Regulation of the cardiovascular function by CO2 laser stimulation in anesthetized rats
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- Friedemann, T., Shen, X., Bereiter-Hahn, J. et al. Lasers Med Sci (2012) 27: 469. doi:10.1007/s10103-011-0973-3
Physical stimulation of body surface points is known to affect various organ functions. In traditional Chinese medicine, so-called acupoints were defined. These points can be physically stimulated to effectively treat various diseases. Here we describe for the first time the effect of CO2 laser stimulation at the acupoints Neiguan (PC-6), Quchi (LI-11), Zusanli (ST-36), and Taichong (LR-3) on heart rate and mean arterial blood pressure in anesthetized rats. CO2 laser stimulation increased the skin surface temperature to 54°C. Our results revealed that the laser stimulation at the left or right PC-6 and LR-3 increased heart rate and mean arterial pressure. There was no response of heart rate and mean arterial pressure during and after stimulation of the left LI-11, but laser stimulation at the right LI-11 slightly increased heart rate and mean arterial pressure. On the other hand, laser stimulation at the left and right ST-36 decreased heart rate and mean arterial pressure. The effects on mean arterial pressure were more pronounced than those on heart rate. After full spinal cord transection, all heart-rate and mean-arterial-pressure responses were attenuated or completely abolished. These results suggest that CO2 laser stimulation at either the left or right PC-6, ST-36, and LR-3, as well as at the right LI-11 can modulate the cardiovascular functions in anesthetized rats, and its modulatory site might be supraspinal.
KeywordsCO2 laserBlood pressureHeart rateSpinalizationRat
LU-5 (Lung 5)
PC-6 (Pericardium 6)
LI-11 (Large intestine 11)
ST-36 (Stomach 36)
LR-3 (Liver 3)
Central nervous system
Infrared laser stimulation
Mean arterial blood pressure
Standard error of the mean
The present study aims to investigate the effect of LS at different acupoints and to determine the neuronal pathways responsible for the cardiovascular regulatory effect of LS.
Animals and surgical methods
Sixteen adult male Sprague-Dawley (SD) rats weighing between 250 and 300 g were kept in a climate-controlled environment with food and water ad libitum. For all surgical operations, such as cannulation of trachea, artery, vein, and full spinal cord transection, the animals were initially anesthetized with an intraperitoneal injection of 50 mg/kg pentobarbital. The dura overlying the C1 and C2 spinal segment was removed to expose the spinal cord and a complete transection of the spinal cord was preformed with a sharp hook. The success of the transaction was confirmed by pinching the tail. After the spinal cord transaction, the rats were allowed to recover for 15 min. All experiments were stopped 1 h after spinalization or if the BP was no longer stable. During the entire time period of data recording, a 1% pentobarbital PBS solution was continuously injected via a cannula inserted into the right jugular vein (rJV). A microinjection pump was used to maintain a constant flow speed of 0.75-1 ml/h for stable anesthetic condition, as described previously . All animal experiments were carried out in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals in China.
To obtain a stable baseline recording, the rats were kept in the supine position for 30–60 min after the operation. In eight rats, a full spinal cord transection was carried out at the C2 level.
Electrocardiogram (ECG) was measured according to Einthoven; the negative electrode was placed underneath the skin of the right forelimb and the positive electrode was inserted under the skin of the left hind limb. Another electrode was placed subcutaneously at the right hind limb and connected to the ground. Both electrodes were connected to a differential amplifier (EX4-400 Differential Amplifier), which fed the signal to a computer-linked main amplifier (PowerLab/16, AD Instruments, Australia). The ECG was recorded and analyzed with LabChart 6 Pro. From the recorded data, heart rate (HR) was extracted.
