Lasers in Medical Science

, Volume 27, Issue 2, pp 469–477

Regulation of the cardiovascular function by CO2 laser stimulation in anesthetized rats

Authors

  • Thomas Friedemann
    • Shanghai Research Center for Acupuncture and Meridians
    • Institute for Cell BiologyGoethe-University
  • Xueyong Shen
    • Shanghai University of Traditional Chinese Medicine
  • Jürgen Bereiter-Hahn
    • Institute for Cell BiologyGoethe-University
    • Shanghai Research Center for Acupuncture and Meridians
    • Institute for BiophysicsGoethe-University
Original Article

DOI: 10.1007/s10103-011-0973-3

Cite this article as:
Friedemann, T., Shen, X., Bereiter-Hahn, J. et al. Lasers Med Sci (2012) 27: 469. doi:10.1007/s10103-011-0973-3

Abstract

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.

Keywords

CO2 laserBlood pressureHeart rateSpinalizationRat

Abbreviations

Chize

LU-5 (Lung 5)

Neigua

PC-6 (Pericardium 6)

Quchi

LI-11 (Large intestine 11)

Zusanli

ST-36 (Stomach 36)

Taichong

LR-3 (Liver 3)

BP

Blood pressure

CNS

Central nervous system

CVD

Cardiovascular disease

ECG

Electrocardiogram

HR

Heart rate

LS

Infrared laser stimulation

MAP

Mean arterial blood pressure

SEM

Standard error of the mean

Introduction

According to the World Health Organization, approximately 17.1 million people died from cardiovascular disease (CVD) in 2004. During the last decades, patients used complementary and alternative medicine for the treatment of CVD with increasing frequency, including acupuncture [1]. Clinical studies already have provided evidence that acupuncture may have therapeutic effects on hypertension, coronary heart disease, arrhythmia, angina pectoris, and myocardial infarction [25]. Also, studies performed on anesthetized animals, thus after elimination of emotional responses [5], have demonstrated beneficial effects of acupuncture on myocardial ischemia [6, 7], arrhythmia [8], and hypertension [9, 10]. It was shown previously that manual acupuncture applied to various areas of the body could modulate blood pressure (BP) [11, 12] and heart rate (HR) of anesthetized animals via a somato-sympathetic reflex [13], and the central components were supraspinally located. Recently, Hotta et al. [14] demonstrated that mechanical stimulation at skin results in modulation of cardiac somato-autonomic reflexes. According to Chinese medicine, stimulation of acupuncture points (acupoints) also includes application of high temperature by moxibustion. In animal experiments, thermal stimulation was shown to modulate cerebral blood flow [15] and heart rate [16]. Temperatures exceeding 50°C in the skin can be achieved by infrared light using CO2 laser for thermal stimulation of nociceptors in monkey skin [17]. This raised the question whether CO2 laser stimulation (LS) can also modulate BP and HR. Four different acupoints were chosen, which are frequently used in clinical daily routine to treat cardiovascular disease by moxibustion or acupuncture [18]. Two of them are located in the forelimb (Neiguan (PC-6), Quchi (LI-11)), one in the hind limb (Zusanli (ST-36)), and one in the hind paw (Taichong (LR-3)) (see Fig. 1). The present study is based on the hypothesis that stimulation of different acupoints elicits different cardiovascular reflex responses because different acupoints might belong to different segmental levels, as was speculated by Kimura and coworkers [19], thus they might exert different therapeutic effects. To investigate whether there is laterality in HR and mean arterial blood pressure (MAP) response, stimulation of each acupoint was preformed at the left and right side of the body. Kimura et al. [19] examined the effects of pinching the skin at various segmental levels on BP and HR in anesthetized rats and observed supraspinal and propriospinal somato-cardiovascular reflex responses. This raises the question whether the reflex center, in case of LS, is located in propriospinal or supraspinal structures.
https://static-content.springer.com/image/art%3A10.1007%2Fs10103-011-0973-3/MediaObjects/10103_2011_973_Fig1_HTML.gif
Fig. 1

Location of the acupoints PC-6, LI-11, LR-3, ST-36, and LU-5 in the rat and their known function in regulation of cardiovascular system in humans

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.

