Medulla oblongata mechanism of inhibitory effect of thermal stimulation to nociceptive colorectal distention in rats

Liang Li; Pei-Jing Rong; Xin-Yan Gao; Hui Ben; Hong Cai; Bing Zhu

doi:10.15806/j.issn.2311-8571.2016.0022

ACUPUNCTURE & MOXIBUSTION

Year : 2016 | Volume : 2 | Issue : 2 | Page : 65-71

Medulla oblongata mechanism of inhibitory effect of thermal stimulation to nociceptive colorectal distention in rats

Liang Li, Pei-Jing Rong, Xin-Yan Gao, Hui Ben, Hong Cai, Bing Zhu
Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing 100700, China

Date of Submission	29-Jun-2016
Date of Acceptance	05-Jul-2016
Date of Web Publication	8-Sep-2020

Correspondence Address:
Bing Zhu
Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing 100700
China

Source of Support: None, Conflict of Interest: None

DOI: 10.15806/j.issn.2311-8571.2016.0022

Abstract

Objective: To discuss mechanism of moxibustion (thermal stimulation) effect and best moxibustion stimulus parameter.
Methods: Experiments were performed on 48 male Sprague-Dawley rats. Unit discharges from individual single neuron were recorded extracellularly with glass-microelectrode in Subnucleus Reticularis Dorsalis (SRD). Visceral-intrusive stimulation is done by colorectal distension. Thermal stimulation with different temperature (40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 52°C) and different stimulus area (diameter of circle : 1.0 cm, 1.5 cm, 2.0 cm, 2.5 cm, 3.0 cm, 3.5 cm, 4.0cm) was applied around RN12 during nociceptive colorectal distension.
Results: SRD neurons could be activated by visceral stimulation within noxious range. Under low temperature of stimulus, especially under 45°C of pain threshold to ordinary people, visceral nociceptive afferent facilitated thermal stimulus from the body surface. While after thermal stimulation reached a harmful degree, the thermal stimulus will inhibit visceral nociceptive afferent. Moreover, statistics show that the higher the temperature is, the smaller the size of stimulation area is needed, and they correlate with each other negatively.
Conclusion: Visceral nociception could be inhibited by somatic thermal stimulation with specific parameter at medulla level. According to our finding, best thermal stimulation temperature is around 48°C and the best size of stimulation area is around 3.14-7.07cm² (with 2.0-3.0cm diameter).

Keywords: Thermal stimulation, Subnucleus Reticularis Dorsalis, Stimulus Parameter

How to cite this article:
Li L, Rong PJ, Gao XY, Ben H, Cai H, Zhu B. Medulla oblongata mechanism of inhibitory effect of thermal stimulation to nociceptive colorectal distention in rats. World J Tradit Chin Med 2016;2:65-71

How to cite this URL:
Li L, Rong PJ, Gao XY, Ben H, Cai H, Zhu B. Medulla oblongata mechanism of inhibitory effect of thermal stimulation to nociceptive colorectal distention in rats. World J Tradit Chin Med [serial online] 2016 [cited 2023 Dec 1];2:65-71. Available from: https://www.wjtcm.net/text.asp?2016/2/2/65/294527

Introduction

In ancient time, the Father of Western Medicine said: disease, can be treated with metal (needle or knife) if cannot be treated with medicine; can be treated with fire if cannot be treated with metal; And it is cannot be treated with anything if cannot be treated with fire. And in traditional Chinese medicine, there were similar expressions in classics. At present, the mechanism of acupuncture analgesia have already been studied wildly and deeply. But research about the mechanism of moxibustion lag far behind.

In our study, we tried to use the SRD neuron as our research model to observe influences of moxibustion applied with different area and temperatures on the activity of these neurons in order to expound the best parameter of moxibustion treatment; if thermal sensitized acupoint is coursed under pathological circumstances; the neurological mechanism of effects of moxibustion and thermal sensitized acupoint.