Laser stimulation at 10.6 μm was achieved by use of the 0.5-W CO2 Laser Surgical System ML015-CA (SWOT, China) in continuous-wave mode. For a spot size of 3 mm in diameter, the power density was 71 mW/mm2. The irradiation was applied for 30 s at acupoints PC-6, LI-11, ST-36, and LR-3, giving a total energy density of 2.1 J/mm2. The positions of these points and their functions [21, 22] in the rat are indicated in Fig. 1. The figure also shows Chizi (LU-5), which is needed to locate LI-11. To properly locate the stimulating laser beam, a red pilot beam of a 2-mW diode laser was used. An irradiation time of 30 s was used since it did not produce any irritation to the skin, and the same responses could be obtained for repeated stimulation. After only 1 min of irradiation the skin started to turn red and show blisters. Though longer irradiation times or higher output power produced stronger effects, we stayed at this energy density for allowing repeated application on a single rat. Before LS was applied, the acupoints were shaved with an electrical shaver, paying attention not to hurt the skin. Afterwards, the CO2 laser was pointed perpendicularly onto the acupoints with a spot size of the laser of 3 mm in diameter. The distance between the laser head and the acupoint was adjusted so that the skin surface temperature during the stimulation reached 54°C within 4 to 5 s; this was achieved reproducible at a distance of 14 cm. The temperature was confirmed in three ways: (1) by a thermistor probe (1 mm) attached to the skin; (2) by an infrared thermometer; and (3) by an infrared camera (ThermaCAM P30, FLIR Systems); the 54°C were reached within the 30-s illumination. With this procedure of stimulation, superficial skin effects could be detected without thermal stimulation of subcutaneous tissue. The test interval between two different acupoints was at least 10 min. A new stimulation was only preformed if the BP and HR showed a stable baseline for 5 min.
The mean values of HR and MAP determined for 1 min before stimulation were used as pre-stimulus control values and set to 100%. Presented average data are means ± standard error of the mean (SEM). During 30 s of CO2 laser stimulation, the means of HR and MAP were calculated at 10-s intervals. In addition, means of HR and MAP were determined during 2-min post-stimulation periods at 30-s intervals; the values were expressed as percentage of the pre-stimulus control value. Changes of mean of HR and MAP during and after CO2 laser stimulation were then compared with pre-stimulus control values using Student’s t test followed by one-way repeated ANOVA analysis. Statistical differences between groups were determined using a one-way repeated ANOVA followed by Bonferroni post-hoc analysis. Statistical significance was accepted on the basis of p < 0.05.
Modulation of HR and MAP by laser stimulation (LS) in CNS-intact rats
Relative changes (in % of control) in HR and MAP in response to 30 s of LS application to different acupoints of CNS-intact rats: Data represent mean relative responses of n = 8 ± SEM (four rats; two trials/rat). Differences in the effects that are mentioned in the text were considered as significantly different on the basis of p < 0.05. Asterisks indicate significant difference of right versus left
+2.2 ± 0.3
+3.8 ± 0.5*
+9.4 ± 1.1
+7.0 ± 0.4
+1.1 ± 0.4*
+3.5 ± 1.6*
+1.7 ± 0.3
+1.6 ± 0.3
+6.8 ± 0.7
+6.4 ± 0.7
−3.4 ± 1.0
−3.2 ± 1.0
−14.1 ± 3.6
−19.6 ± 2.3
Effects of LS on HR
LS was applied to the four different surface points (see Fig. 1) for a time period of 30 s. If the HR responded to the stimulation, the onset occurred with a delay of 10 to 15 s.
When LS was applied to PC-6 for 30 s, the mean HR increased with a delay of about 10 s (Fig. 2a); statistical analysis revealed a slightly more pronounced, but significant, effect when the right PC-6 was stimulated compared to the left PC-6 (see Table 1). Stimulation of LI-11 hardly affected HR (Fig. 2b). Only right LI-11 stimulation produced a very small though significant increase (see Table 1). In all cases, after LS was turned off, the HR returned to its control level within 1 min. Also, stimulation of LR-3 produced only a small increase in HR with no significant difference between left and right LR-3 (Fig. 2c, Table 1).
Effects of LS on MAP
Simultaneously with the determination of effects on HR, those on MAP were determined. The results on MAP were qualitatively similar to those on HR. The onset developed with a delay, and quantitatively the effects were more pronounced.
PC-6 stimulation led to a sharp increase in MAP and returned to the base level within a few seconds when LS was turned off (Fig. 3a) with no significant difference whether left or right PC-6 was stimulated (Table 1). For LI-11 stimulation MAP showed no significant change on stimulation of left LI-11, but a slight increase could be detected in response to right LI-11 stimulation (Table 1). The slight decrease of MAP shown in Fig. 3b was not always observed; on average, there was no significant change. Stimulation of LR-3 (Fig. 3c) resulted in similar effects as stimulation of PC-6 with no left/right difference (cf. Table 1).
