| Needling or pressing on auricular point is a common somato-stimulating therapy for treating visceral disorders. The auricular branch is the only peripheral branch of the vagus nerve. The fibers from the epibranchial placodes to the ganglia of the vagus and the course of vagus and other nerves have been observed in human being. However, in the previous studies the mechanism underlying the effects of auricular acupuncture on visceral function was conclusively attributed to the segmental innervations of sympathetic nervous system or the central integration between somatic and visceral input, omitting the direct visceral innervations of auricular concha.There were numerous clinic reports introducing parasympathetic excitation responses, such as cough, cardiac deceleration, even asystole and depression of blood pressure, induced by ear-cough-reflex and auriculo-cardiac-reflex following cerumen cramming in auditory canal, or stimulation of foreign body on ear, or speculum inspection, or douching ear canal. The previous study showed that acupuncture at auricular concha produced depressor and cardiac deceleration. After arterial atropine injection, the auricular acupuncture no longer inhibited the blood pressure and heart rate (HR)> indicating that the inhibitory effects were mediated by the excitation of parasympathetic nervous system after the stimulation of auricular concha.According to classical anatomy, the auricular branch of vagus is the branch innervating ear skin area, and its central projection affiliates glossopharyngeal nerve and facial nerve. This nerve belongs to somatic afferents and terminates in the nucleus of spinal tract of trigeminal nerve. Excitatory signals of somatic afferents producing visceral effect relay at spinal level and reach the nucleus of the tractus solitary (NTS) indirectly, a public relay station of visceral afferents. In NTS, the signals can be transmitted to other neurons projecting to visceral motor nuclei or reticular formation, which synapse with other neurons with efferent fibers innervating visceral ganglions. It was hypothesized in the present study that acupuncture at auricular concha might excite the auricular branch of vaguscontaining the primary visceral afferents projecting to NTS, and stimulate further the neurons in NTS and subsequently activate the motor nuclei of visceral such as the dorsal motor nucleus of vagus (DMV) so as to regulate the visceral function. Since the stimulation of parasympathetic nervous system can produce different effects on the cardiac system and gastrointestinal system, i.e. restraining the cardiac function and exciting the gastric movement, we investigated the function of the two systems simultaneously to detect the role of parasympathetic system in the mediation of the effects of auricular stimulation on the function of visceral organs.1 Electrophysiological experiment: The. influence of acupuncture at rats' auricular concha on the discharges of NTS and DMV neurons and on the cardiac and gastric functionThe male healthy Sprague-Dawley rats weighing 300-350g were anesthetized with 10% urethane (lgkg"1 i.p.) in the present study. During the operation and the whole experiment, the body temperature of the animals was kept at 37±1 °C by a Time Temperature Controller . The anminals were fed with 5% glucose saline during 12h fasting before the observation on gastric pressure.The extracellular discharge signals responding to the auricular stimulation were recorded with glass microelectrode and compared with those responding to the stimulation at the acupoints on body including ST36, S25 and P6. At the same time, mean arterial blood pressure (MAP), heart rate (HR) and gastric pressure (GP) were also recorded. Acupuncture was administered for 30s in all animals. After intravenous injection of atropine (0.18mg-kg"'), known to block cholinergic effect, the acupuncture stimulation was administered again to identify the involvement of vagus in the mediation of the effects of the auricular acupuncture.1.1 Results on intact ratsDuring 30 seconds of auricular acupuncture, the discharges of totally 363 NTS neurons in 71 rats were recorded. There were 222 neurons responding to acupuncture at ipsi-lateral auricular concha with a discharge increment of 3.50±0.32 spikes/sec which was 304.17±66.50% in percentage (PO.001); there were 159 neurons responding to acupuncture at contra-lateral auricular concha with a discharge increment of 3.45±0.56spikes/sec which was 415.73±103.36% in percentage (P<0.001); there were 100 neurons responding to acupuncture at ST36points with a discharge increment of 0.83±0.25 spikes/sec, i.e. 10.09±2.19% (P<0.01) in percentage; the discharge in 88 neurons responding to acupuncture at S25 points was increased by 0.60±0.20 spikes/sec, i.e. 10.77±2.64%(P<0.01) in percentage; the discharge in 