Acupuncture stimulation and phototherapy (laser treatment) are very commonly used in pain clinics. Good results have been reported with these techniques, either alone or in combination with other treatments, such as nerve block therapy or drug therapy.1–12 However, the effectiveness of acupuncture stimulation and phototherapy remains controversial.1,13–16 The beneficial effects of acupuncture stimulation and phototherapy are most likely attributed to increased blood flow because of physical stimulation. In addition to analgesic activity, improved microcirculatory flow has been reported.4,5 However, few studies17–20 have directly examined changes in peripheral blood vessels, either quantitatively or objectively. We therefore evaluated whether the microcirculation was improved by acupuncture stimulation and phototherapy in rabbits. The microcirculation was assessed by means of the rabbit ear chamber (REC) method.21,22
The REC method developed at our laboratory allows a single blood vessel to be observed by intravital microscopy, both directly and noninvasively in real-time. This method has been confirmed to be useful for observing the effects of various interventions on peripheral hemodynamics.21 Vasomotor activity, neural control, and responses of vessels to drugs in an REC are similar to those of in situ vessels 6 wk after attachment of a clear window to a rabbit's ear.21,22 We have used this model to study the peripheral microcirculation during wound healing,23 systemic agglutination anaphylaxis,24 acute severe hemorrhage caused by colloid resuscitation,25 various inspired oxygen concentrations,26 and various arterial pressures of CO2.27
In the present study, we assessed the responses of arteriolar blood flow to acupuncture stimulation and phototherapy under direct visualization to examine the effects of these treatments.
All experiments were in accordance with the National Institutes of Health guidelines on the use of experimental animals. Approval from the Animal Use Committee of Tokyo Women's Medical University was obtained before initiating the experiments.
Forty randomly selected Japanese domestic white rabbits (body weight, 2.5–3.0 kg) were studied. Transparent round chambers made of acrylic resin (REC) were inserted in the ear lobes, as described previously.21,22 In short, the REC was composed of a disk with a central round table and three peripheral pillars, a cover plate, and a holder ring. The disk was designed to leave a cavity measuring 50 μm in thickness and 6.4 mm in diameter between the central round table and the cover plate. A cover plate was fixed onto the pillars with the use of a holding ring. New microvessels arose from the blood vessels of the dermis and covered the entire cavity within 6 wk. Several studies reported that newly developed vessels in RECs respond to various drugs given systemically, similar to vessels in vivo.21–25
The RECs were observed microscopically at a magnification of 100× more than 6 wk after insertion of the ear chamber. Microcirculatory changes were recorded using a microscope-closed video camera (DXC 750; SONY, Tokyo, Japan) with a shutter speed of 1/10,000 s.
After IV injection of pentobarbital 30 mg/kg, the trachea of the rabbit was intubated, and spontaneous respiration was maintained. Anesthesia was maintained by the IV injection of pentobarbital at 30 mg · kg−1 · h−1. Oral temperature was continuously monitored (CTM-303; TERUMO, Tokyo, Japan), and body temperature was kept constant (38°C–39°C) with the use of a heating pad.
Rabbits were randomly assigned to receive acupuncture stimulation (acupuncture group, n = 10), near-infrared lamp irradiation (lamp group, n = 10), near-infrared low-powered laser irradiation (laser group, n = 10), or no irradiation (control group, n = 10). Stainless steel disposable acupuncture needles (length, 30 mm; diameter, 0.20 mm) (Seirin CO, Shizuoka, Japan) and a near-infrared lamp (Super Lizer HA-30, Tokyo Medical Research CO, Tokyo, Japan) were used. This irradiation system uses a superiodine lamp as the light source. An optical filter was used to provide only short-wavelength near-infrared irradiation (600–1600 nm). Near-infrared rays more deeply penetrate the body than ordinary infrared rays (including far-infrared rays). Because an infrared lamp was used as the light source, high-energy radiation (up to 1800 mW) can be delivered. A low-output gallium-aluminum-arsenic semiconductor laser device (Flat 10, Nippon Infrared Industries CO, Tokyo, Japan) was used to produce the near-infrared low-powered laser radiation. A contact-type laser with a wavelength of 830 nm and a maximum output of 100 mW could be activated when the probe came in contact with the skin. In the control group, a dummy device with the same external appearance as the Super Lizer and a contact probe were used. In the acupuncture group, five needles were placed around the crus of the helix at the center of the outer ear, which is controlled by the vagus nerve and corresponds to the cavum conchae in humans.28,29 The needles were left in place for 20 min, without electrical stimulation. A typical region of stimulation of the auricle is indicated by the open circle in Figure 1. The stimulated region was located around 4 cm from the center of the REC. The lamp group repeatedly received 1 s of near-infrared irradiation (1540 mW) followed by 4 s of treatment cessation. The diameter of the irradiated area was 7 mm, and the energy power density was 40 mW/mm2. The laser group continuously received 60 mW of laser irradiation. The diameter of the irradiated area was 1.4 mm, and the energy power density was 39 mW/mm2. In the lamp, laser, and control groups, the same site as that of the acupuncture needles in the acupuncture group was irradiated or applied the contact probe for 10 min.