Also, the MAP responded during the stimulation of ST-36 with a decrease (Fig. 3d) as seen for the HR (Figs. 2d and 4a). While the signal in case of PC-6 and LR-3 returned to the base level within a few seconds, at least 2 min were needed after LS of ST-36 was turned off. The time course of averaged data is shown in Fig. 4b, indicating that even a small persisting component seems to be present.
Modulation of HR and MAP by laser stimulation (LS) in spinalized rats
Relative changes (in % of control) in HR and MAP in response to 30 s of LS application to different acupoints of spinalized rats: Data represent mean relative responses of n = 8 ± SEM (four rats; two trials/rat). Differences in the effects that are mentioned in the text were considered as significantly different on the basis of p < 0.05. Asterisks indicate significant difference to unstimulated acupoints
+1.5 ± 0.7*
+0.8 ± 0.2*
+2.0 ± 0.9*
Effects of LS on HR
Changes in HR in response to LS application to the left PC-6 were completely abolished in the spinalized rats. Only stimulation of the right PC-6 produced a slight (but nevertheless significant) increase that continued to further increase to 2.3 ± 0.9% after LS was terminated. For all three of the other acupoints (LI-11, LR-3, ST-36), no significant changes in HR could be detected, independent of whether the left or right side was stimulated.
Effect of LS on MAP
As for HR, also MAP responses nearly complete disappeared after CNS transection. Only in response to stimulation of the left and right PC-6 a tiny increase in the averaged MAP values could be detected.
In the present study, we investigated the effects of LS at various body surface points on cardiovascular function in anesthetized rats. Since the temperature could reach values of more than 50°C, this stimulus may be considered as a cutaneous nociceptive thermal stimulation. The points were selected on the basis of traditional Chinese medicine for the treatment of heart diseases (PC-6, LI-11, LR-3, ST-36; see Fig. 1). The acupoints were heated up to about 54°C by the infrared light of a CO2 laser, which was achieved within 4–5 s. In rats with intact spinal cord, LS produced more or less pronounced immediate and transient effects on cardiovascular function as measured by HR and MAP. A cumulative effect we did not observe. In spinalized rats, only very small (if any) responses could be detected.
All the cardiovascular responses developed with a delay of 10–15 s. This could be due to energy absorption and heat transmission within the skin. In the hairy skin of monkeys, heating the surface from 38–53°C with a CO2 laser required about 6 s until the temperature at a depth of about 0.5 mm was near equilibrium . These authors, nevertheless, suggest a slow transduction mechanism or slow release of chemicals to account for the delayed response in nociception. Delayed response to heat of transient receptor potential channels (TRPV2, which has a threshold temperature at about 53°C) in Aδ and Aß fibers was also proposed by Lumpkin and Caterina . The energy of the CO2 laser is mainly absorbed by water molecules in the epidermis and varies between different locations and different animal. On the average CO2 laser, radiation has a penetration depth of approximately 20–50 μm [24–27]. Nevertheless, the heat that is produced in the tissue will spread out into superficial layers of the dermis due to heat transduction. Therefore, we may say that we stimulate only the epidermis and upper layers of the dermis and that the observed delay in cardiovascular responses could partially be caused by the slow temperature rise in deeper layers of epidermis and upper dermis due to heat transduction.
The effects on HR in rats with intact spinal cord were with only a few percent very small, but nevertheless statistically significant on the basis of p < 0.05. The most pronounced changes of HR were observed during LS of PC-6 and ST-36 (see Table 1), with PC-6 stimulation increasing and ST-36 stimulation decreasing the HR. Since all changes in HR were very small, a physiological significance is questionable. On the other hand, the changes of MAP in response to LS at the same time are in the range of up to 20%. As for the HR also for MAP, the most pronounced changes were observed during LS of PC-6 and ST-36, and again, PC-6 stimulation increased while ST-36 stimulation decreased MAP. In particular, the finding of reduced MAP in response to LS is in line with increased nerve discharges leading to reduced blood pressure in response to manual stimulation of ST-36 in rat ; also, in clinical studies, blood pressure could be reduced by stimulation of ST-36 . Of particular interest could be the fact that after termination of ST-36 stimulation the HR remained reduced.
The opposite modulations of HR and MAP by stimulation of PC-6 versus ST-36 may be due to different spinal segmental innervations. It had been previously suggested that cardiovascular responses depend on the segment where afferent fibers enter the spinal cord [19, 30]. The authors demonstrated that stimulation of somatic afferent fibers, which enter the spinal cord at different segments, elicited different MAP and HR responses as well as cardiosympathetic reflexes. Another possibility could be that the distribution of thermosensitive receptors is different at these acupoints leading to activation of different fiber groups.