Methods

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 [20]. 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.

A cannula, which was inserted into the trachea, was connected to a ventilator (small animal ventilator, Hugo Sachs Elektronik-Harvard Apparatus GmbH, Germany) for artificial respiration. The end-tidal CO2 concentrations were kept at about 3.0% by controlling respiratory rate and volume (Gas Analyzer, AD Instrument, Australia). The body temperature was kept between 37 and 37.6°C, using a homoeothermic blanket (DC Temperature Controller 41-90-8D-01, FHC, USA). The right carotid artery was cannulated for measuring blood pressure, which was converted into a digital signal by an analogue-to-digital converter (PowerLab/16, AD Instruments, Australia) for analysis. The mean arterial pressure (MAP) was calculated according to
$$ {\text{MAP}} = {{\text{P}}_{\text{dias}}} + {1}/{3}\left( {{{\text{P}}_{\text{sys}}} - {{\text{P}}_{\text{dias}}}} \right). $$

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.

ECG measurement

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.

Stimulation procedure

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.

Data analysis

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.

Results

Modulation of HR and MAP by laser stimulation (LS) in CNS-intact rats

In the first series of experiments, effects of LS were investigated in rats with intact central-nervous system (CNS). LS at various acupoints elicited different cardiovascular reflex responses. Figures 2 and 3 illustrate the effects for selected examples; analyzed, averaged maximum responses are summarized in Table 1.
https://static-content.springer.com/image/art%3A10.1007%2Fs10103-011-0973-3/MediaObjects/10103_2011_973_Fig2_HTML.gif
Fig. 2

Effect of acupoints laser stimulation on HR (intact spinal cord). Four different acupoints (a-d) were stimulated on the left side (a) and right side (b). Hatched bars indicate periods of LS

https://static-content.springer.com/image/art%3A10.1007%2Fs10103-011-0973-3/MediaObjects/10103_2011_973_Fig3_HTML.gif
Fig. 3

Effect of acupoints laser stimulation on MAP (intact spinal cord). Four different acupoints (a-d) were stimulated on the left side (a) and right side (b). Hatched bars indicate periods of LS

Table 1

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

Acupoint

HR

MAP

Left

Right

Left

Right

PC-6

+2.2 ± 0.3

+3.8 ± 0.5*

+9.4 ± 1.1

+7.0 ± 0.4

LI-11

No response

+1.1 ± 0.4*

No response

+3.5 ± 1.6*

LR-3

+1.7 ± 0.3

+1.6 ± 0.3

+6.8 ± 0.7

+6.4 ± 0.7

ST-36

−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).

Interestingly, LS applied to ST-36 produced reduction of HR (Fig. 2d) with no significant difference between stimulation of left or right ST-36 (Table 1). After LS was turned off, HR stayed reduced for more than 2 min. The time course of averaged data is shown in Fig. 4a.
https://static-content.springer.com/image/art%3A10.1007%2Fs10103-011-0973-3/MediaObjects/10103_2011_973_Fig4_HTML.gif
Fig. 4

Effect of ST-36 laser stimulation on HR (a) and MAP (b) (intact spinal cord). Data represent averages of n = 8 determinations (±SEM) at the left (a) and right (b) acupoint. Hatched bars indicate periods of LS

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

In a second set of experiments, the effect of LS applied to the same acupoints was investigated in rat with spinal cord transection at C2 level. After spinalization, HR as well as MAP hardly responded anymore during or after the LS application; averaged responses are summarized in Table 2.
Table 2

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

Acupoint

HR

MAP

Left

Right

Left

Right

PC-6

No response

+1.5 ± 0.7*

+0.8 ± 0.2*

+2.0 ± 0.9*

LI-11

No response

No response

No response

No response

LR-3

No response

No response

No response

No response

ST-36

No response

No response

No response

No response

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.

Discussion

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 [17]. 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 [23]. 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 [2427]. 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 [28]; also, in clinical studies, blood pressure could be reduced by stimulation of ST-36 [29]. 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 [19]. 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 [31]. 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. [30] 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, 3234]. 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.

Acknowledgements

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.

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