fixed.Baihui (GV 20) was punctured subcutaneously for 4-5 mm with the tip backwards after the scalp was lifted, and then the needle was retained.Neiguan (PC 6), Shenmen (HT 7) and Taichong (LR 3) on the left side were punctured obliquely for 2-3 mm on Monday, Wednesday and Friday, and Neiguan (PC 6),Shenmen (HT 7) and Taichong (LR 3) on the right side were punctured obliquely for 2-3 mm on Tuesday,Thursday and Saturday.During the needle retaining, all the 3 acupoints were successively needled for 1 min with a small-amplitude twirling manipulation.The needles were retained for 4 min, and then all the needles were removed and the needled areas were pressed with dry cotton swab.The rats were put back into the cage for feeding.The treatment was performed once a day, and 6-day was a course of treatment.A total of 12-day intervention was continuously performed.

1.4.2 Paroxetine group

Paroxetine hydrochloride solution was intragastrically administered at 5 mL/(kg·bw) after the rats were grasped and fixed.The injection was evenly performed within 1 min after the intragastric needle was inserted into the stomach.The rats were continuously grasped for 4 min and then put back into the cage for rearing after the gavage needle was withdrawn.Intragastric treatment was performed once a day, for 6 d as a course of treatment.A total of 12-day intervention was continuously performed.

1.4.3 Grasping group

Each rat in the grasping group was subjected to grasping stress for 4 min, using the same grasping and fixing methods as in the acupuncture group and paroxetine group, and then put back into the cage for rearing.The grasping was performed once a day, and a total of 12-day grasping was continuously performed.

1.5 Observation indicators and detection methods

1.5.1 General condition

The activities and mental states of rats in each group were observed.

1.5.2 Water maze test

The preliminary experiment was conducted for 1 d,followed by positioning navigation experiment for 4 d and space exploration experiment for 1 d.The platform was fixed in the first quadrant, and the third quadrant was used as the site for entering water.The navigation experiment recorded the time spent on searching the platform after the rats entered water (i.e., the escape latency).If the rats did not find the platform within 2 min, the experimenter guided them to the platform,and the escape latency was recorded as 2 min.The space exploration experiment was carried out on the next day at the end of the positioning navigation experiment.The platform was removed to explore the times of rats crossing the original platform position and the effective areas within 2 min after the rats entered water.

1.5.3 Resting-state EEG

Resting-state EEG testing was performed in a quiet room.Anesthesia was performed before data collection.Before anesthesia, rats’ scalp hair was shaved, and they were fasted for 8 h and prohibited from taking water for 6 h.The grouped and numbered rats were randomly grasped and weighed, and then anesthetized by intraperitoneal injection of mixed anesthetic solution(10 g of chloral hydrate crystals were dissolved in normal saline for injection to a volume of 100 mL,stored at 4 ℃ away from light) at 5 mL/(kg·bw).The detailed anesthesia methods and assessment of rat status after anesthesia were referred to the abdominal anesthesia method by Tong S,et al[12].The anesthetized rats were placed on the table top.Two recording electrodes were inserted respectively at 1 cm above the midpoint of the connection line between rat inner canthus and 5 mm away to the left and the right,respectively.The insertion depth was kept at 5 mm.A needle was inserted at the left root of ear for 5 mm and the reference electrode was inserted, with the ground wire inserted into the root of the rat tail.The EEG data collected during the experiment were processed using the BrainVision Analyzer 2.1 (USA), and the band with the frequency within 0.1-40 Hz was selected.The original EEG and the power spectrum value after the Fourier transform of the α-wave, β-wave, δ-wave and э-wave of rat’s bilateral cerebral hemispheres in each group were analyzed and processed.The power spectrum was a graph in which the frequency was plotted on the abscissa and the square of the amplitude was plotted on the ordinate, and the signal energy was accorded with the frequency.

1.6 Statistical methods

Statistical processing was performed using SPSS 21.0 version software.If the measurement data were in a normal distribution, they would be expressed as mean ± standard deviation (), and the comparison among multiple groups was analyzed by one-way analysis of variance; the least significant difference (LSD)t-test was used to compare the differences between two groups.The Dunnettt-test was used for the data with heterogeneity of variance.The rank sum test was used when the data were in an abnormal distribution.P＜0.05 indicated a statistically significant difference.

