All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of China Academy of Chinese Medical Sciences. Experiments were performed on 48 adult male Sprague-Dawley rats weighing between 250 and 300 g. The animals were maintained on a 12:12-h light-dark cycle (8:00-20:00) with free access to food and water. The rats were fasted overnight with free access to water in proxima luce and anesthetized with an intraperitoneal injection of urethane (1.0-1.2 g/kg). The trachea was cannulated but not immobilized only to keep respiratory tract unobstructed and a catheter was inserted into one of the jugular veins for infusion of the necessary solutions or anesthetics. The abdomen was opened by midline section, and a small longitudinal incision was made in the duodenum about 2-3 cm from the pylorus. A small balloon made of flexible condom rubber was inserted via incision of the duodenum into the pyloric area of rat and kept in position by tying the connecting catheter to the duodenum, and another catheter (inner diameter of 1 mm) was also inserted into the same hole by incision in order to drain digestive juices secreted from stomach. Then the cut skin of the abdomen was closed by suture before experiments. The balloon was filled with warm-water about 0.2-0.5 mL, which gave about 80-150 mmH₂O pressures. Pressure in the balloon was measured by a transducer (TP-400T, Nihon Kohden) for low pressure through a thin polyethylene tube (1.5 mm in outer diameter) and then input on a polygraph (RM-6000, Nihon Kohden) amplifier and led into a data acquisition system (Power-Lab) for further analysis. Demi-fasting gastric motor activity was recorded as a control for at least 1 h before either the acupuncture or somatic stimulation.

The changes of gastric motility induced by the stimulation were compared with the background activity in intragastric pressure and/or waves of gastric contraction recorded before any stimulation. If the changes of gastric motility during stimulation were 20% more or less than the background activity, the response was then considered to have an excitatory or inhibitory regulation, respectively. Systemic blood pressure was continuously monitored from a common carotid artery and heart rate was continuously monitored, rectal temperature kept constantly around 37°C by means of a feedback-controlled heating blanket.

Cutaneous stimulation was obtained by pinching a localized skin area of about 5 mm² with a forceps, pinching area distributed in forelimb, lateral abdomen, dorsum and hindlimb. And an additional hot water bath (50°C) on the tail was given. Manual acupuncture with a needle of 0.3 mm in diameter was inserted into the skin and underlying muscles at different acupoints of the body, and it was rotated clockwise and anti-clockwise at 2 Hz for 30 s. Based on the comparative anatomical localization in rats as compared with that in human, selected points included 29/45 cephalocaudal acupoints in stomach-Meridian, and Li-11 (quchi) acupoint of Large-intestine-meridian in forelimb, Cv-6 (qihai), Cv-12 (zhongwan) acupoints of conception-vessel in abdomen, Bl-13 (feishu, T₃ spinal segment), Bl-17 (geshu, T₇), Bl-21 (weishu, T₁₂), Bl-27 (xiaochangshu, S₁) acupoints of Bladder-meridian in dorsum, Sp-6 (sanyingjiao) of spleen-meridian in hindlimb and the auriculo-concha, non-acupoint in tail (the location of acupoints is shown in Figure 2, Figure 3, Figure 4).

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Figure 2 Gastric motility in response to cutaneous natural stimuli. ^aP < 0.05, ^bP < 0.01 vs control gastric motility of pre-stimuli.

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Figure 3 The gastric motility in response to craniocaudal stomach-meridian acupoints stimuli.

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Figure 4 Gastric motility in response to acu-stimulation.

This experiment was performed in ten SD rats including six for mineral oil of injection in intragastric mucous membrane and four for the control of saline injection. Fifteen minutes after the surgery was completed, Evans blue dye (Sigma) dissolved in sterile water (5 mg per 100 mL) was injected through the arterial catheter. Ten minutes after the dye injection, 0.3-0.5 mL (volume depending on injected points of the stomach) of 10% mustard oil (Sigma, dissolved in mineral oil) was injected into the intragastric mucosa and visceral peritoneum covering the stomach in several areas, in order to induce a chemical inflammation of the gastric mucous membrane. Skin color changes were observed for 1-2 h, and the area of dye extravasation was sketched on the body charts for assessment. Dye extravasation was quantified by counting the number of blue dots in the skin over frontal and dorsal body areas. In control rats, a similar volume of physiological saline was injected into the intragastric mucous membrane. Evans blue dye injection was the same as mustard oil experimental animals.

The rats were kept in supine position; gastric motor activity was first analyzed visually to detect cyclic waves of contractions. When gastric background activity is well recorded, the response of gastric motor to skin stimulation and acupuncture is tested separately. A standard protocol was employed for the determination of effects of acupuncture and somatic stimuli on the gastric motility response. A gastric background activity was recorded for 5-10 min followed by a test of their responses to manual acupuncture at various acupoint or natural stimulation on the skin was applied for 30 s. After the acupuncture or somatic stimulation the gastric motility response was recorded for another 5-10 min. After the series of experiments was finished, rats were sacrificed under an overdose deep anesthesia (urethane, > 2 g/kg ip).