Materials and Method

1. Animals

Experiments were performed on Sprague-Dawley rats weighing between 220-300g provided by the experimental animal center of Academy of Military Medical Science. The animals were anesthetized with an intraperitoneal injection of urethane (1.0~1.2 g-kg^–1). All the animal experiments in the present study were approved authoritatively in accordance with the Animal care and use principles of China Academy of Chinese Medical Sciences.

2. Surgery Preparation

Following an intraperitoneal injection of 100μg atropine sulfate, a tracheal cannula was inserted and the animals were paralyzed by intravenous injection of gallamine triethiodide (Flaxedil) and artificially ventilated.

The animals were mounted in a stereotaxic frame with the head fixed in a ventro-flexed position by means of a metallic bar cemented to the skull, and the foramen magnum was then exposed by removing the overlying musculature, atlantooccipital membrane, and dura mater dryness. The heart rate and body temperature were monitored in real time. The body temperature of rats was maintained between 36-38° C by feedback-control heating apparatus (RWDCL-8).

Visceral-intrusive stimulation is done by colorectal distension. A condom was used to make a 4–6 cm-long air sac, and it was tied to a 4 mm-diameter rubber tube. The tube was connected to a sphygmomanometer-pressure transducer with a T-tube. During the experiment, the air sac was inserted into the rat’s colorectum with a depth of 4 cm. CRD stimulation is carried out by pressure supplied by a 20–80 mm Hg sphygmomanometer for 20 s or longer. Previous research^[1],[2] indicated that the pressure bigger than 40 mm Hg is visceral-intrusive stimulation. In order to prevent possible sensitization triggered by over stimulation in the colorectum, the interval between two CRD stimulations should be at least more than 10 minutes.

3. Extracellular Recording

Unitary extracellular recordings were made with glass micropipettes filled with 5% NaCl and Pontamine Sky Blue solution. The micropipettes were inserted on the left side of the medulla, 1.0-2.0 mm caudal to the obex, and 0.5-l .5 mm lateral to the midline. Meanwhile, stainless steel electrodes were inserted on the tail of rats to emit harmful electronic stimulation (4-6mA, 0.66Hz, 2ms duration) to find SRD neuron. Cell electricity was amplified by oscilloscope (VC-10 Japan) and microelectrode amplifier (Photonix 8301) then processed by Electrophysiological recording system (Power- Lab) from microelectrode.

4. Procedures of experiment

First, neuron activity was recorded for 2 minutes, then 50s of CRD stimulation was given to the rats. At the same time, 5s reaction of neuron to CRD was recorded. Thermal stimulation was added after 10s of CRD stimulation for 30s, and the reactions were observed. After thermal stimulation, another 10s neuron’s reaction to CDR was recorded. In order to control the temperature of thermal stimulation accurately, water in different temperature bottled in different size of wild-mouth bottle (40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 52°C) was used instead of moxibustion around RN12 (Zhongwan). Also, different sizes of area were stimulated: 0.785cm² (Φ1.0cm), 1.766cm² (Φ1.5cm), 3.14cm² (Φ2.0cm), 4.906cm² (Φ2.5cm), 7.065cm² (Φ3.0cm), 9.616cm² (Φ3.5cm), 12.56cm² (Φ4.0cm). There were in total 49 combinations of heat temperature and sizes of area^[3].

5. Histological location

After single-cell recording, the location was recorded by sending 20μA negative direct current to the glass microelectrode. The hearts of rats were retained and fixed by infusion. Then sections of icing brain tissue were obtained and observed by HE dying. The Rat Brain in Stereotaxic Coordinates, Sixth Edition (Paxions & Watson, 2007) was taken as reference to locate the microelectrode.