2 Results

2.1 General condition

The activities and mental status of rats in each group were observed before daily intervention.For rats in the blank control group, the hair was bright and tidy; there was no increase in alertness, irritability, horripilation,gathering and scrunching; the rats were active and agile.For rats in the model group, the hair was erected and disordered in the early stage of modeling, and the alertness was increased and they were easy to be irritated.The hair color was dull in the later stage, and the rats preferred to be gathering and scrunching; the activity was decreased, which suggested that the rats showed obvious stress damage after incarceration plus electric shock, indicating that the models were established successfully.The manifests of rats in the grasping group were similar to those in a model group.Rats in the paroxetine group and acupuncture group showed transient increase in alertness and piloerection during the modeling period.After the modeling, there was no irritability or gathering and scrunching; the hair was basically normal, and the activity and agility were good.

2.2 Water maze test results

2.2.1 Comparison of positioning navigation

Compared with the blank control group, the average escape latency of rats in the model group was significantly prolonged (P＜0.05), suggesting that stress had an effect on the learning and memory acquisition process in rats.There was no significant difference in the average escape latency between the grasping group and model group (P＞0.05), suggesting that the grasping did not affect the learning and memory function of the rats.Compared with the grasping group, the average escape latencies of rats in the paroxetine group and acupuncture group were significantly shortened(P＜0.05), which suggested that both paroxetine and acupuncture could improve the effects of stress on learning and memory of the rats.The average escape latency of rats in the acupuncture group was not significantly different from that in the paroxetine group(P＞0.05), (Table 1).

There were mainly 4 ways for the rats to search for the hidden platforms: marginal, tendency, random and orthoscopic modes during the positioning navigation experiment.It can be seen from Figure 1 that rats in the blank control group mostly presented with orthoscopic mode, showing good learning and memory ability; rats in the model group and grasping group mainly searched by marginal and random modes, which suggested that the rats had poor learning and memory; rats in the acupuncture group and paroxetine group tended to use orthoscopic or tendency mode, showing relatively good learning and memory ability.

Table 1.Comparison of daily latency of the rats during positioning navigation experiment (, s)

Table 1.Comparison of daily latency of the rats during positioning navigation experiment (, s)

Note: Compared with the blank control group, 1) P＜0.05; compared with the grasping group, 2) P＜0.05

Group n Day 1 Day 2 Day 3 Day 4 Blank control 10 117.82±3.37 48.73±13.99 26.64±12.49 10.64±4.13 Model 10 119.60±1.27 73.70±29.391) 48.70±26.821) 32.40±19.021)Grasping 10 119.64±1.21 80.73±22.96 47.09±19.49 27.91±13.57 Paroxetine 10 120.00±0.00 57.50±27.632) 27.10±9.972) 12.80±5.902)Acupuncture 10 119.60±1.26 49.40±22.762) 24.90±14.262) 9.10±3.932)

Figure 1.Schematic diagram of rats to search the platform during positioning navigation experiment

2.2.2 Comparison of space exploration

It can be seen from Table 2, compared with the blank control group, the times of rats crossing the plateform or the effective area in the target quadrant decreased significantly in the model group (P＜0.01), which suggested that stress had an effect on the learning and memory ability of rats in the model group.There were no significant differences in all indicators between the grasping group and model group (allP＞0.05), which suggested that grasping did not affect the spatial learning and memory of the rats.Compared with the grasping group, the times of rats crossing the platform and the effective area in the paroxetine group and acupuncture group increased significantly (bothP＜0.05),suggesting that paroxetine and acupuncture improved the spatial learning and memory in the rats.There were no significant differences in all indicators between the acupuncture group and paroxetine group (allP＞0.05).

2.3 EEG detection

2.3.1 Bilateral cerebral hemisphere EEG and power spectrum of rats in each group

Rats in the blank control group mainly showed highfrequency and low-amplitude wave (α-wave), (Figure 2),while waves of rats in the model group and grasping group were dominated by low-frequency δ-wave and э-wave (Figure 3 and Figure 4), while high-frequency β-wave was dominant in the paroxetine group and acupuncture group (Figure 5 and Figure 6).In Figure 2-Figure 6, 1 indicated the left cerebral hemisphere, and 2 indicated the right cerebral hemisphere.

