Abstract
OBJECTIVE: To evaluate the effect of needling Baihui (GV20), Neiguan (PC6), Shenmen (HT7) and Taichong (LR3) on cerebral cortical blood oxygen level in rats with insomnia identified as liver-Qi stagnation pattern in terms of Traditional Chinese Medicine.
METHODS: Sixty Wistar rats were randomly divided into blank group (n = 10), the rest of the rats were clamped tail as stimulation combined with injected intraperitoneally p-chlorophenylalanine (PCPA) to establish sleep deprivation model. After the successful replication of the model, they were randomly divided into model group, grabbing group, Western Medicine group, acupuncture group and sham acupuncture group, with 10 rats in each group. The model group was given normal saline; The grasping group was grasped in the same way as the two treatment groups; Western medicine group was given estazolam solution; The acupuncture group was treated with the acupuncture method of "soothing the liver Qi and regulating mind" needling Baihui (GV20), Neiguan (PC6), Shenmen (HT7) and Taichong (LR3); The sham acupuncture group were needled with four non-acupoint sites. After 7 d of treatment of rats in each group, sodium pentobarbital sleep experiment was used to evaluate the sleep latency (SL) and sleep time (ST). The percentage of rats entering the open arm (OE%) and the percentage of time entering the open arm (OT%) in each group were monitored by elevated cross maze, and the vertical score, horizontal crossing times, central grid score and modification times were monitored by open field test, functional near infrared spectroscopy (fNIRs) was used to monitor the changes of oxygenated hemoglobin (Oxy-Hb), deoxyhemoglobin (Deoxy-Hb) and total hemoglobin (Total-Hb) in the cerebral cortex of rats in each group under light stimulation and dark stimulation, the statistically significant channel combinations were selected from 8 light sources and 12 detectors (S-D). According to the position of the light source detector covering the cerebral cortex, Preliminarily determine the key brain areas of insomnia (the preliminary experiment found that 6S-8D and 7S-9D were the key channels of insomnia under light stimulation, and the corresponding brain areas were prefrontal lobe and occipital lobe respectively; 7S-7D channel under dark stimulation, and the corresponding brain area was occipital lobe); The hemodynamic map of cerebral cortex was constructed through the absolute value of whole brain blood oxygen level. Further identify key brain regions for insomnia.
RESULTS: Compared with the blank group, ST, OE%, OT%, the vertical score, horizontal crossing times, central grid score, the concentration of Deoxy-Hb in prefrontal and occipital lobes were decreased significantly (P<0.01), and the concentrations of SL, modification times, Oxy-Hb and Total-Hb were increased significantly (P<0.01); There was no difference in the above indexes between the model group and the grabbing group (P>0.05); After treatment, ST, OE%, OT%, the vertical score, horizontal crossing times, central grid score and Deoxy-Hb concentration in acupuncture group and Western medicine group increased significantly, while SL, modification times, Oxy-Hb and Total-Hb concentrations were decreased significantly (P<0.01, P<0.05), Compared with the Western Medicine group, the values of OE% and OT% were more significantly higher in the acupuncture group (P<0.05), while the rest of the indexes were not significantly different between the two groups (P>0.05); Compared with the acupuncture group, ST, OE%, OT%, the vertical score, horizontal crossing times, central grid score and the concentration of deoxy Hb in the sham acupuncture group decreased significantly (P<0.01), and the concentrations of SL, modification times, Oxy-Hb and Total-Hb increased significantly (P<0.01).
CONCLUSIONS: The needling method for soothing the liver Qi and regulating mind could improve the abnormal behavior of insomnia rats with liver Qi stagnation, and its effect on the improvement of abnormal mood caused by insomnia with liver Qi stagnation is better than that of Western Medicine, and the mechanism of action may be related to the regulation of blood oxygen metabolism in the prefrontal and occipital lobes of the cerebral cortex by acupuncture.
Keywords: sleep initiation and maintenance disorders; acupuncture; spectroscopy, near-infrared; hemoglobins; brain function
1. INTRODUTION
Insomnia is one of the common physiological and psychological disorders that affect the physical and mental health of the human body.1 Patients with insomnia have varying degrees of altered brain function, often accompanied by abnormal manifestations of affective and cognitive functions.2 Studies have shown that insomnia can lead to abnormalities in brain regions such as the prefrontal lobe, anterior cingulate, amygdala, hippocampus, and thalamus, which have relatively reduced metabolic functions during wakefulness and enhanced metabolic functions during sleep,3,4 which is consistent with the findings of the neurocognitive and neurobiological models of high arousal in insomnia and is closely related to cognitive functions such as learning, memory, and poor mood.5 Insomnia belongs to the category of “sleeplessness” in Chinese medicine. The onset of insomnia is most closely related to the heart and liver, and affects the brain and mind, and liver depression is a common clinical evidence of insomnia.6 Acupuncture in Chinese medicine can effectively improve the symptoms of insomnia caused by abnormal emotional and cognitive functions, and has a good regulatory effect on the abnormal activation of brain areas in insomnia.7 Based on the previous clinical practice, the project group concluded that needling Baihui (GV20), Neiguan (PC6), Shenmen (HT7) and Taichong (LR3) has significant effects on the treatment of insomnia.8 The results of previous experimental studies found that needling Baihui (GV20), Neiguan (PC6), Shenmen (HT7) and Taichong (LR3) can not only improve the insomnia state of rats,9 but also improve the abnormal expression of 5-HT1A, 5-HT2A receptor protein in hippocampus and GABAA receptor protein in hypothalamus to varying degrees. However, the above studies only explored the mechanism of insomnia by acupuncture from the microscopic molecular biology point of view, but not from the macroscopic function construction point of view, and did not observe the mechanism of insomnia brain function by acupuncture in real time. Therefore, based on the effectiveness of acupuncture in the treatment of insomnia and the "neurovascular coupling mechanism" (changes in the functional activity of local neurons in the brain cause corresponding changes in oxygen consumption in the region),10 functional near infrared spectroscopy (fNIRs) was used to reveal and explore the regulatory effect of needling Baihui (GV20), Neiguan (PC6), Shenmen (HT7) and Taichong (LR3) on cerebral cortical blood oxygen level in rats with insomnia identified as liver-Qi stagnation pattern from the perspective of brain function.