6. Data collection and analysis

Software such as Power-lab data acquisition system Chart 5.0, and SPSS13.0 were used for data collection and analysis. The volume of neuronal discharge per second and the activation/inhibition rate were calculated. The mean and standard deviation before and after the thermal intervention were calculated as the descriptive statistics and represented by x̄ ± SE. The activation/inhibition rate was represented by x̄ ± SE%. One-Way ANOVA was used for the comparison between groups. P<0.05 was considered as statistically significant.

Results

1. The common features of SRD neuron activities

There are 105 neurons on the dorsal side of medulla oblongata in 48 male adult SD rats, among which there are 89 SRD neurons and 16 spinal trigeminal nucleus. [Figure 1] shows the Pontamine sky blue location of part of the SRD neurons.

Figure 1: ▴ shows the Pontamine sky blue location of part of the SRD neurons

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Over-threshold electronic stimulation (2ms duration) on any part of the body can activate SRD neurons. This activation is featured by two peaks of stimulation, before which there is a incubation period. The incubation period of stimulating the basilar part on the tail is shorter than that of stimulating 10cm from the tip of the tail. The two incubation periods of the basial tail are 12-40ms and 200-250 ms (after 50 single square wave stimulation), and those of the tip of tail are 23-60ms and 560-650ms. Statistics show that the time difference of early incubation period between the two location’s stimulation is 9.5±0.6ms, and it can be deduced that the peripheral fiber conduction velocity is 10.5±0.6m/s, which is in line with the conduction velocity of Aδ neurotransmitter fibers. Besides, the time difference of the latter incubation period between the two location’s stimulation is 145±10.2ms, and it can be deduced that the peripheral fiber conduction velocity is 0.7±0.05m/s, which is in line with the conduction velocity of C type neurotransmitter fibers. So it can be concluded that the early peak is activated by Aδ neurotransmitter fibers and the latter peak by C type neurotransmitter fibers. This is in accordance with the results drawn by Villanueva^{[4],[5],[6],[7]}.

All SRD neurons could not be activated by non-nocuous stimulation (such as sound, light, and proprioceptive), but they can be activated by nocuous mechanical stimulation (such as to pinch the skin with toothed forceps) or hot water with 48°C on general areas of the body.

2. Activation of CRD on SRD neuron

Like what was mentioned before, stimulation of CRD ≥40 mmHg is harmful to organs. In the experiment, CRD stimulation on 8 SRD neurons were observed. It has been recorded that the intensity of neuron activities increased from 2.85±1.72spikes/s to 10.53±3.81spikes/s, and the increase rate is 202.1±5.89%. The following Figure shows that there are significant difference (P<0.001) after 80mmHg CRD stimulation compared to the background, indicating that nocuous CRD can activate SRD neurons. [Figure 2]

Figure 2: Activation of SRD under 80mmHg CRD stimulation (***P<0.001)

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3. Influence on SRD neurons reaction under the simulation of CRD and different kinds of thermal stimulation

During stimulation with CRD, We examined SRD neurons’ responses to thermal stimulation with 40°C. Results showed that 40°C-Φ1.0cm (n=15), 40°C-Φ1.5cm (n=20) don’t have any influence on the activity of SRD induced by CRD ; at the ranges of 40°C-Φ2.0cm (n=16), 40°C-Φ2.5cm (n=14), and 40°C-Φ3.0cm (n=11), the reaction of SRD can be slightly amplified with no statistical difference (P>0.05). However, when the simulation area reach the size of 03.5cm (n=11) and 04.0cm (n=8), 11±5.12% and 10.21±3.56% increase rate can be seen in SRD neuron activity, and compared with control group, there is statistical difference (P<0.05). However, there are no statistical difference showed between the two stimulus intensities (P>0.05). [Figure 3].

