I. Introduction
Baihui (GV20) is the 20th acupoint of the Du meridian (the government vessel) located on the highest place of the head where all the yang meridians meet1. And GV20 is a principle acupoint of “Seven Acupoints for Stroke” which are used to treat the symptoms of stroke1. GV20 is used to treat neurological and psychiatric diseases such as headache, dizziness, anxiety and stroke2. In previous study using transcranial doppler (TCD), both Cerebrovascular Reactivity (CVR) and Corrected Velocity at PETCO2 40 mmHg (CV40) were significantly increased in both Middle Cerebral Arteries (MCA) and Anterior Cerebral Arteries (ACA) after GV20 acupuncture treatment3. It showed that GV20 acupuncture has the effect on improving CBF by reducing peripheral vascular resistance, increasing CBF velocity and CVR.
A previous study4 that evaluate the effect of GV20 acupuncture on CBF of MCA in normal subjects using TCD showed the results of a signigicant increase in mean velocity, systolic velocity, diastolic velocity, and decrease in pulsatility index, which suggested the GV20 acupuncture have a specific effect on CBF. According to a systemic review and meta-analysis study2, in an animal model of focal cerebral ischemia, GV20 based scalp acupuncture improved infarct volume and neurological function score, and it suggested potential neuroprotective role of GV20 acupuncture. Previous studies2-4 showed that GV20 acupuncture has the effect on improving CBF by reducing peripheral vascular resistance, increasing CBF velocity and CVR.
Sishencong (EX-HN1) is composed of four acupoints located 1 B-cun(proportional methods) anterior, posterior and bilateral to GV20, which are often used together with GV20 for stroke, dementia, headache. Previous studies5-9 showed that acupuncture at GV20 and EX-HN1 improves the cerebral blood flow, brain activity and alleviate the symptoms of cerebrovascular disease. Electroacupuncture treatment including GV20 and EX-HN1 in cervical vertigo patients improved blood supply of vertebro-basliar artery and its clinical effect is better than the effect of western medicine treatment5. Moreover, Acupucture treatment including GV20 and EX-HN1 improved balance fuction in stroke patients6 and have a significant effect on the improvement of cognitive function in vascular dementia7,8. It has been confirmed through functional infrared spectroscopy (fNIRS) imaging that the application of intra- dermal acupuncture at GV20 and EX-HN1 with Neurodevelopmental treatment to cerebral palsy children improves brain activity compared to applying Neurodevelopmental treatment alone9.
In addition, acupuncture at certain acupoints has been reported to affect the specific brain regions and cerebral arteries10. Moreover, when comparing the functional brain areas stimulated by a single acupoint and acupoint combinations using fMRI, acupuncture treatment with acupoint combinations act on wider brain areas than a single acupoint. These studies11,12 support a synergistic effect of acupoint combinations.
Therefore, this study aimed to compare the changes on CVR between GV20 and combination of GV20 and EX-HN1 in the cerebral arteries using TCD. Furthermore, this study is expected to provide a clinical evidence for the use of GV20 and EX-HN1 in clinical practice to improve cerebral blood flow.
II. Materials and Methods
1. Study design
The study was designed as a randomized crossover trial. Participants visited the study center four times with the interval of one-week to receive acupuncture treatment. TCD was performed on both MCAs at the first and second visits, and both ACAs at the third and fourth visits. Before the first and third visits, participants were randomly allocated to two groups (Group A and Group B, n=5 per group).
Group A sequentially received two phases of intervention of acupuncture at GV20 single point (the control group) and combination of GV20 and EX-HN1 acupoints(the experimental group), whereas Group B received interventions in the reverse order. The phases were separated by a one-week washout interval13-15 to eliminate the residual effect by the former phase (Fig. 1). For outcome assessor blinding, the randomizer, acupuncturist, and TCD performer-statistician (assessor) were separated.
As a randomization method, this study used block randomization to equalize the number of subjects between groups. Group A or B, 5 of each, were listed. Paired with the list, the random numbers were generated using Excel program and then listed in descending order. The randomizer determined the group allocations in this order according to the subjects’ screening sequence numbers which is given in order of registration.