2. MATERIALS AND METHODS
2.1. Experimental animal
A total of 60 specific pathogen free (SPF) 2-3 months old male Wistar rat with weighing (200 ± 20) g were provided by the Scientific Research and Experimental Center of Gansu University of Traditional Chinese Medicine [Laboratory Animal Quality Certificate No. 62001000000571, License No. SCXK (Gan) 2015-0005]. The mice were divided into 5 per cage and housed in animal facility under SPF condition in Scientific Research and Experimental Center of Gansu University of traditional Chinese Medicine, China Academy of Chinese Medical Science with standard rodent chow and fresh sterilized water. The temperature was maintained at (22 ± 2) ℃ with a relative humidity of 45%-70% and a 12-h light/dark cycle. All experiments were conducted according to international guidelines on animal research and ethics. Feeding conditions: the temperature of the feeding environment was controlled at (20 ± 2) ℃ and the relative humidity was controlled at 55% ± 10%, and the light and dark cycle limit of 12∶12 was given every day (the starting and ending time of light is 7:00-19:00, and the starting and ending time of darkness is 19:00-7:00) (Ethics No. 2021-007), and the animals were disposed of in strict accordance with the “Guiding Opinions on the Good Treatment of Laboratory Animals” issued by the Ministry of Science and Technology in 2006.11
2.2. Main reagents and instruments
Elevated cross maze, open field box, electronic balance (Sartorius Scientific Instruments Co. Ltd., Gottingen, Germany); fNIRs detection and analysis system (LLC NIRx Medical Technologies, New York, NY, USA), rat stereotaxic fixation instrument (LLC NIRx Medical Technologies, New York, NY, USA); Chloral hydrate (Tianjin Damao Chemical Reagent Factory, 20180716 Tianjin, China); Pentobarbital sodium (Tianjin Damao Chemical Reagent Factory, DYY-6D, Tianjin, China); Eszolam (Shandong Xinyi Pharmaceutical Ltd., 200504 Shandong, China); Disposable sterile acupuncture needles (Suzhou Medical Supplies Factory Co. Ltd., Suzhou, China).
2.3. Model preparation
Clamped tail as stimulation combined with injected intraperitoneally p-chlorophenylalanine (PCPA) was used for model replication.10,12 Each group of rats was placed in the same cage, and the distal 1/3 of the rat tails were clamped separately with hemostatic forceps for 30 min/time, every 3 h/time, 4 times/d. The stimulation intensity was controlled so as not to break the skin and bleed for 16 d. From the 15th day of modeling, the rats in the modeling group were injected intraperitoneally with PCPA suspension (1 mL/100 g) at 450 mg/kg from 8:00 to 10:00 a.m. daily for 2 d. 100 g), 1 time/d, for 2 d.
2.4. Grouping and intervention methods
The rats were divided into blank group (10 rats) and modeling group (50 rats) by random number table method, and after successful modeling, they were then randomly divided into modeling group, grabbing group, Western Medicine group, acupuncture group and sham acupuncture group, 10 rats in each group.
Blank group: no intervention.
Model group: no intervention.
Grasping group: the grasping treatment was started on the second day after model replication, and the grasping method, strength and duration were the same as the Western Medicine group and acupuncture group.
Western medicine group: gavage with eszopiclone (0.2 mg/kg) starting on day 2 after model replication,13 1 time/d for 7 d.
Acupuncture group: On day 2 after model replication, refer to Experimental Acupuncture-Moxibustion14 and The Rat Brain in Stereotaxic Coordinates: 15 select Baihui (GV20), Neiguan (PC6), Shenmen (HT7) and Taichong (LR3) on both sides.
The above four acupuncture points are all operated by using flat tonic and flat diarrhea (twisting amplitude 180°, frequency 60-80 times/min), each point operated for 1 min, 1 time/d, continuous treatment for 7 d.
Sham acupuncture group: on the 2nd day after successful replication of the model, rats were selected from the tail root and the upper, middle and lower 1/3 of the tail,16 each point was operated for 1 min, 1 time/d, and treated for 7 d.
2.5. Observation index and detection method
2.5.1 Macroscopic signs observation
Observe the changes of mental, activity, sleep, diet and coat color of the rats in each group during the experiment.