Figure 3: Effect of thermal stimulation with the intensity of 40°C-Φ2.0cm and 40°C-Φ4.0cm on SRD neuron's discharges induced by CRD

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Similar to 40°C, thermal stimulation of 42°C-Φ1.0cm (n=11), 42°C-Φ1.5cm (n=13), 42°C had no effect on the activity of SRD induced by CRD. At the ranges of 40°C-Φ2.0cm (n=11) and 40°C-Φ2.5cm (n=15) the reaction of SRD can be slightly amplified with no statistical difference (P>0.05). However, at the ranges of Φ3.0cm(n=10), Φ3.5cm (n=9) and Φ4.0cm (n=7), increase rate of 10.56±4.32%, 9.38 ±4.58% and 9.27±3.94% can be seen respectively on SRD neuron activity. Compared with control group, there is statistical difference (P<0.05), while inter-group comparisons show that there are no statistical difference (P>0.05).

At 44°C, thermal stimulation of 44°C-Φ1.0cm(n=15) and 44°C-Φ1.5cm(n=11) had no effect on the activity of SRD induced by CRD, and there is no statistical difference compared to control group. At the range of 44°C-Φ2.0cm (n=10), thermal stimulation can increase 10.25±2.14% of SRD activity induced by CRD, and compared with control group there is statistical difference (P<0.05). Moreover, thermal stimulation of 44°C-Φ2.5cm(n=11) can raise 19.12 ±3.12% of SRD neurons activity induced by CRD; at the range of 44°C-Φ3.0cm(n=8), a 18.52±3.24% increase rate was seen; at the range of 44°C-Φ3.5cm(n=8), an increase rate of 22.85±2.45% was witnessed, and at the range of 44°C-Φ4.0cm(n=9), SRD neurons activity rose 20.14±1.45%. Statistics show that all these increase had statistical significance with P<0.05. [Figure 4] A & [Figure 4]B.

Figure 4: A shows 44°CΦ-3.0cm thermal stimulation on SRD neurons under CRD and B shows the effect of heat stimulus at 44°C of various sizes of area on SRD neuron reaction. (compared to blank group, *P< 0.05, **P<0.01)

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At 46°C, thermal stimulation of 46°C-01.Φcm(n=14), 46° C-Φ1.5cm(n=11), 46°C-Φ2.0cm(n=16) and 46°C-Φ2.5cm (n=12) had no effect on activity of SRD induced by CRD, and there is no statistical difference compared to control group. At the range of 46°C-Φ3.0cm(n=10), thermal stimulation decreased SRD neurons’ discharges by 7.85±2.54%; at the range of 46°C-Φ3.5cm(n=9), thermal stimulation decreased reaction intensity of SRD neurons by 9.55±3.25%; and at the range of 46°C-Φ4.0cm(n=8), a 8.27±1.51% reduction rate was seen. Statistics show that all these increase had statistical significance with P< 0.05.

At 48°C, thermal stimulation of 48°C-Φ 1.0cm(n=17) can slightly increase activity of SRD neurons induced by CRD, but there is no statistical difference compared to control group. However, when the size of stimulus area is larger than Φ1.5cm(n=19), all 6 groups of thermal stimulation could inhibit activities of SRD neurons induced by CRD [Figure 5] B. To be specific, thermal stimulation of 48°C-Φ1.5cm decreased the activity of SRD neurons by 20.34 ±5.31%; at the range of 48°C-Φ2.0cm(n=11), a 25.85±4.54% decrease rate was seen. Differences of the two groups are statistically significant (P<0.01). At the ranges of 48°C-Φ 2.5cm(n=10), 48°C-Φ3.0cm(n=10), 48°C-Φ3.5cm(n=6) and 48°C-Φ4.0cm(n=7), SRD neurons’ activity decreased 41.31 ±3.21%, 39.25±1.28%, 42.57±4.10% and 42.85±2.41% respectively. Statistics show that all these four groups have statistical difference compared to control group (P<0.001). [Figure 5] A & [Figure 5]B.