These randomization results were transferred from the randomizer to the acupuncturist as an allocation concealment using the envelope method. When the randomizer finished the randomization, the group allocation results put into opaqe sealed envelopes and was delivered to the acupuncturist. The acupuncturist opened the envelopes according to the subjects’ screening sequence numbers and performed acupuncture according to the group allocation. And the allocation result was not informed to the TCD performer. Through this process, the TCD performer-statistician (assessor) was blinded to the group allocation until the analysis was finished.
The study was approved by the Institutional Review Board at the Hospital of Korean Medicine, KyungHee Medical Center (KOMCIRB 2021-03-001-001) and registered with Clinical Research Information Service, a service of the Korea Centers for Disease Control and Prevention (KCT0006324).
2. Participants
Ten healthy male volunteers aged 20 to 29 years were enrolled in this study. As age increases, the CBF velocity and CVR significantly decrease16. In addition, as estrogen decreases vascular resistance, the CBF velocity of women is significantly higher than that of men. And the CBF is affected by changes in estrogen secretion according to a woman’s menstrual cycle17.
Because age and gender act as physiological determinants of cerebral blood flow rate, the participants were limited to males in 20s.
Participant were recruited from June 2021 to October 2021.
Participants were included only when they met the following inclusion criteria:
- Inclusion Criteria
1) Males aged 20 to 29 years
2) Physically and mentally healthy confirmed by a physician
3) Good protocol compliance and agreeing to sign an informed consent document
Participants would be excluded if they had one of the following conditions:
- Exclusion Criteria
1) History of cerebrovascular disease, cardiovascular disease, diabetes mellitus, hypertension, endocrinologic disease, or psychiatric problems.
2) Any diseases during the study period.
3) Consuming caffeine, alcohol and drugs for 24 hours before the study.
All participants were informed of the procedures and signed a written informed consent form prior to enrollment.
3. Sample Size Calculation
The sample size was calculated to enhance the reliability of the results. Sample size calculation was performed using G*power, version 3.1.9.4. The sample size was calculated by referring to the results of a previous study using GV203. The main outcome was the change in the CO2 reactivity of the Rt.MCA before and after GV20 acupuncture. According to the previous study3, the CO2 reactivity of the Rt.MCA was 1.98±0.41%/minute in the before acupuncture group and 3.08±0.74%/minute in the after acupuncture group. Taking into consideration a two-sided significance level of 5% (α) and a test power of 80% (β), the required sample size was 6. Based on the result, in order to consider the dropout rate of 10% and to verify the results of the previous study, the sample size was set to 10 as in the previous study.
4. Acupuncture Treatment
All procedures of acupuncture treatment were performed by an experienced Korean Medical Doctor with more than 3 years of clinical experience. The control group received acupuncture treatment at GV20, and the experimental group received acupuncture treatment at GV20 and EX-HN1. The acupuncture point, Baihui (GV20), is located on the head, 5 B-cun superior to the anterior median line. When the ears are folded, GV20 is located at the midpoint of the connecting line between the auricular apices. Sishencong (EX-HN1) are four points located 1 B-cun anterior, posterior and bilateral to GV20. The locations of the GV20 and EX-HN1 were determined according to the WHO standard Acupuncture Point Locations18. A stainless steel acupuncture needles (diameter 0.25 mm, length 40 mm; DongBang Acupuncture, Seoul, Korea) were used for GV20, EX-HN1 acupuncture. The acupoints were stimulated manually until the participant felt the De-Qi sensation (aching, dullness, heaviness, numbness, radiating, tingling and spreading sensations)15. The needles were inserted into the skin approximately 5mm deep and removed after 20 minutes. All procedures were conducted in accordance with the Revised Standards for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA19, Table 1) guidelines.
Table 1
Acupuncture Rationale and Needling Details according to STRICTA Guideline
5. Measurements
The research protocol was based on previous studies that used TCD to identify the relationship between blood flow in the cerebral arteries and acupuncture points3,20-22. For each process, CBF velocity and the CO2 reactivity of both MCAs and both ACAs were measured using the - a Multi-Dop X4 system TCD device (Compumedics DWL, Singen, Germany). All procedures of TCD measurement were performed by a trained TCD perfomer with more than 2 years of experience.