2.5.2 Sodium pentobarbital sleep experiment
The sleep latency (SL) and sleep time (ST) were monitored by intraperitoneal injection of pentobarbital sodium. After the last treatment, pentobarbital sodium (45 mg/kg) was injected intraperitoneally, and the rats were placed in supine position on a flat plate with their tails perpendicular to the plate to start timing. If they did not turn within 1 min, it was regarded as the disappearance of righting reflex, and the injection time to the disappearance of righting reflex was counted as SL; Keep timing until the rats automatically turn over from supine position and do not turn over within 1min, it is ST.17
2.5.3 Elevated cross maze experiment
Elevated cross-maze experiment: the rats were acclimatized in the laboratory for 5 min before the test, then placed in the central area of the maze with their heads facing either closed-arm direction, while starting a timer and recording their activity for 5 min.18 The test area was wiped with alcohol after each rat to prevent the odor from disturbing the next animal.
Monitoring metrics: percentage of entries into the open arm (OE%): number of entries into the open arm / total number of entries into the open and closed arms×100%; percentage of time spent in the open arm (OT%): time spent in the open arm / total time spent in the open and closed arms ×100%.
2.5.4 Open field test
Open field test: the rats were placed in the laboratory for 10 min before the start of the experiment for adaptation, and quickly placed in the central zone position of the open field after the start of the experiment, with the rats’ heads fixed to one side, and the scores of the rats were recorded within 3 min,10 and the test area was wiped with alcohol after each rat was tested to prevent the odor from disturbing the next animal.
Measurement: number of horizontal cross-frame, central frame score, vertical score, and number of modifications.
2.6. fNIRs monitoring
Studies have shown that Oxy-Hb changes are the most sensitive markers in the detection of fNIRs. Therefore, most current fNIRs studies use HbO2 as a characteristic indicator.19 In this study, Oxy-Hb was used as the main indicator, and Deoxy-Hb and Total-Hb were used as secondary indicators. The NIRStar13-0 software was applied to collect Oxy-Hb, Deoxy-Hb and Total-Hb concentrations in resting state 180s in each group of rats, and the absolute values of blood oxygen levels were used to construct a cortical hemodynamic map, and the key brain regions for insomnia were further identified by referring to the Stereotaxic Atlas of the Rat Brain and the analysis software matlab planimetric control.20
2.7. Statistical Methods
SPSS 22.0 software (IBM SPSS Inc., Chicago, IL, USA) was used for statistical analysis. If the data conforms to the normal distribution, the measurement data is expressed in the form of mean ± standard deviation ($\bar{x}±s$). If the variance is homogeneous, one-way analysis of variance is used between groups, and least significant difference test is used for further comparison between groups. If the data does not conform to the normal distribution, the measurement data are expressed in the form of median or quartile, and the W-H rank sum test is used. The test level α = 0.05, and the test results were considered statistically significant at P <0.05. fNIRs data were subjected to principal components and partial least squares cluster analysis method using R3.5.1 software (Alcatel Lucent, Paris, France).
3. RESUILS
3.1. Comparison of general conditions of rats in each group
Compared with the blank group, the rats in the model group were depressed after 1 week of modeling, with slow weight gain, dim hair color, their modification behavior increased, and showed high vigilance to external stimuli; after two weeks of modeling, the rats in the modeling group showed obvious symptoms related to liver depression and insomnia, such as significantly lower weight, reduced sleep duration, increased sensitivity to external stimuli, fatigue and weakness, preference for quietness, dry and disorganized hair, lack of luster, and reddish paw nails. The grasping group was similar to the above group; the two treatment groups recovered to different degrees after treatment, with increased sleep duration, slow increase in body weight, gradual normalization of diet and water intake and activity, and gradual appearance of glossy hair.
3.2. Comparison of sleep test results of pentobarbital sodium in rats of each group
Compared with the blank group, SL in the model group was significantly increased and ST was significantly shortened (P <0.01). Compared with the model group, there was no significant difference in the above indexes in the grasping group (P >0.05). Compared with the grasping group, SL was significantly shortened and ST was significantly increased in the acupuncture group and Western medicine group (P<0.01). There was no significant difference between the two treatment groups (P >0.05); Compared with the acupuncture group, SL was significantly increased and ST was significantly shortened in the sham acupuncture group (P <0.01) (Table 1).
Table 1.
Comparison of pentobarbital sodium sleep test results of rats in each group after treatment ($\bar{x}±s$, t/min)
| Group | n | SL | ST |
|---|---|---|---|
| Blank | 10 | 2.7±0.7 | 102.2±5.2 |
| Model | 10 | 4.4±0.7a | 78.1±14.2a |
| Grabbing | 10 | 4.5±0.8 | 74.7±2.4 |
| Western Medicine | 10 | 2.9±0.6b | 94.8±13.6b |
| Acupuncture | 10 | 3.0±0.5b | 88.1±3.9b |
| Sham acupuncture | 10 | 4.4±1.0c | 69.3±4.5c |
Notes: blank group: rats without insomnia modeling with liver-Qi stagnation and acupuncture treatment; model group: rats with insomnia modeling with liver-Qi stagnation; grabbing group: rats with insomnia modeling with liver-Qi stagnation and grasping stimulation; Western Medicine group: rats with insomnia modeling with liver-Qi stagnation and eszopiclone treatment; acupuncture group: rats with insomnia modeling with liver-Qi stagnation and acupuncture treatment; sham acupuncture group: rats with insomnia modeling with liver-Qi stagnation and sham acupuncture treatment. Single factor analysis of variance was used for inter group comparison. SL: sleep latency; ST: sleep time. Compared with the blank group, aP<0.01; compared with the grabbing group, bP<0.01; compared with acupuncture group, cP<0.01.