The data obtained before and after intervention in the same group was compared statistically by an independent t test. P < 0.05 was considered as a statistical significance. All data are expressed as mean ± SE.

Injection of mustard oil into intragastric mucous membrane resulted in massive mucous inflammation evidenced on histological examination, which showed that the intragastric mucosa had become edematous, dilated blood vessels and ulceration of the endogastric lining. In all six rats that received mustard oil injections into intragastric mucosa (Figure 1), small blue dots appeared in the skin over the whole abdomen, but mainly in peri-midline upper- and middle- abdomen, middle-back, a few in thigh and groin. The number and distribution of blue dots varied considerably among rats. The dots started to appear about 20 min after injection of mustard oil, and the majority of dots were visible within 50 min. The dots were very small, usually ranging from 1-3 mm in diameter. However, several dots appeared in the state of stick-like in length of 3-6 mm (Figure 1). In contrast, two of four control rats that received saline injections into the intragastric mucous membrane showed no skin color changes at all. The remaining two control rats showed only 3-5 small blue dots over the middle-abdomen (Figure 1), perhaps this extravasation restricted to abdominal skin in some of the control rats might be associated with the abdominal surgical incision. It may speculate that cutaneous innervations of blue dots had same distribution as gastric segmental innervations.

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Figure 1 Schematic drawing of number and location of blue dots in rats. Dye extravasation in skin after inflammation of intragastric mucous membrane and rare small blue dots over abdominal area of control rats.

Gastric motor characteristics were observed in 38 rats under normal anesthetic condition. When the intrapyloric balloon pressure was increased from 0 to about 80-200 mmH₂O levels, the rhythmic wave of contractions in pyloric area were observed in 32 rats, at least in the periodic course during recording. Others showed no cyclic contraction during recording. Among the total, the contractile amplitude of rhythmic wave was about 5-50 mmH₂O, whereas strong contractions exceeded to 300 mmH₂O. When the pressure in an intragastric balloon was maintained at about 100 mmH₂O by expanding the volume of the balloon with warm-water, rhythmic contractions occurred at a rate of 4-6 per minute. With regard to gastric motor characteristics, it was noteworthy both the changes of intragastric pressure and rhythmic contraction in present study. Generally, the intragastric pressure represents the index of gastric tone motility and rhythmic contraction represents then gastric peristalsis induced by circular muscle contractions, similar to slow-wave of gastric motor activity.

Effects of noxious mechanical or hot cutaneous stimulation of various sites on gastric motility were studied in 14 rats. As illustrated in Figure 2, pinching of the abdominal skin always produced strong inhibition (10/11 rats, 100.09 ± 2.73 vs 87.82 ± 3.68, P < 0.05) in the pyloric pressure; the maximum response of decrease was obtained about 10-s after the onset of stimulation. Pinching the middle dorsal skin in some, but not all cases, produced slight or moderate inhibition or only post-inhibition (n = 11, 102.3 ± 3.36 vs 100 ± 3.33, P < 0.05). Interestingly, pinching in fore- or hind-limb induced mainly moderate increase of gastric motility (n = 8, 104.56 ± 1.29 vs 113.78± 5.37, P < 0.05; n = 9, 97.13 ± 4.63 vs 104.88 ± 5.03, P < 0.05; respectively), meanwhile, hot water bath (50°C) on the tail could produce moderate augmentation of gastric motility.

Figure 3 shows the gastric motility in response to majority craniocaudal stomach-meridian acupoint stimuli was systemically observed in four rats. Manual acu-stimulation in 9 acupoints of face (St-2, St-3, St-4, St-5 and St-7), neck (St-9) and cephalic-chest (St-12, St-13 and St-15), induced significantly moderate increase of gastric motility. This facilitation could be observed about 2-4 s after application and continued to augment during 30 s acu-stimulation. On seven acupoints of lower-chest (St-18) and whole-abdomen (St-19, St-21, St-23, St-25, St-26 and St-28), acu-stimulation produced moderate-strong suppressive responses on gastric motility, which started to suppress about 2 s delay of onset and lasted throughout the period of application of the acu-stimulation. The inhibitory magnitude on gastric motility was arranged from fadein to fadeout among these cephalocaudal acupoints. Acu-stimulation in hindlimb (St-30, St-31, St-32, St-34, St-35, St-36, St-37, St-39, St-40, St-41 and St-45) evoked moderate augmentation of gastric motility. However, in between acupoints of St-16 (in the 5^th intercostal space) and St-30 (slightly above the inguinal groove), acupuncture could not bring about clear response of excitation/inhibition on gastric movement in most cases. It was noteworthy that, from the above observations, manual acu-stimulation applied in the Stomach-meridian acupoints from the lower-breast to the groin suppressed generally the gastric motor activity, whereas acu-stimulation in other acupoints of Stomach-meridian (i.e. face, neck, cephalic-chest and hindlimb) brought about exciting responses in different magnitudes on gastric motility. These results suggested the effective regularity of site-special acupoint on gastric motility. Next study focused at acupoint stimuli in different meridians and different parts of the body, and compared their effects on responses of gastric motility.