Variables that can affect CBF, i.e., blood pressure, heart rate, and end-tidal carbon dioxide (PETCO2), were measured using various modules on the Cardiocap S/5 monitor (Datex-Ohmeda, Helsinki, Finland). The mean blood pressure was determined and the heart rate was continuously recorded via an oximetry device attached to the participant’s finger to exclude the possibility that CBF changes were caused by BP, HR changes. Further, PETCO2 was continuously obtained via a Cardiocap S/5 monitor-connected nasal prong placed in the participant’s nostril, and each participant was instructed to breathe only through the nose during the procedure. A snapshot function in the Cardiocap S/5 monitor program was used to obtain the mean heart rate and PETCO2 at specific time points during the procedure. These variables were monitored and recorded on a computer that was connected to the Cardiocap S/5 monitor program.
When the visit, each participant was asked to sit in a comfortable position. The Cardiocap S/5 monitor and bilateral probe holder (LAM-Rack; Compumedics DWL) was positioned (Fig. 2.) to measure the CBF velocities of MCAs or ACAs through the temporal windows using a 2 MHz pulsed-Doppler probe. The strongest wave pattern was captured at depths ranging from 40 to 60 mm for MCAs, and from 65 to 80 mm for ACAs. All measurements were initiated after making the participant rest for 5 minutes. The blood pressure was checked 3 times every 2 minutes. After starting the first measurement of CBF velocity, the participant had rest for 2 minutes and performed CCR (Closed Circuit Rebreathing) for 1 minute. The CCR was the method to breath that participants inhaled their own exhaled air again using a 5 liter reservoir bag. After performing CCR, the participant was given a 1 minute break and the first TCD measurement was completed. Then, acupuncture was performed at GV20/GV20+EX-HN1 for 20 minutes. Subsequently, the same procedure was repeated for the post-acupuncture measurements (See Fig. 2. for the entire process).
For TCD measurement, the sample and gain values were corrected and saved if the CBF wave patterns remained constant. The mean blood flow velocity was calculated continuously as the time-averaged maximum velocity over the cardiac cycle, as computed from the envelope of maximum frequencies. The mean blood flow velocities were obtained at rest under stable normocapnic conditions, and near the end of the CCR period under hypercapnic conditions. All TCD spectras were recorded for subsequent review.
6. Calculations
CO2 reactivity refers to the percent change in mean blood flow velocity per millimeter of mercury change in PETCO2, as calculated by the following formula23:
Where Vrest is the blood flow velocity at rest, obtained during the most stable period under stable normocapnic conditions; Vhypercapnia is the blood flow velocity in the latter half of the 1 minute CCR period; and ΔPETCO2 is the change in PETCO2 between baseline and maximal CCR.
CBF velocity is dependent on the arterial CO2 tension, and corrected the blood flow velocity was calculated at 40 mmHg of CO2 tension (CV40, cm/s) using the following formula24:
CV40 (Corrected Velocity at PETCO2 40 mmHg) =V1 · eb(PCO2 40mmHg-P1CO2)
Where b represents CO2 reactivity; V1 represents velocity at P1CO2; and PETCO2 represents the end-tidal CO2 partial pressure.
The rate of change of CO2 reactivity was calculated as the percentage(%) of CO2 reactivity after acupuncture compared to CO2 reactivity before acupuncture.
Rate of change of CO2 reactivity=100×CO2 reactivityafter/CO2 reactivitybefore
CO2 reactivityafter is the CO2 reactivity after acupuncture and CO2 reactivitybefore is the CO2 reactivity before acupuncture
The rate of change of CV40 was calculated as the percentage (%) of CV40 after acupuncture compared to CV40 before acupuncture.
Rate of change of CV40=100×CV40after/CV40before
CV40after is the CV40 after acupuncture and CV40before is the CV40 before acupuncture
7. Statistical analysis
Statistical analysis was performed using the Statistical Package for the Social Sciences version 25.0 for Windows (SPSS, Chicago, Illinois, United States). As a result of a normality test using Shapiro-Wilk, the normality was not satisfied in some data. Wilcoxon signed-rank test were used to made statistical comparisons of the study parameteres between before and after GV20, EX-HN1 acupuncture and between GV20 single point acupuncture and combination acupuncture of GV20, EX-HN1. P-values under 0.05 were considered statistically significant. Data was summarized as the mean±standard deviation.