3.3. Comparison of results of elevated cross maze
Compared with the blank group, the values of OE% and OT% in the model group were significantly lower (P <0.01); Compared with the model group, there was no significant difference in OE% and OT% in the grasping group (P >0.05); Compared with the grasping group, the values of OE% and OT% in the Western Medicine group and acupuncture group were significantly higher (P <0.01); Compared with the Western Medicine group, the values of OE% and OT% were more significantly higher in the acupuncture group (P <0.05); Compared with the acupuncture group, the values of OE% and OT% in the sham acupuncture group were significantly lower (P <0.01) (Table 2).
Table 2.
Comparison of elevated cross maze results of rats in each group after treatment ($\bar{x}±s$)
| Group | n | OE% | OT% |
|---|---|---|---|
| Blank | 10 | 65.9±5.4 | 66.5±4.3 |
| Model | 10 | 40.0±5.3a | 46.8±3.9a |
| Grabbing | 10 | 42.1±5.3 | 50.0±2.7 |
| Western Medicine | 10 | 57.8±3.9b | 60.7±6.1b |
| Acupuncture | 10 | 64.3±2.3bc | 68.2±6.0bc |
| Sham acupuncture | 10 | 43.7±4.2d | 43.1±5.7d |
Notes: blank group: rats without insomnia modeling with liver-Qi stagnation and acupuncture treatment; model group: rats with insomnia modeling with liver-Qi stagnation; grabbing group: rats with insomnia modeling with liver-Qi stagnation and grasping stimulation; Western Medicine group: rats with insomnia modeling with liver-Qi stagnation and eszopiclone treatment; acupuncture group: rats with insomnia modeling with liver-Qi stagnation and acupuncture treatment; sham acupuncture group: rats with insomnia modeling with liver-Qi stagnation and sham acupuncture treatment. Single factor analysis of variance was used for inter group comparison. Compared with the blank group, aP<0.01; compared with the grabbing group, bP<0.01; compared with Western Medicine group, cP<0.05; compared with acupuncture group, dP<0.01.
3.4. Comparison of open field experimental results
Compared with the blank group, the number of central grid scores, horizontal crossing times and vertical score in the open field experiment of model group rats were significantly reduced, and the number of modifications times was significantly increased (P<0.01), indicating that chronic tail clamping + intraperitoneal injection of PCPA can reduce the rats excitability, exploration and cognitive function and increase the alertness of rats; Compared with the model group, there was no significant difference in the above indexes in the grasping group (P>0.05); Compared with the grasping group, central grid scores, horizontal crossing times and vertical score in the Western Medicine group and acupuncture group increased significantly, and the modification times decreased significantly (P <0.01, P <0.05). There was no significant difference between the two treatment groups (P >0.05), suggesting that acupuncture can improve the liver depression manifestation of the insomnia rats; Compared with the acupuncture group, the times of standing up, the times of horizontal cross grid and the score of central grid in the sham acupuncture group decreased significantly, and the times of modification increased significantly (P <0.01) (Table 3).
Table 3.
Comparison of open field test results of rats in each group after treatment ($\bar{x}±s$)
| Group | n | Vertical score | Horizontal crossing times | Center grid score | Modification times |
|---|---|---|---|---|---|
| Blank | 10 | 11.2±1.5 | 26.7±4.1 | 19.6±1.7 | 1.7±0.8 |
| Model | 10 | 5.9±1.9a | 16.8±2.2a | 14.3±1.2a | 3.6±1.1a |
| Grabbing | 10 | 6.4±1.7 | 17.1±2.2 | 13.7±1.6 | 3.3±1.1 |
| Western Medicine | 10 | 10.9±1.4b | 24.9±1.7b | 65.7±5.5b | 1.6±1.1b |
| Acupuncture | 10 | 11.1±1.4b | 24.8±2.7b | 19.3±2.4b | 1.8±1.0c |
| Sham acupuncture | 10 | 5.5±1.3d | 16.9±2.0d | 13.4±1.5d | 3.6±1.1d |
Notes: blank group: rats without insomnia modeling with liver-Qi stagnation and acupuncture treatment; model group: rats with insomnia modeling with liver-Qi stagnation; grabbing group: rats with insomnia modeling with liver-Qi stagnation and grasping stimulation; Western Medicine group: rats with insomnia modeling with liver-Qi stagnation and eszopiclone treatment; acupuncture group: rats with insomnia modeling with liver-Qi stagnation and acupuncture treatment; sham acupuncture group: rats with insomnia modeling with liver-Qi stagnation and sham acupuncture treatment. Single factor analysis of variance was used for inter group comparison. Compared with the blank group, aP<0.01; compared with the grabbing group, bP<0.01; compared with the grabbing group, cP<0.05; compared with acupuncture group, dP<0.01.