After full spinal cord transection at the C2 level, a very small response could still be detected for the HR when the right PC-6 was stimulated, but no response on stimulation of left PC-6. Also, in rats with intact spinal cord laterality could be detected with significantly higher increase in HR when the right PC-6 was stimulated. In addition, stimulation of right LI-11 resulted in laterality with increased HR and MAP compared to stimulation of left LI-11. These observations are in favor of a combination of spinal and supraspinal component of the reflex response. In the supraspinal reflex pathway, somatic afferent information ascending to the brain stem will be integrated and descended equally to both the left and right sympathetic (or parasympathetic) efferent neurons resulting in equal left and right cardiac responses . The observation that LS at the right PC-6 elicited an increase in HR in spinalized rats also does not support a pure supraspinal reflex pathway. Stimulation of the acupoint PC-6 might activate somatic afferent nerve fibers in the median nerve, which enter the spinal cord at the 5th to 8th cervical (C5-8) segments and the 1st thoracic segment. The cardiac sympathetic efferent nerves emerge from the spinal cord at the 1st–6th thoracic (T1-T6) segments in rats . Therefore, some afferent nerve fibers from the median nerve enter the spinal cord at the same segment at which cardiac sympathetic efferent nerves emerge from the spinal cord. Kimura et al.  showed that spinal reflex responses of the cardiac sympathetic nerve were only evoked if the stimulation was delivered to afferent nerves close to the cardiac sympathetic outflow segment. Furthermore, it was reported that in spinalized rats, the HR response was stronger if a noxious mechanical stimulation was applied to the right side of the thorax, rather than at the left, and this phenomenon was explained by the strong influence of the cardiac sympathetic nerve on the right side of the heart, especially on the sinus node [19, 32–34]. Taking these results together, LS at PC-6 seems to activate somatic afferents in the median nerve, which leads to an activation of spinal and supraspinal reflex pathways in CNS-intact rats. The activation of a supraspinal reflex pathway produces an HR response that could be the same for stimulation of the left and right PC-6, but the additional activation of a spinal reflex pathway might increase HR if stimulation were applied to the right PC-6, due to strong influence of the cardiac sympathetic nerve at the right side of the heart. The spinal reflex pathway might also account for the increase of the HR during stimulation of the right PC-6 in spinalized rats. The fact that after spinal cord transection at the C2 level no HR response was found during the stimulation of the left PC-6 indicates that the increase in HR elicited via LS in CNS-intact rats at this acupoint was caused by a supraspinal reflex response.
In conclusion, the presented results indicate that LS applied to various acupoints in the fore and hind limbs and hind paw modulate cardiovascular function in CNS-intact pentobarbital-anesthetized rats. Among the tested acupoints, LS stimulation only at ST-36 reduced HR and MAP and, in addition, the effects were the most pronounced ones; this suggests that LS at ST-36 might be suitable for the treatment of hypertension in patients. The persistent effect of LS on HR supports the view of ST-36 as an acupoints for the treatment of CVD.
The nearly complete elimination of LS responses (except for the right PC-6) after C2 transection suggests a modulatory site for the cardiovascular response is mainly supraspinal. LS at the acupoints PC-6 and LI-11 increased the HR significantly more if the stimulation was applied at the right side of the body and after spinal transection at the C2 level a HR increase was still observed if the right PC-6 was stimulated. These findings suggest that the HR reflex response activated by LS at the right PC-6 and LI-11 might consist of a spinal and supraspinal reflex arc and that both reflexes are added to an overall HR response in CNS-intact rats. The stimulation of cardiovascular function as determined by HR and MAP in response to PC-6 activation and inhibition in response to ST-36 activation is also in favor of two different supraspinal reflex arcs triggered by LS at those acupoints.
We gratefully acknowledge the very helpful comments from Dr. Harumi Hotta on a previous version of the manuscript. TF is very grateful to Dr. Weimin Li for introducing him into the scientific research on the autonomic nervous system, and to the “Freunde und Foerderer” of Goethe University for travel support. The project was partially supported by the 973 Program of China (2009CB522901), the Shanghai Science and Technology Developing Foundation (08DZ1973000), and the Key Program of State Administration of Traditional Chinese Medicine of China.