During the baseline period after surgical preparation, we selected arterioles with diameters of 20–100 μm, displayed on a video television screen. Blood-vessel diameter, blood flow velocity, and blood flow rate for up to 60 min (measured at 10-min intervals) after acupuncture or irradiation treatment were compared with the baseline values. To analyze blood flow velocity, the play speed of the video recorder was set at 1/60 of a second. The distances between two erythrocytes at the center of the blood vessel were measured 10 times, and the values were averaged. Blood flow rate was calculated by multiplying the blood flow velocity by the blood vessel cross-sectional area.
All data are expressed as means ± sd. Statistical comparisons were performed using repeated-measures analysis of variance followed by Fisher's protected least significant difference test. A value of P < 0.05 was considered to indicate statistical significance.
As shown in Figure 2, arteriolar diameter significantly increased to 131% ± 14% in the acupuncture group (P < 0.005), 129% ± 19% in the lamp group (P < 0.005), and 128% ± 11% in the laser group (P < 0.005), when compared with a pretreatment value of 100%. Arteriolar diameter reached the maximal value at 10 min after the end of lamp and laser irradiation, and 20 min after the end of acupuncture stimulation. The arteriolar diameters in the three treated groups increased significantly when compared with the value in the control group (P < 0.005). Blood flow velocity (Fig. 3) and blood flow rate (Fig. 4) significantly increased to 131% ± 14% and 220% ± 66% in the acupuncture group (P < 0.005), 130% ± 17% and 206% ± 66% in the lamp group (P < 0.005), and 126% ± 15% and 216% ± 48% in the laser group (P < 0.005), when compared with the respective pretreatment values (100%). Blood flow velocity and rate showed similar trends to the changes in arteriolar diameter. Arteriolar blood flow significantly increased after acupuncture stimulation and phototherapy. There were no nonresponders in the acupuncture, lamp, or laser groups. The effect of treatment persisted for 40–50 min after the end of stimulation and irradiation. The time to the onset of response and the duration of response did not differ significantly among the three treated groups. In the acupuncture group, however, the time to response was slightly delayed, and the duration of response tended to be longer. Microscopic views of microvessels observed with the REC method are shown in Figure 5. Arteriolar diameter increased after acupuncture stimulation (Fig. 5a, lower panel), near-infrared lamp irradiation (Fig. 5b, lower panel), and low-powered laser irradiation (Fig. 5c, lower panel). Arterioles were unchanged after use of the dummy device (Fig. 5d, lower panel).
Several methods have been used to examine the relations of acupuncture stimulation or laser irradiation to blood flow. Ernst and Lee6 attributed the increase in skin-surface temperature after acupuncture stimulation to the inhibition of sympathetic-nerve activity. Maeda et al.7 reported that acupuncture stimulation decreases plasma endothelin-1 concentrations, thereby causing vasodilation and an increase in skin temperature. In studies using pulsed Doppler ultrasonography, Litscher et al.8 found that acupuncture stimulation increases blood flow velocity in the middle cerebral artery, and Li et al.9 demonstrated that electrical stimulation of the median nerve improves local myocardial ischemia in anesthetized cats. Boutouyrie et al.10 showed that acupuncture stimulation causes vasodilation of the radial artery. In studies using positron emission tomography and radioactive isotopes, Hsieh et al.11 found that blood flow of the hypothalamus, midbrain, and cerebellum increases after acupuncture stimulation. Uchida et al.12 showed that acupuncture stimulation increases cerebral blood flow as measured with a laser Doppler blood flowmeter in anesthetized rats. This increase in cerebral blood flow was independent of arterial blood pressure. Banzer et al.30 reported that laser needle stimulation does not promote peripheral tissue oxygenation but improves the microcirculation. Ihsan31 histopathologically demonstrated that low-level laser therapy increases capillary diameter and elevates levels of fibroblast growth factor. These studies thus provided indirect evidence that acupuncture stimulation and laser treatment improve blood flow, whereas in our study direct evidence was obtained under real-time direct visualization. Litscher monitored the time course of the peripheral microcirculation by laser Doppler after stimulation of the fingertips of healthy volunteers with a laser needle and reported an increase in flow rate within 1 min after the start of stimulation. After the increase, the flow rate remained constant for 20 min during stimulation.18,19 In our study, arteriolar blood flow began to increase 10 min after the start of acupuncture stimulation, further increased after 20 min and continued to increase until 20 min after the completion of stimulation. Discrepancies among studies in the response of arteriolar blood flow might be attributed to the differences in the stimulation technique or stimulation point.