Table 2.Comparison of the space exploration experiment(, time)

Table 2.Comparison of the space exploration experiment(, time)

Note: Compared with the blank control group, 1) P＜0.01;compared with the grasping group, 2) P＜0.05

Group n Times crossing platform Times crossing the effective areas Blank control 10 7.55±1.37 10.36±1.50 Model 10 2.10±0.991) 3.10±0.991)Grasping 10 1.82±0.87 2.91±1.38 Paroxetine 10 6.90±1.372) 9.90±1.792)Acupuncture 10 7.10±1.372) 9.30±2.402)

Figure 2.EEG and power of bilateral cerebral hemispheres in the blank control group

Figure 3.EEG and power of bilateral cerebral hemispheres in the model group

Figure 4.EEG and power of bilateral cerebral hemispheres in the grasping group

Figure 5.EEG and power of bilateral cerebral hemispheres in the paroxetine group

Figure 6.EEG and power of bilateral cerebral hemispheres in the acupuncture group

2.3.2 Resting EEG data of rats in each group

It can be seen from Table 3, compared with the blank control group, the α-wave power spectrum values of the bilateral cerebral hemispheres in the model group decreased significantly (P＜0.01), suggesting that‘incarceration plus electric shock’ can make α-wave distribution abnormally changed in rat’s bilateral cerebral hemispheres.The α-wave power spectrum values of the bilateral cerebral hemispheres in the grasping group were insignificantly different from those in the model group (P＞0.05), suggesting that the grasping intervention could not affect the release of α-wave in the rat brain.Compared with the grasping group, α-wave power spectrum values of the bilateral cerebral hemispheres in the paroxetine group and acupuncture group increased significantly (P＜0.01),suggesting that paroxetine positively regulated the abnormally changed α-wave in the rat’s cerebral hemisphere.There was no significant difference in α-wave power spectrum between the acupuncture group and paroxetine group (P＞0.05).Compared with the blank control group, the power wave values of β-wave, δ-wave and э-wave in the bilateral cerebral hemispheres of the model group increased significantly(P＜0.01), suggesting that ‘incarceration plus electric shock’ induced abnormal β-wave, δ-wave and э-wave in the rats, to aggravate the abnormal changes in the distribution of the normal β-wave, δ-wave and э-wave.There were no significant differences in the indicators between the grasping group and model group (P＞0.05),suggesting that the grasping intervention could not affect the distribution of β-wave, δ-wave and э-wave in rat’s brain.Compared with the grasping group, the power spectrum values of β-wave, δ-wave and э-wave in the bilateral cerebral hemispheres of the paroxetine group and acupuncture group were significantly lower(P＜0.01), suggesting that paroxetine positively regulated the abnormally changed β-wave, δ-wave and э-wave in rat‘s cerebral hemispheres.There were no significant differences in each indicator between the acupuncture group and paroxetine group (P＞0.05).

Table 3.Comparison of EEG power spectrum values of bilateral cerebral hemispheres (, μV2)

Table 3.Comparison of EEG power spectrum values of bilateral cerebral hemispheres (, μV2)

Note: Compared with the blank control group, 1) P＜0.01; compared with the grasping group, 2) P＜0.01

Group n Site α-wave β-wave δ-wave Э-wave Blank control 12 Left side 2.50±0.56 1.37±0.61 8.25±2.24 0.73±0.34 Right side 2.50±0.57 1.42±0.53 8.30±2.70 0.79±0.52 Model 12 Left side 1.51±0.471) 2.77±0.601) 17.37±3.971) 3.80±1.511)Right side 1.58±0.72 3.07±1.15 17.37±3.24 3.83±0.62 Paroxetine 12 Left side 2.60±0.602) 1.42±0.592) 8.21±2.402) 0.82±0.482)Right side 1.47±0.641) 2.88±0.761) 17.24±4.891) 3.84±0.961)Grasping 12 Left side 1.65±0.82 2.79±0.96 17.29±2.19 3.81±0.81 Right side 2.55±0.722) 1.40±0.482) 8.25±2.422) 0.81±0.642)Acupuncture 12 Left side 2.64±0.522) 1.46±0.292) 8.24±3.322) 0.84±0.512)Right side 2.60±0.512) 1.45±0.252) 8.24±2.212) 0.84±0.632)

3 Discussion

PTSD is clinically characterized by mental,psychological, and behavioral abnormalities.The disease is mainly located in the heart and brain, and the heart and brain are connected through the Governor Vessel.The regulation of the heart-mind is the basic rule for treating such diseases.PTSD belongs to the category of emotional disease in traditional Chinese medicine.The theory of traditional Chinese medicine believes that the incidence of emotional illness is mostly caused by qi stagnation, which is related to the liver failing to maintain the normal flow of qi and dysfunction of the qi movement.Therefore, emotional disorders are predominantly treated by targeting the liver[13].This is also the embodiment of the theory that the liver qi stagnation disturbs the heart-mind.Modern research has found that ‘the liver maintains the free flow of qi’ is the core of regulating the stress response[14].The liver-soothing and mind-regulating acupuncture method is based on the treatment of liver and emphasizing the regulation of qi.Qi movement all over the body will be unblocked, thus to adjust the mind, via soothing the liver.This is also the characteristic of this study.Our previous clinical study has confirmed that liver-soothing and mind-regulating acupuncture method can effectively treat the symptoms associated with PTSD[15].