III. Results
1. Changes after GV20 Acupuncture
1) Changes of the CO2 reactivity of both MCAs and ACAs after GV20 Acupuncture
There was significant increase in the CO2 reactivity of both MCA and ACA after GV20 acupuncture treatment compared with baseline (Table 2).
Table 2
Comparing the CO2 Reactivity (%/minute) before and after GV20 Acupuncture or Acupuncture at GV20 and EX-HN1
| Acupuncture | Before | After | p-value* | The rate of CO2 reactivity | p-value** | |
|---|---|---|---|---|---|---|
| Right MCA | GV20 | 3.85±1.62 | 4.92±1.16 | 0.013 | 136.08±33.89 | 0.017 |
| GV20+EX-HN1 | 4.02±1.37 | 6.80±1.79 | 0.005 | 178.39±53.45 | ||
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| Left MCA | GV20 | 3.57±1.10 | 4.27±0.65 | 0.037 | 127.45±32.78 | 0.017 |
| GV20+EX-HN1 | 3.52±0.97 | 6.46±2.14 | 0.005 | 191.38±67.68 | ||
|
|
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| Right ACA | GV20 | 3.78±1.77 | 4.66±1.39 | 0.022 | 133.95±37.43 | 0.013 |
| GV20+EX-HN1 | 3.43±1.55 | 4.92±1.40 | 0.009 | 158.42±56.57 | ||
|
|
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| Left ACA | GV20 | 4.23±1.91 | 5.08±2.20 | 0.037 | 122.80±24.93 | 0.028 |
| GV20+EX-HN1 | 3.71±1.77 | 6.21±3.56 | 0.005 | 168.00±35.74 | ||
2) Changes of the CV40 of both MCAs and ACAs after GV20 Acupuncture
There were no significant changes in the CV40 between before and after GV20 acupuncture in both MCAs and ACAs (Table 3).
Table 3
Comparing the CV40 (cm/s) before and after GV20 Acupuncture or Acupuncture at GV20 and EX-HN1
| Acupuncture | Before | After | p-value* | The rate of CV40 | p-value** | |
|---|---|---|---|---|---|---|
| Right MCA | GV20 | 65.89±17.95 | 66.07±14.59 | 0.959 | 102.40±11.24 | 0.878 |
| GV20+EX-HN1 | 66.45±21.24 | 66.48±19.47 | 0.878 | 101.20±7.63 | ||
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| Left MCA | GV20 | 68.26±14.67 | 71.29±13.78 | 0.386 | 105.61±11.29 | 0.799 |
| GV20+EX-HN1 | 65.84±12.57 | 68.25±14.04 | 0.241 | 104.24±13.25 | ||
|
|
||||||
| Right ACA | GV20 | 63.00±11.93 | 65.66±15.24 | 0.646 | 104.63±15.92 | 0.508 |
| GV20+EX-HN1 | 62.30±9.82 | 64.06±12.77 | 0.285 | 102.34±5.42 | ||
|
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| Left ACA | GV20 | 69.76±8.75 | 69.06±9.28 | 0.646 | 99.10±6.36 | 0.959 |
| GV20+EX-HN1 | 65.79±6.37 | 66.17±6.88 | 0.799 | 101.02±10.85 | ||
3) Changes of the mean blood pressure and heart rate after GV20 Acupuncture
There was no significant difference in the mean blood pressure and heart rate before and after GV20 acupuncture (Table 4).
Table 4
Comparing the Mean Blood Pressure and Heart Rate before and after GV20 Acupuncture or Acupuncture at GV20 and EX-HN1
| Acupuncture | Before | After | p-value* | |
|---|---|---|---|---|
| GV20 | Mean blood pressure (mmHg) | 103.98±8.35 | 105.18±7.71 | 0.067 |
| Heart rate (bpm) | 73.32±7.21 | 71.92±6.76 | 0.115 | |
|
|
||||
| GV20+EX-HN1 | Mean blood pressure (mmHg) | 104.22±8.09 | 104.65±9.12 | 0.503 |
| Heart rate (bpm) | 72.38±8.66 | 70.53±6.58 | 0.062 | |
2. Changes after Acupuncture at GV20 and EX-HN1
1) Changes of the CO2 reactivity of both MCAs and ACAs after Acupuncture at GV2O and EX-HN1
There was significant increase in the CO2 reactivity of both MCAs and ACAs after acupuncture treatment at GV20 and EX-HN1 compared with baseline (Table 2).