3.5. Schematic diagram of resting cerebral cortical blood flow of rats in each group
3.5.1 The results of fNIRs detection in the cerebral cortex of rats in each group under light stimulation
3.5.1.1 Localization of key brain regions
Referring to the stereotactic atlas of rat brain and the plan view of analysis software MATLAB, by comparing the blood flow status of cerebral cortex between the blank group and the model group, it is further determined that the key brain areas of insomnia are prefrontal lobe and occipital lobe (Figure 1).
Figure 1. Localization of brain area.
The blue area indicates that the cerebral cortex is inhibited, and the red area indicates that the cerebral cortex is activated. A: blood flow state of the prefrontal lobe of rats in the blank group, B: blood flow state of the prefrontal lobe of rats in the model group, C: blood flow state of the occipital lobe of rats in the blank group, D: blood flow state of the occipital lobe of rats in the model group.
3.5.1.2 Schematic diagram of resting cerebral cortical blood flow of rats in each group
Compared with the blank group, the concentrations of Oxy-Hb and Total-Hb in the prefrontal and occipital lobes of the key brain areas in the model group increased significantly, and the concentration of Deoxy-Hb decreased significantly (P <0.01); Compared with the model group, there was no significant difference in the above three kinds of hemoglobin in the upper brain area of the grasping group (P >0.05); Compared with the grasping group, the concentrations of Oxy-Hb and Total-Hb in prefrontal lobe and occipital lobe in Western Medicine group and acupuncture group were significantly lower, and the concentration of Deoxy-Hb was significantly higher (P <0.01); Compared with the Western Medicine group, there was no significant difference in the concentrations of three kinds of hemo-globin in the upper brain area of the acupuncture group (P >0.05); Compared with the acupuncture group, the concentrations of Oxy-Hb and Total-Hb in prefrontal lobe and occipital lobe in the sham acupuncture group were significantly increased, and the concentration of Deoxy-Hb was significantly decreased (P <0.01) (Figure 2).
Figure 2. Schematic diagram of cerebral cortical blood flow in resting state of rats in each group.
The closer to dark red, the higher the concentration of oxy-Hb, indicating that the activity of cerebral cortex is relatively active; The closer to dark blue, the lower the concentration of oxy-Hb, suggesting that the activity of cerebral cortex is inhibited to a certain extent. A: blank group; B: model group; C: grasping group; D: western medicine group; E: acupuncture group; F: sham acupuncture group. Blank group: rats without insomnia modeling with liver-Qi stagnation and acupuncture treatment; model group: rats with insomnia modeling with liver-Qi stagnation; grabbing group: rats with insomnia modeling with liver-Qi stagnation and grasping stimulation; Western Medicine group: rats with insomnia modeling with liver-Qi stagnation and eszopiclone treatment; acupuncture group: rats with insomnia modeling with liver-Qi stagnation and acupuncture treatment; sham acupuncture group: rats with insomnia modeling with liver-Qi stagnation and sham acupuncture treatment.
Figure 2 is a schematic diagram of cerebral cortical blood flow from top to bottom with Oxy-Hb as the standard at the 150th second of resting state of rats in each group. In the blank group, there were uniform blue areas in the prefrontal and occipital lobes, suggesting that the neurons’ activity in this area was inhibited to a certain extent, the local blood flow decreased and the oxygen consumption was low, indicating that the activity of cerebral cortex was inhibited to a certain extent; In the model group, several red areas appeared in the key brain areas, suggesting that the neuronal activity in this area was activated to a certain extent, the local blood flow increased and the oxygen consumption increased, suggesting that the activity of the cerebral cortex was relatively active, indicating that the model was copied successfully; Even blue and green areas appeared in the midbrain area of the key brain area in the Western Medicine group, and blue areas appeared in the key brain area in the acupuncture group, indicating that the cerebral cortex was inhibited to a certain extent, and estazolam and acupuncture had a benign regulatory effect on the abnormal activation of the insomnia brain area. Figure 4 is the same as.
Figure 4. schematic diagram of cerebral cortical blood flow in resting state of rats in each group.
The closer to dark red, the higher the concentration of oxy Hb, indicating that the activity of cerebral cortex is relatively active; the closer to dark blue, the lower the concentration of oxy Hb, suggesting that the activity of cerebral cortex is inhibited to a certain extent. A: blank group; B: model group; C: grasping group; D: Western Medicine group; E: acupuncture group; F: sham acupuncture group. blank group: rats without insomnia modeling with liver-Qi stagnation and acupuncture treatment; model group: rats with insomnia modeling with liver-Qi stagnation; grabbing group: rats with insomnia modeling with liver-Qi stagnation and grasping stimulation; Western Medicine group: rats with insomnia modeling with liver-Qi stagnation and eszopiclone treatment; acupuncture group: rats with insomnia modeling with liver-Qi stagnation and acupuncture treatment; sham acupuncture group: rats with insomnia modeling with liver-Qi stagnation and sham acupuncture treatment.