Figure 5: A shows the effect of thermal stimulation of 48°C-Φ3.0cm on activityof SRD induced by CRD; and B shows the effect of thermal stimulation under 48°C with different sizes of area on activityof SRD induced by CRD (compared to control group **P<0.01, ***P<0.001

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At 50°C, the results is similar to the resunlts with 48°C. Thermal stimulation of 50°C-Φ1.0cm(n=10) reduced the activity of SRD induced by CRD by 18.68±3.71%, and there is statistical difference compared to control group (P<0.05). However, when the size of stimulus area is larger than Φ1.5cm(n=19), 6 groups of thermal stimulus could restricted the activities of SRD neurons. At the ranges of 50°C-Φ1.5cm (n=9), 50°C-Φ2.0cm(n=9), 50°C-Φ2.5cm(n=8), 50°C-Φ3.0cm(n=9), 50°C-Φ3.5cm(n=6), and 50°C-Φ4.0cm(n=6), SRD neurons’ activity decreased 36.1±4.31%, 46.23±5.32%, 56.22±4.23%, 52.52±2.32%, 51.25±3.54% and 46.57±6.12% respectively. Statistics show that all these six groups have inhibiting effect on SRD neurons and there were statistical difference compared to blank group (P<0.001).

At 52°C, thermal stimulation of 52°C-Φ1.0cm(n=10) significantly inhibit activity of SRD induced by CRD by 17.24±3.57%, and there is statistical difference compared to control group (P<0.05). However, when the size of stimulus area is larger than Φ 1.5cm(n=19), 6 ranges of thermal stimulus all could inhibit the activities of SRD neurons. At the ranges of 52°C-Φ1.5cm(n=10), 50°C-Φ2.0cm(n=9), 52°C-Φ2.5cm(n=10), 52°C-Φ3.0cm(n=10), 52°C-Φ3.5cm(n=6), and 52°C-Φ4.0cm(n=5), SRD neurons reaction intensity decreased 40.67±3.12%, 51.74±4.52%, 50.57±4.58%, 52.82 ±3.57%, 47.82±1.52%, and 49.87±3.22% respectively. Statistics showed that all these six groups have inhibiting effect on SRD neurons and there were statistical difference compared to blank group (P<0.001).

Viewing from the results, it can be concluded that under low temperature of stimulus, especially under 45°C of pain threshold to ordinary people, viseral nocicepetive afferent facilitated thermal stimulus from the body surface and further activated SRD neurons. While after the thermal stimulation reached a harmful degree, the thernal stimulus will inhibit viseral nocicepetive afferent. Moreover, statistics show that the higher the temperature is, the smaller the size of stimulating area is needed, and they correlate with each other negatively. From [Figure 6], it can be seen that at the degree of 48°C with Φ2.0cm~3.0cm, the effective rate can reach to 50%.

Figure 6: Line charts about SRD intensity under different ranges of thermal stimulation induced by CRD

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Discussion

1. SRD neurons and nociceptive information processing

According to former research, much more attention has been drawn to the SRD’s function to process periphery afferent. First, all SRD neurons could accept peripheral afferent from all over the body, which ensures that the study can be conducted by stimulating different sizes of areas on rats. Second, SRD neurons could be activated by nociceptive mechanical, thermal, chemical, and visceral stimulation while they do not react to sound, light and proprioceptive stimulus. Third, it can respond to any levels of stimulus sensitively. Forth, it has an obvious, lasting aftereffect on natural stimulus. There is a large chance that SRD is the information network in medulla spinal-reticular formation-thalamus, and it is a critical path to transmit warm pain signals.