Baihui (GV 20) can adjust yin and yang, calm the mind,and refresh brain to open orifices.It is widely used in the treatment of sleep disorders, anxiety disorders,depressive disorders, schizophrenia and other diseases[16].Neiguan (PC 6) is the Luo-Connecting point of the Pericardium Meridian, and it is also one of the Eight Confluent Points with the Eight Extraordinary Meridians.It can regulate qi and blood, smoothe the emotion, and calm the mind, thus producing a good regulating effect on mental and emotional diseases.It is clinically synergied with other acupoints to treat emotional disorders[17].Shenmen (HT 7) is the Yuan-Primary point of the Heart Meridian, and can cure all heart-related disorders.Studies have found that acupuncture at Shenmen (HT 7) can effectively reduce the expression of arginine vasopressin (AVP) in the hypothalamic paraventricular nucleus of rats with stress and anxiety, and inhibit the activity of the hypothalamic pituitary adrenal axis (HPAA), which in turn achieves anti-stress and anxiolytic effects[18].Taichong (LR 3) is the Yuan-Primary point and the Shu-Stream point of the Liver Meridian.It has the effect of soothing the liver,regulating the spirit, promoting qi and adjusting the emotions.The four acupoints were used together to achieve the function of soothing liver to promote qi,refreshing brain to open orifices and calming the mind.

The Morris water maze experiment can effectively evaluate the learning and memory ability of animal models.The positioning navigation experiment is mainly for detecting the spatial learning ability of animals, and the space exploration experiment is mainly for evaluating the spatial memory ability of animals[19].In this experiment, the average escape latency, the search platform searching trajectory and the times crossing the platform and effective areas during space exploration period of the PTSD rats were observed, and the results showed that the learning and memory ability of the PTSD rats were damaged, while the acupuncture intervention improved the stress-caused damage.This confirmed the effectiveness of liver-soothing and mind-regulating acupuncture method, and also provided a behavioral basis for the reliability of restingstate EEG data analysis.

Studies have shown that abnormal brain network connectivity is closely related to the clinical manifestations of patients with neuropsychiatric diseases[20].Resting-state EEG is an objective indicator of brain activity, as it can directly reflect the release of nerve impulses.The amplitude represents the intensity of the potential, and the high amplitude suggests that the neuron number participating in the synchronous discharge is large and the direction is consistent, and it is closer to the recording electrode[21], and vice versa.Some studies have found that α-wave is the main waveform of people in a calm state and distributed throughout the brain[22].The higher the α-wave level,the closer the brain is to the ‘zero-load’ state, i.e.a lower level of cerebral cortex activation[23].Therefore,the power spectrum of the α-wave is inversely proportional to the brain activity[24], and the low power spectrum of α-wave suggests that brain activity increases and the individual is alert.The β-wave is considered to be an excitatory expression of the cerebral cortex.An increased β-wave indicates that the person is nervous and extremely sensitive to the surrounding; the increased δ-wave suggests cognitive impairment; the increased э-wave indicates a serious cognitive impairment.Relevant data have showed that acupuncture has a significant intervention effect on the EEG result.For example, Liu YZ,et al[25]treated patients with depression with warm needling to reduce the symptoms of depression by adjusting the frequency and amplitude of EEG.Zhang Y,et al[26]found that scalp acupuncture in the abnormal discharge area guided by EEG plus body acupuncture intervention could effectively improve the abnormal power spectrum of α-wave, β-wave, δ-wave and δ-wave during seizure interval to regulate the EEG activity in patients with epilepsy.

In this study, we found that there was a certain degree of cognitive impairment in PTSD by observing the resting-state EEG of the stress model rats: the power spectrum value of α-wave decreased significantly,and the power spectrum values of β-wave, δ-wave and э-wave increased significantly.The liver-soothing and mind-regulating acupuncture method could significantly increase the α-wave power spectrum value of rat‘ bilateral cerebral hemispheres, and decrease the power spectrum values of β-wave, δ-wave and э-wave,which revealed the function mechanism of liversoothing and mind-regulating acupuncture method in improving the cognitive function of PTSD rats, and further confirmed the clinical effectiveness of liversoothing and mind-regulating acupuncture method in the treatment of PTSD.