3.5.2 Results of fNIRs detection in rat cerebral cortex under dark stimulation in each group
3.5.2.1. Localization of key brain regions
Referring to the stereotactic atlas of rat brain and the plan view of the analysis software MATLAB, by comparing the blood flow status of cerebral cortex between the blank group and the model group, it is further determined that the key brain area of insomnia is the prefrontal lobe (Figure 3).
Figure 3. Determination of brain area.
The blue area indicates that the cerebral cortex is in an inhibited state, and the red area indicates that the cerebral cortex is in an activated state. A: blood flow state of the occipital lobe of rats in the blank group, B: blood flow state of the occipital lobe of rats in the model group. Blank group: rats without insomnia modeling with liver-Qi stagnation and acupuncture treatment; model group: rats with insomnia modeling with liver-Qi stagnation; grabbing group: rats with insomnia modeling with liver-Qi stagnation and grasping stimulation; Western Medicine group: rats with insomnia modeling with liver-Qi stagnation and eszopiclone treatment; acupuncture group: rats with insomnia modeling with liver-Qi stagnation and acupuncture treatment; sham acupuncture group: rats with insomnia modeling with liver-Qi stagnation and sham acupuncture treatment.
3.5.2.2. Schematic diagram of resting cerebral cortical blood flow of rats in each group
Compared with the blank group, the concentrations of Oxy-Hb and Total-Hb in the prefrontal lobe of the key brain areas of insomnia in the model group were significantly increased, and the concentration of Deoxy-Hb was significantly decreased (P <0.01); compared with the model group, there was no significant difference in the above three kinds of hemoglobin in the upper brain area of the grasping group (P >0.05); compared with the grasping group, the concentrations of Oxy-Hb and Total-Hb in the key brain areas of insomnia in the Western Medicine group and acupuncture group decreased significantly, and the concentration of Deoxy-Hb increased significantly (P <0.01); compared with the Western Medicine group, there was no significant difference in the concentrations of three kinds of hemoglobin in the upper brain area of the acupuncture group (P >0.05); compared with the acupuncture group, the concentrations of Oxy-Hb and Total-Hb in the sham acupuncture group increased significantly, and the concentration of Deoxy-Hb decreased significantly (P <0.01) (Figure 4).
4. DISCUUSSION
PCPA injection is currently recognized as the preparation method of insomnia model,21 and tail clamping stimulation is consistent the TCM theory about the pathological process of liver Qi stagnation due to emotional disorders.22 Long-term tail-clamping stimulation + intraperitoneal injection of PCPA can replicate the signs of liver Qi stagnation insomnia in Chinese medicine, and is one of the common and effective methods for preparing liver Qi stagnation insomnia models.12 In this study, we used long-term tail-clamping stimulation + intraperitoneal injection of PCPA to replicate the liver Qi stagnation insomnia rat model, and found that liver-depressed rats showed signs of liver Qi stagnation, such as significantly reduced excitability and activity; the elevated cross-maze experiment showed that rats showed anxiety and depression; the open-field experiment showed that rats had significantly reduced excitability, exploratory and spatial cognitive functions, and increased alertness; the pentobarbital sodium experiment showed that rats had prolonged sleep latency and shortened sleep time. After needling Baihui (GV20), Neiguan (PC6), Shenmen (HT7) and Taichong (LR3), the above behavioral indicators were improved, and the acupuncture was better than the Western Medicine group in improving anxiety and depression, and other behavioral experiments achieved the same effect as the Western Medicine group.
As one of the Chinese medical methods, acupuncture has been widely used in the clinical treatment of insomnia due to its unique advantages, and the clinical efficacy of acupuncture in treating insomnia has been found to be significant.23 Baihui (GV20) belongs to the Directing Vessel, which enters the brain and has the effect of awakening the brain and opening the orifices, calming the mind and fixing the will; Shenmen (HT7) is the original point of the Heart meridian, which can calm the mind and tranquilize the mind; Neiguan (PC6) is a point of the Hand Convulsive Yin Pericardium meridian, which has the effect of opening the blood vessels, regulating the heart Qi and awakening the brain and mind. Taichong (LR3) is the original point of the Liver meridian, which nourishes Yin and Blood and nourishes the Liver and Kidney. The above four acupoint work together to treat insomnia with definite clinical efficacy.8 Previous experimental studies have explored the central neural mechanism of acupuncture intervention in insomnia from a molecular biological perspective, but have not yet studied the effect of acupuncture on the function of brain regions related to liver Qi stagnation insomnia from a macroscopic brain function level.