The acu-stimulation at 16 acupoints in the different parts of the body and one non-acupoint in tail on responses of gastric motility was systemically observed. A total of 27 rats were studied. Most of them (22/27) exhibited the rhythmic wave of contractions in the periodic course, and the pyloric pressure maintained at about 80-200 mmH₂O. Gastric motility in the most rats showed various responses to acu-stimulation applied to some representative acupoints in several meridians and different parts of the body. An individual example of various kinds of responses was illustrated in manual acu-stimulation applied to acupoints either St-2 (sibai), St-3 (juliao) of Stomach-meridian and the auriculo-concha in face, Li-11 (quchi) in forelimb, St-13 (qihu) in upper-breast, Bl-13 (feishu) in upper-dorsum, Bl-27 (xiaochangshu) in lower-dorsum, or St-32 (futu), St-36 (zusanli), Sp-6 (sanyinjiao) in hindlimb and non-acupoint in tail, which gave rise to slight-moderate facilitation of gastric motility with a rapid onset, followed by a tonic motor that lasted throughout the period of acu-stimulation. These responses were followed in several cases by after-effects of promotion; this was particularly clear when the auriculo-concha, Li-11, St-36 and Sp-6 acupoints were acu-stimulated but was also obvious in the other cases (except from Bl-27). However it was interesting to emphasize that, the effects in both abdomen and middle-dorsum on modulation of gastric motility differed basically from those in the head, upper chest/dorsum and fore-/hind-limbs. The same stimulus applied to the acupoints of St-18 (rugen, lower breast) and St-21 (liangmen), Cv-6 (qihai) and Cv-12 (zhongwan) in the abdomen, and Bl-17 (geshu) and Bl-21 (weishu) in the middle dorsum, resulted in high suppression of gastric tonic motility with a rapid onset, followed by a obvious inhibition of rhythmic wave of contractions. These suppressions lasted throughout the period of acu-stimulation and followed in most cases after inhibition.

At a quantitative level, the facilitative/inhibitory responses on the gastric motility were evaluated before 20 s, during 30 s and after 40 s of acu-stimulation. The mean values are shown in Figure 5. Acu-stimulation in acupoints of head-neck, limbs upper chest-dorsum and lower-dorsum induced markedly augmentation of gastric motility (acupoints on head-neck such as St-2: n = 16, 105.19 ± 1.36 vs 112.25 ± 2.02 and St-3: n = 14, 101.5 ± 1.75 vs 109.36 ± 1.8; acupoints on limbs such as Sp-6: n = 19, 100.74 ± 1.54 vs 110.26 ± 3.88; St-32: n = 17, 103.59 ± 1.64 vs 108.24 ± 2.41; St-36: n = 16, 104.81 ± 1.72 vs 110.81 ± 2.74 and Li-11: n = 17, 106.47 ± 2.61 vs 114.77 ± 3.77, P < 0.05-0.001 respectively). Notable inhibitory responses of gastric motility induced by acu-stimulation applied in acupoints of whole abdomen and middle-dorsum were significantly different as compared with the control before acu-stimulation (abdomen acupoints such as Cv-12: n = 11, 109.36 ± 2.09 vs 101 ± 2.21; Cv-6: n = 18, 104.39 ± 1.42 vs 91.83 ± 3.22 and St-21: n = 12, 107 ± 2.97 vs 98.58 ± 2.81; acupoints on middle-dorsum such as Bl-17: n = 19, 100.63 ± 1.4 vs 92.21 ± 2.07 and Bl-21: n = 19, 103.84 ± 1.48 vs 97.58 ± 2.16, P < 0.001 respectively). Acu-atimulation in the tail of non-acupoint could also increase gastric motor. Such regular regulatory effects both of facilitation/inhibition on gastric motility did appear to be somatotopically organized in the acupoints of the whole body and suggested the effective regularity of site-special acupoints on gastric motility.

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Figure 5 The quantitative analysis of gastric motility in response to acu-stimulation. ^aP < 0.05, ^bP < 0.01, ^dP < 0.001 vs control gastric motility of pre-stimuli.