The most essential feature of SRD neurons is that nociceptive afferent from different area converge to one single neuron. Besides, SRD neurons have special summation ability, which means that under a certain level, the activity of SRD neurons would increase along with the increase of stimulus intensity, while once exceed the level, the neurons’ reaction will reach “saturation” even start to decrease. This phenomena cannot be illustrated by the saturation of afferent information, as SRD activities could be further activated by higher level stimulus. The saturation as well as negative feedback inhibition are both the results of the integrative function of SRD neurons, and it is a neurological function of medulla spinal-SRD-spinal cord adjustment circuit. The mechanism of the negative feedback inhibition has been discussed under the research of spinal dorsal horn neurons. That nociceptive responses can be inhibited by harmful stimulus on either part of the body was referred as diffuse noxious inhibitory controls (DNIC)^[8],[9]. The integrative function of negative feedback inhibition of SRD neurons closely related to central verve functions. They share similarities in periphery stimulus nature, nociceptor, afferent fibers, and convergent neurons transmission and they can perform negative inhibition on harmful spinal stimulus by following the route of ventral lateral nerve tract—brain stem—DLF.

To take SRD convergent neurons as study subjects can best analyze the dose-effect activation on these neurons in different stimulus sizes and intensities of heat-moxibustion. Also, the lines of reactions can be drawn to calculate the optimal index of heat-moxibustion stimulus, which can be taken as clinical reference.

Scientific research shows that half of the SRD neurons that are connected to thalamus send single synapsis fiber projection on medulla spinal^[10]. This is in accordance with the conclusions drawn by Scheibel, who have proved that most of the axones of brain stem giant cell reticular nucleus are long and can project long distance stimulus along the brain stem. This discovery puts a challenge in front of traditional physiology^[11]. It was believed that the reticular formation is composed by cell-chain with short axones and they can be activated like domino. Nonetheless, different from Golgi method^[11],[12], early phase trace method did not discover that neurons of brain stem reticular formation that related to medulla oblongata can emit lateral fiber to diencephalon.^[13] The difference between them might be due to the sensitivity of trace agents and research locations.

SRD was basically composed by cells with similar reactive nature which responds to the pain signals of medullathalamus. The neurons on the dorsal side of SRD have manifested the nature of converging pain sensations of all over the body and pitching fiber projection to many parts of central nervous system, and these parts correlate with kinesis. Specifically, they are the deep dorsal horns and dorsal sides of frontal horns of all spinal cords, nuclear of brain stem nervous motorius, reticular nucleus of giant cells and cellules and tissues in prosencephalon that are connected to kinesis including lateral side of thalamus, parafascicular nucleus, and ventral side of zone incerta. A successive areas of deep sides dorsal horns that responded to harmful skin and organ stimulation can emit fiber projection to SRD^[14]. SRD can respond to the signals from spinal marrow by excitatory and inhibitory regulation. Ethology study shows that incubation period can be prolonged in tail immersion test if glutamic acid is injected into SRD^[15]. An intriguing phenomena is that SRD can be directly affected by many areas of the cerebral cortex^[16]. Many corticofugal impacts can be simultaneously transmitted to cranial and caudal side of central nerve system by SRD lateral branch.

It can be assumed that SRD is one of the material bases in medulla oblongata to regulate pain signals. The functional meaning of SRD lies in the fact that it can form a neural circulate of spinal marrow-SRD-spinal marrow, then coordinate the harmful signals from periphery tissues and regulate the pain signals negatively through DINC. The harmful information from other areas will converge into one neuron of SRD, and thus be excluded from the process of pain differentiation. This may implies its nonspecific function in participating in emotions induced by pain.