One of the mechanisms underlying the local response of blood flow to acupuncture stimulation may involve a reaction via the axon reflex. Kashiba and Ueda32 immunohistochemically showed that electro-acupuncture stimulation at high voltage (10 V) and 10 Hz for 30 min stimulates the release of substance P, a neurotransmitter of the axon reflex, and calcitonin gene-related peptide from the peripheral terminals of primary sensory neurons. These findings suggested that blood flow results from vasodilation induced by calcitonin gene-related peptide. Our results indicated that the axon reflex was one factor related to the increase in peripheral blood flow.
Nitric oxide (NO) has been implicated in vasodilation caused by acupuncture stimulation. Loaiza et al.33 reported that the diameter of arterioles in the knee joint capsule increased by about 25% after acupuncture stimulation to the quadriceps femoral muscles in anesthetized rats. To examine the mechanism of vasodilation, they studied the effect of NO, a powerful vasodilator released from endothelial cells and nerves. Vasodilation disappeared after pretreatment with N(omega)-nitro-l-arginine methyl ester (L-NAME), an inhibitor of NO synthetase. This finding suggested that NO is involved in the vascular response to acupuncture stimulation. Tsuchiya et al.4 studied the effects of acupuncture on local NO levels and blood flow in a single hand and forearm, with a 1-wk interval between treatments. The NO level in plasma obtained from the arm treated by acupuncture significantly increased 5 min and 60 min after treatment. Blood flow was measured with a laser tissue-blood flowmeter. Blood flow in the palmar subcutaneous tissue of the arm undergoing acupuncture also increased, and this increase correlated with the rise in the NO level. Acupuncture increases the NO level in treated regions and thereby enhances the local circulation. These regulatory effects might contribute to the pain relief provided by acupuncture. Maegawa et al.5 studied the effects of laser irradiation on the microcirculation of the rat mesentery. The main mechanism responsible for an 80% increase in blood flow was reported to involve effects on calcium ions (Ca2+) in vascular smooth-muscle cell membranes; laser irradiation apparently decreased intracellular Ca2+ and relaxed smooth muscle. To further examine the effect of NO on vascular response, they performed laser irradiation after treatment with L-NAME. There was no increase in arteriolar diameter, suggesting that NO contributes to the increase in blood flow induced by laser irradiation. The increase in peripheral blood flow in response to acupuncture stimulation and phototherapy in our study thus most likely involved NO.
The mesentery is widely used to directly assess the microcirculation. Observation of the mesenteric microcirculation is, however, invasive because it requires open surgery. Consequently, animal models of the mesenteric circulation do not exactly reproduce the physiologic situation. In contrast, the REC method is noninvasive and permits real-time and repeated measurements of the hemodynamics of the same peripheral blood vessels under direct vision because the wound completely heals 6 wk after surgery for installation. In addition, the REC method allows changes in the microcirculation to be followed for a prolonged period after repeated stimulation because the same animals can be successively observed for a long period.21,22 We, therefore, used the REC method to directly observe the peripheral microcirculation in our study.
The REC method has also been used to study the nonlocal effects of acupuncture. Asano20 reported that the placement of a single acupuncture needle (32 gauge, silver) in the back of rabbits, corresponding to Geshu (B17) in humans, enhanced microvascular blood flow in association with increased vasomotion.
In summary, acupuncture stimulation and phototherapy were directly confirmed to increase the diameter and blood flow velocity of peripheral arterioles. Acupuncture stimulation and phototherapy, associated with minimal systemic and local side effects, can enhance the microcirculation and may be a useful supportive treatment for diseases caused by poor peripheral blood flow.
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© 2009 International Anesthesia Research Society
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