2) Changes of the CV40 of both MCAs and ACAs after Acupuncture at GV2O and EX-HN1
There were no significant changes in the CV40 between before and after acupuncture treatment at GV20 and EX-HN1 in both MCAs and ACAs (Table 3).
3) Changes of the mean blood pressure and heart rate after Acupuncture at GV2O and EX-HN1
There was no significant difference in the mean blood pressure and heart rate before and after acupuncture treatment at GV20 and EX-HN1 (Table 4).
3. Comparison of the parameters before acupuncture between the Control group (GV20 single point) and the Experimental group (combination of GV20 and EX-HN1 acupoints)
1) Comparison of the CO2 reactivity before acupuncture between the control group (GV20 single point) and the experimental group (combination of GV20 and EX-HN1 acupoints)
There was no significant difference in the CO2 reactivity before acupuncture treatment between control group (GV20 single point) and experimental group (combination of GV20 and EX-HN1 acupoints) in both MCAs and ACAs. (Rt. MCA: 3.85 to 4.02, p=0.721 ; Lt.MCA: 3.57 to 3.52, p=0.799 ; Rt. ACA: 3.78 to 3.43, p=0.074 ; Lt.ACA: 4.23 to 3.71, p=0.074)
2) Comparison of the CV40 before acupuncture between control group (GV20 single point) and experimental group (combination of GV20 and EX-HN1 acupoints)
There was no significant difference in the CV40 before acupuncture treatment between control group (GV20 single point) and experimental group (combination of GV20 and EX-HN1 acupoints) in both MCAs and ACAs. (Rt. MCA: 65.89 to 66.45, p=0.959 ; Lt.MCA: 68.26 to 65.84, p=0.508 ; Rt. ACA: 63.00 to 62.30, p=0.799 ; Lt.ACA: 69.76 to 65.79, p=0.285)
3) Comparison of the mean blood pressure and heart rate before acupuncture between control group (GV20 single point) and experimental group (combination of GV20 and EX-HN1 acupoints)
There was no significant difference in the mean blood pressure and heart rate before acupuncture treatment between control group (GV20 single point) and experimental group (combination of GV20 and EX-HN1 acupoints). (Mean blood pressure: 103.98 to 104.22, p=0.968 ; Heart rate: 73.32 to 72.38, p=0.614)
4. Comparison between the Control group (GV20 single point) and the Experimental group (combination of GV20 and EX-HN1 acupoints)
1) Comparison of the rate of Change of the CO2 reactivity between the control group (GV20 single point) and the experimental group (combination of GV20 and EX-HN1)
In both MCAs and ACAs, the increase rate of CO2 reactivity before and after acupuncture at combination of GV20 and EX-HN1 was significantly higher than the increase rate of CO2 reactivity before and after acupuncture at GV20 single point (Table 2).
2) Comparison of the rate of Change of the CV40 between the control group (GV20 single point) and the experimental group (combination of GV20 and EX-HN1)
There were no significant changes in the rate of change of CV40 between acupuncture at GV20 single point and combination of GV20 and EX-HN1 in both MCAs and ACAs (Table 3).
IV. Discussion
Cerebrovascular reactivity (CVR) is the change in cerebral blood flow in response to a vasodilatory or vasoconstrictive stimulus25, and it represents the compensatory potential of the vessels regulating blood flow to the brain. Several studies have reported decreased cerebrovascular reactivity in patients with cerebral ischemia26. Furthermore, the CVR of the ischemic side was more impaired than that of the unaffected side, which means that the arterioles are already chronically dilated and the range of automatic regulatory function is reduced27.