2. Clinical application of moxibustion

In ancient China, the emergence of moxibustion is earlier than acupuncture. In “Shuo Wen Jie Zi”, moxibustion is explained as “burning”, to burn with fire. The silk manuscripts “Eleven Moxibustion Meridians on Legs and Arms” and “Eleven Moxibustion Meridians of Yin and Yang” which excavated from the 3rd Mawangdui Han Dynasty Tomb in Changsha city, Wuhan Province in 1973 are the earliest literature that comprehensively introduce the 11 meridians routes on human body and their indications. Also, they pioneered to record moxibustion, and only moxibustion method is introduced in the book. According to the books, all kinds of complicated meridian diseases including cardiac pain, difficulty in urination, insane can be treated by moxibustion on corresponding meridians. Wang Xi in “Waizhi Miyao” Tang Dynasty has specifically described moxibustion rather than acupuncture, which indicates his value on moxibustion. In Song Dynasty, Dou Cai wrote in “Bianque Xinshu-Wangshi Zhifa” that to save life, moxibustion should be firstly used, then pills and medicine, and the third Fuzi. In Qing Dynasty, all doctors have emphasized on moxibustion, it is also said in “Yixue Rumen” that people should perform moxibustion in every season to strengthen qi and resist diseases and that if medicine and acupuncture are impotent, moxibustion can be used.

Except for moxa, materials like sulphur, wick, mulberry twig, peach branchlet, beeswax, and pill lozenges are all often used. Also, there are thunder fire miraculous moxa roll, Taiyi miraculous moxa roll, oil-lamp moxibustion (lighting rush and oil on the skin of patients), canister moxibustion (insert bamboo or reed tube into patient’s eat to treat ear diseases) and so on. The methods of moxibustion are various, including direct contact moxibustion, indirect contact moxibustion, needle warming moxibustion, mild moxibustion, sparrow-pecking moxibustion and so on. Indirect moxibustion also can be subdivided into several types: ginger moxibustion, garlic moxibustion, Fuzi-cake moxibustion, Douchi-cake moxibustion, Pepper-cake moxibustion and so on. Clinically, indirect moxibustion and moxa cone are mostly used. In general, the heat, size and time of moxibus- tion are critical factors, and the amount of moxa and size of area can be adjusted. It is better that the skin keeps red and warm, but should not be scorched (except for blistering moxibustion).

As to the size of stimulation, the diameter of moxa cone often used is 1.0cm and that of moxa stick is 1.2 cm^[17]. They can at least warm the skin area of 3cm diameter. As a special moxibustion, long snake moxibustion is also widely applied in clinical treatment. The size of area it covers is the largest among all kinds of moxibustion, the whole spine length and the 6cm width. Pastille moxibustion takes pastille as material, which covers the least size of area. According to experiment, with 2.0~3.5cm diameter size of stimulation, well treatment effect would be achieved.

When a 2mg moxa cone is used, along with the increase of moxa cone, the highest temperature will rise and duration will extend. For example, one moxa cone can reach 100°C at highest, and the temperature can keep above 40°C for 20s; while 20 moxa cones can reach 150°C at highest, and the temperature can keep above 40°C for 350s. Okazaki^[18], M have observed the effect of single and multiple moxa cones on skin. He also regards that in single moxa cone, the temperature can go up with the increase of moxa cone diameters (0.5~2cm). On skin, the temperature of single moxa cone can rise to 105°C and the temperature of 3 moxa cones can rise to 125°C. Under skin, the temperature of single moxa cone can reach 56°C, which is similar to the temperature of three moxa cones. It can be seen that the moxibustion stimulation often exceed the feeling threshold of pain (45°C)^[19]. In clinical treatment, long snake moxibustion and blister moxibustion are commonly used, which can exceed the pain threshold of human beings. Viewing from the most clinical experiments, while these stimulation can achieve well effect, they can also generate physical damage.

Visceral nociception could be inhibited by somatic thermal stimulation with specific parameter at medulla level. According to our finding, best thermal stimulation temperature is around 48°C and the best size of stimulation area is around 3.14-7.07cm² (with 2.0-3.0cm diameter).

Acknowledgment

This scientific work was supported by National Basic Research Program of China (973 Program, no. 2011CB505201, 2009CB- 522902), and National Natural Science Foundation of China (no. 81102649). All authors approved the paper and there are no conflicting financial interests for this paper.

Funding

This work was supported by National Basic Research Program of China (973 Program, no. 2011CB505201, 2009CB522902), and National Natural Science Foundation of China (no. 81102649).

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