Therefore, in this experiment, the fNIRs technique was used to monitor the changes of blood oxygen concentration in insomnia-related brain regions from the perspective of blood oxygen response, which in turn reflects the neuronal activity in the relevant brain regions. It was found that Oxy-Hb and Total-Hb concentrations increased and Deoxy-Hb concentrations decreased in the prefrontal and occipital lobes of insomniac rats under light stimulation; the study used matlab to establish hemodynamic planes, and further found that abnormal activation and response were found in the spatial distribution of prefrontal and occipital lobes, the key activating brain regions of insomnia, through channel time series and hemodynamic planes localization, which is consistent with the results of Liang et al.24 Similarly, it was found that the prefrontal Oxy-Hb and Total-Hb concentrations increased and Deoxy-Hb concentrations decreased in insomnia rats during dark stimulation, and it was also found that there was abnormal activation and response in the spatial distribution of insomnia key activating brain regions, which was consistent with the results of Grounauer et al.25
The prefrontal lobe was abnormally activated in both of these cases, and the prefrontal lobe is an important brain region involved in cognitive-emotional regulation and is closely related to negative emotions caused by sleep, and anxiety state, decreased arousal, and increased alertness as a negative emotion are directly related to prefrontal function,26 so it is hypothesized that the increased prefrontal activity after the onset of insomnia may be due to abnormalities in cognitive-emotional related loops caused by insomnia. Occipital lobe function is abnormally activated by light stimulation, and the occipital lobe is the visual center of the brain, which is responsible for some language and movement functions in addition to visual perception functions, and is closely related to the brain's short-term memory and spatial integration functions,27 and in addition to visual impairment, memory function is also reduced when the occipital lobe is damaged. Thus, it is suggested that abnormal activation of the occipital lobe is not only caused by visual stimulation, but may also be caused by abnormal spatial integration functions after insomnia. It was found that the thalamus is involved in fiber projections from the occipital to the prefrontal cortex,28,29 transmits visual information,30 and regulates higher functions of the prefrontal cortex, and that prefrontal firing activity is diminished after damage to the medial thalamic nucleus,31 leading to the speculation that the occipital and prefrontal lobes are activated after light stimulation, possibly as a superimposed effect of prefrontal and occipital functions.
From the above results, it is clear that both acupuncture and eszopiclone could improve insomnia symptoms and mood-cognitive function by improving prefrontal and occipital neuron function in insomnia patients, which is consistent with the findings of Shi and Huang et al;32,33 the improvement of mood by acupuncture was better than that of the Western medicine group, probably due to the improvement of prefrontal neuron activity by acupuncture than that of the Western medicine group.
REFERENCES
- [1]. Feng H, Liu Y, Xu H, Liu YH, Chen GL, Liu WJ. Effects of acupuncture and estazolam on episodic memory and sleep structure in patients with chronic insomnia: a randomized controlled study. Zhong Guo Zhen Jiu 2020; 40: 707-12. [DOI] [PubMed] [Google Scholar]
- [2]. Dzierzewski JM, Dautovich N, Ravyts S. Sleep and cognition in older adults. Sleep Med Clin 2018; 13: 93-106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [3]. Li S, Tian J, Li M, et al. Altered resting state connectivity in right side frontoparietal network in primary insomnia patients. Eur Radiol 2018; 28: 664-72. [DOI] [PubMed] [Google Scholar]
- [4]. Wang ZY, Huo JW, Li JQ, Yan CQ, Liu CZ, Liu QQ. Resting state fMRI study on local consistency of chronic primary insomnia. Yi Xue Ying Xiang Xue Za Zhi 2020; 30: 2179-82 + 87. [Google Scholar]
- [5]. Dzierzewski JM, Dautovich N, Ravyts S. Sleep and cognition in older adults. Sleep Med Clin 2018; 13: 93-106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [6]. Liu XW, Wang WD. Observation on the efficacy of Xiaoyaosan decoction combined with Chinese psychological TIP therapy for insomnia. Shi Jie Shui Mian Yi Xue Za Zhi 2019; 6: 280-3. [Google Scholar]
- [7]. Zhao ZQ, Li YP, Xiang XX, Zhuang JH, Zhao ZX. Pathogenesis of chronic insomnia and cognitive-behavioral therapy. Zhong Guo Lin Chuang Yao Li Xue Yu Zhi Liao Xue 2021; 26: 482-6. [Google Scholar]
- [8]. Li XR, Hou X, Ye YJ, Bu XM, An CL, Yan XK. Comparison of the efficacy of the buried thread method and acupuncture based on the formula of “dredging the liver and regulating the spirit” in the treatment of insomnia with liver depression and Qi stagnation. Zhong Guo Zhen Jiu 2020; 40: 1277-80+85. [DOI] [PubMed] [Google Scholar]
- [9]. Li XR. Effects of "Dredging the liver and regulating the mind" acupuncture on the expression of 5-HT1AR and 5-HT2AR in hippocampus and GABAAR-α1 in hypothalamus of insomnia rats. Lanzhou: Gansu University of Chinese Traditional Medicine, 2021: 19-29. [Google Scholar]