In this study, the CO2 reactivities of both MCA and ACA were significantly increased in both acupucture treatment at GV20 single point and combination of GV20 and EX-HN1. And the increase rate of the CO2 reactivity was significantly higher in both MCA and ACA when acupuncture treatment was performed at GV20 and EX-HN1 compared to GV20 alone. However the CV40 of both MCA and ACA has no significant change in both acupucture treatment at GV20 single point and combination of GV20 and EX-HN1, and the change rate of CV40 also has no significant difference between the control group and the experimental group.
As the results of this study verified the increase rate of CVR of GV20 and EX-HN1 acupuncture was significantly higher than GV20 acupuncture, the study suggests that the effect of GV20 acupuncture increases when acupuncture along with EX-HN1. And it suggests the potential of a synergistic effect of EX-HN1 on the effect on GV20 acupuncture on CBF.
When compared with the results of the previous study3, the CVR of both MCA and ACA increased significantly after GV20 acupuncture as in the previous study. However, while the CV40 of both MCA and ACA also increased significantly after GV20 acupuncture in the previous study, there was no significant change of CV40 in this study.
The difference between the progress of the previous study and this study is that the cerebral blood flow rate was increased through closed circuit respiration in this study, while the previous study applied hyperventilation-induced hypocapnia to measure the CVR. It is thought that since the degree of change in the partial pressure of carbon dioxide was different according to the change in the breathing method, the degree of change in the CBF velocity also had difference between the studies. Also, when hyperventilation is performed during TCD monitoring, the probe attached on the holder can be shaken, which might make it difficult to measure the blood flow. In addition, some participants appealed that rapid hyperventilation was not easy to do. In this study, the CCR was used to try to improve these problems and the results indicate that it is easier and produce more stable measure values than hyperventilation method. Therefore, the results of this study that applied the CCR are considered to be more reliable. Furthermore, the sample size calculation was performed based on the results on the CO2 reactivity in previous GV20 study3, not base on the results of CV40. Therefore the calculated sample size may not be sufficient to confirm the effect of GV20 acupuncture on CV40.
Acupuncture is thought to improve blood flow by treating endothelial dysfunction28,29. A previous study reported that acupuncture enhanced endothelial function and vascular reactivity by regulating vasoconstrictors and vasodilators29. One of the most significant vasodilating substances secreted by the endothelium is endothelium-derived nitric oxide (EDNO). Reduced NO in impaired endothelial functional status has been reported, which may be due to decreased activity of endothelial NO synthetase (eNOS) and decreased bioavailability of NO29. Several studies have shown that acupuncture increased the production of NO in arterioles, and activated the expression of eNOS28. These results suggest that the effect of acupuncture on endothelial dysfunction might result from inducing synthesis and activation of EDNO30. Also, CBF change by an adjustment of the vessel diameter can be divided into endothelium- dependent and non-dependent. In previous studies, CBF and CVR were reported to be associated with endothelium dependent31,32. Therefore, it can be estimated that acupuncture improves cerebral blood flow and further increases CVR by improving the endothelial function of cerebral blood vessels.
The limitation of this study is that there was an absence of subject blinding, which may have caused placebo bias. And the study is limited to males in 20s, so extended studies of different ages and genders can support the conclusion of this experiment. Also, since the study is limited to healty people with normal cerebral blood flow state, additional studies on patients with hemodynamic disorder such as arteriosclerosis or cerebral ischemia may be necessary.
This study confirmed the difference in cerebral blood flow before and after acupuncture between GV20 alone acupuncture treatment and combination acupuncture treatment of GV20 and EX-HN1, which has been widely used for stroke through an experimental method. Therefore, in clinical practice, this study may be the evidence to apply acupucture at combination of GV20 and EX-HN1 to improve cerebral blood flow. Furthermore, because the CCR method was applied, the results of this study are thought to be more objective and reliable than previous studies.
V. Conclusions
This study demonstrated that GV20 and EX-HN1 acupuncture treatment increased CO2 reactivity in both MCA and both ACA and the increase rate of the CO2 reactivity was significantly higher in GV20 and EX-HN1 than in GV20 acupuncture alone on both MCA and ACA.
These results may clinically support the use of EX-HN1 with GV20 to treat disorders of MCA and ACA circulation, such as ischemic stroke and cerebrovascular insufficiency.
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