- [10]. He LX, Zhu YH, Yang CH, et al. Establishment of a rat model of insomnia with liver stagnation and fire evidence and its evaluation. Zhong Hua Zhong Yi Yao Za Zhi 2018; 33: 3890-4. [Google Scholar]
- [11]. Ministry of Science and Technology . On the release of the “guiding opinions on the good treatment of laboratory animals”. Xu Mu Shou Yi Ke Ji Xin Xi 2007; 04: 35-36. [Google Scholar]
- [12]. Zhu J, Shen G, Wang YJ, Hu W. Establishment and evaluation of a rat model of insomnia with liver-depression evidence. Zhong Yi Za Zhi 2011; 52 : 689-92. [Google Scholar]
- [13]. Lu Y, Liu ZH, Yu XH, Wang LL, Li FH, Ma ZB. Effects of acupunture for smoothing liver and regulating spirit on neurotransmitters in insomnia model rats. Shandong Zhong Yi Za Zhi 2017; 36: 322-5. [Google Scholar]
- [14]. Li ZR. Experimental acupuncture-moxibustion. Beijing: Zhong Guo Zhong Yi Yao Chu Ban She, 2008: 83-6. [Google Scholar]
- [15]. Li YQ. The Rat Brain in stereotaxic coordinates. Wuhan: Huazhong Ke Ji Da Xue Chu Ban She, 2010: 17-19. [Google Scholar]
- [16]. Liu SY, Lu H, Qin QQ, Zhang CH. Design method of sham acupuncture in acupuncture clinical trials. Zhong Guo Zhong Yi Ji Chu Yi Xue Za Zhi 2020; 26: 1531-4. [Google Scholar]
- [17]. Qiao LN, Gao QL, Tan LH, et al. Effects of electroacupuncture single points and acupoint combination on the content of sleep-wake related factors such as interleukin-1β and brain-derived nerve growth factor in the serum of insomnia rats. Zhen Ci Yan Jiu 2018; 43: 651-56. [DOI] [PubMed] [Google Scholar]
- [18]. Cheng SS, Liu P, Wu QW, et al. Effects of electroacupuncture on anxiety state and sympathetic-adrenomedullary system in rats with insomnia model. Zhong Guo Zhen Jiu 2015; 35: 923-6. [PubMed] [Google Scholar]
- [19]. Carius D, Andrä C, Clauß M, Ragert P, Bunk M, Mehnert J. Hemodynamic response alteration as a function of task complexity and expertise-An fNIRS study in jugglers. Front Hum Neurosci 2016; 30: 126-36. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [20]. George P, Charles W, Zhuge QC. Stereotactic atlas of rat brain. Beijing: People's Health Publishing House, 2005: 7- 8. [Google Scholar]
- [21]. Mao DD. Effects of acupuncture on the circadian rhythm of spontaneous activity and the mRNA and protein expression of 5-HT1A and 5-HT2A receptors in hippocampus of insomnia rats. Chengdu: Chengdu University of Traditional Chinese Medicine, 2016: 24-36. [Google Scholar]
- [22]. Liu Q, Li SP, Liao L, Zhu XQ, Liu KY, Liu QQ. Experimental study of harmonizing liver and spleen recipe in the treatment of insomnia. Beijing Zhong Yi Yao 2018; 37: 768-70. [Google Scholar]
- [23]. Zhao T, Xu H, Zhao T, et al. Treatment of insomnia with acupuncture for strengthening brain and calming nerves and its effect on magnetic resonance brain functional imaging. Zhong Guo Zhen Jiu 2021; 41: 767-73. [DOI] [PubMed] [Google Scholar]
- [24]. Liang MJ, Zhou Q, Yang XL, Tang Y, Fang J. Study on resting brain function changes in patients with primary insomnia based on local consistency. Lin Chung Fang She Xue Za Zhi 2014; 33: 10-4. [Google Scholar]
- [25]. Grounauer P, Métraux B. The Somnogen visual training a new CBT to fight insomnia through closed eyes and fNIRS neuroimaging. J Behav Brain Sci 2014; 4: 477-81. [Google Scholar]
- [26]. Evans JW, Szczepanik J, Brutsché N, Park LT, Nugent AC, Zarate CA Jr. Default mode connectivity in major depressive disorder measured up to 10 days after ketamine administration. Biol Psychiatry 2018, 84: 582-90. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [27]. Zhou YL, Cao YZ, Meng Q, Zhou JZ. Relationship between pure dyslexia and occipital lobe lesions in cerebral hemisphere. Lin Chuang Shen Jing Bing Xue Za Zhi 2002; 15: 280-2. [Google Scholar]
- [28]. Huang QL, Tang Y, Wang PD, et al. Resting brain functional magnetic resonance study of limbic system dominated by amygdala in patients with post-traumatic stress disorder. Zhong Guo Jing Shen Ji Bing Za Zhi 2011; 37: 705-9. [Google Scholar]
- [29]. Hao J, Li KC, Wang W, et al. Evaluation of visual attention function in normal elderly by functional magnetic resonance imaging. Zhong Guo Yi Xue Ying Xiang Ji Shu 2005; 1337-41. [Google Scholar]
- [30]. Matsushima A, Tanaka M. Different neuronal computations of spatial working memory for multiple locations within versus across visual hemifields. J Neurosci 2014; 34: 5621-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [31]. Fan LL, Sun BY, Deng B, et al. Effect of damage to the dorsomedial nucleus of thalamus on the electrical activity of neurons in the medial prefrontal cortex of rats. Shanxi Yi Ke Da Xue Xue Bao 2016; 47: 879-83. [Google Scholar]
- [32]. Huang HJ, Wang Y. Evaluation of the effect of acupuncture on brain function in patients with primary insomnia based on whole brain resting fMRI. Zhong Xi Yi Jie He Xin Nao Xue Guan Bing Za Zhi 2019; 17: 3040-3. [Google Scholar]
- [33]. Shi XH, Wang YK, Li T, et al. Gender-related difference in altered fractional amplitude of low-frequency fluctuations after electroacupuncture on primary insomnia patients: a resting-state fMRI study. Brain Behav 2021; 11: 1927-36. [DOI] [PMC free article] [PubMed] [Google Scholar]