81 neurons responding to acupuncture at P6 points was increased by 0.77±0.27 spikes/sec which is 12.90±2.77% in percentage (PO.01). There was no statistical significance between the effects produced by ipsi- and contra-lateral auricular stimulations on the discharge of NTS neurons (P>0.05), the effect of auricular acupuncture was significantly different from that produced by the acupuncture at the body acu-points (PO.001), indicating a better exciting effect of auricular acupuncture than that induced by the acupuncture at the body acu-points.During 30 seconds' acupuncture at 10 rats' auriculars, totally 40 DMV neurons were recorded, 36 of which responded to acupuncture at ipsi-lateral auricular concha with a discharge increment of 4.08±1.99 spikes/sec which was 100.43±28.63% in percentage (PO.05); 33 neurons responded to acupuncture at contra-lateral auricular concha with a discharge increment of 3.72±0.74 spikes/sec which was 112.91±31.13% in percentage (PO.001); 29 neurons responded to acupuncture at ST36 points with a discharge increment of 0.29±0.66 spikes/sec which was 10.84±5.87%in percentage (P>0.05); 20 neurons responded to acupuncture at S25 points with a discharge increment of 0.75±0.72 spikes/sec which was 20.63±7.50%in percentage (P>0.05); 20 neurons responded to acupuncture at P6 points with a discharge increment of 0.43±0.62 spikes/sec which was 22.76±10.72% in percentage (P>0.05). There was no statistical significance between the effects produced by ipsi- and contra-lateral auricular stimulations on the discharge of DMV neurons (P>0.05), the effect of auricular acupuncture was significantly different from that produced by the acupuncture at the body acu-points (.P<0.05), indicating a better exciting effect of auricular acupuncture than that induced by the acupuncture at the body acu-points.During 30 seconds' acupuncture at ipsi-lateral auricular concha in 30 rats, the HR responded with a reduction of 15.60±3.11 beats/sec which was 3.70±0.75% in percentage CP<0.001); when acupuncture at contra-lateral auricular concha in 32 rats, the HR responded with a reduction of 11.63±2.19 beats/sec which was 2.70±0.48% in percentage (PO.001); when acupuncture at ST36 in 31 rats, the HR responded with a increment of 3.87±1.29 beats/sec which was 0.94±0.32% inpercentage (PO.01); when acupuncture at S25 in 27 rats, the HR responded with a increment of 4.44±2.04 beats/sec which was 1.12±0.51% in percentage (PO.05); when acupuncture at P6 in 30 rats, the HR responded with a increment of 1.20 ± 1.45 beats/sec which was 0.27±0.37% in percentage (P>0.05). There was no statistical significance between the effects produced by ipsi-and contra-lateral auricular concha stimulations on HR (P>0.05), the effect of auricular acupuncture was significantly different from that produced by the acupuncture at body acu-points (PO.01), indicating acupuncture at auricular had decrease effect but acupuncture at body acu-points had no or increase effect on HR.During 30 seconds' acupuncture at ipsi-lateral auricular concha in 29 rats, the MAP responded with a reduction of 12.10±1.05 mmHg which was 11.89±1.11% in percentage (PO.001); when acupuncture at contra-lateral auricular concha in 29 rats, the MAP responded with a reduction of 9.89±1.44 mmHg which wasl0.00±1.35% in percentage (PO.001); when acupuncture at ST36 in 27 rats, the MAP responded with a increment of 2.08±1.29 mmHg which was 1.35±1.40% in percentage (P>0.05); when acupuncture at S25 in 27 rats, the MAP responded with a reduction of 6.35±1.32 mmHg which was 6.28±1.38% in percentage (PO.001); when acupuncture at P6 in 27 rats, the MAP responded with a reduction of 2.07±1.07 mmHg which was 1.89±L09% in percentage (P>0.05). There was no statistical significance between the depressor effects produced by ipsi-and contra-lateral auricular concha stimulations on MAP (P>0.05), and the effect of ipsi-auricular acupuncture was significantly different from that produced by the acupuncture at body acu-points (PO.05), and the depressor effect of S25 was similar to that of contra-lateral aucicular, both of them had better depressor effect than ST36 and P6 (PO.05).During 30 seconds' acupuncture at ipsi-lateral auricular concha in 14 rats, the GP responded with a increment of 17.35±3.26 mmH20 which was 17.28±6.39% in percentage (PO.001); when acupuncture at contra-lateral auricular concha in 15 rats, the GP responded with a increment of 19.28±3.77 mmH20 which wasl8.73±5.37% in percentage (PO.001); when acupuncture at ST36 in 18 rats, the GP responded with a increment of 1O.H±1.86 mmH20 which was 10.20±2.54% in percentage (P<0.001); when acupuncture at S25 in 18 rats, the GP responded with a reduction of 15.88±3.75 mmH20 which was 10.50±2.45% in percentage (P<0.001);when acupuncture at P6 in 18 rats, the GP responded with a increment of 11.42±1.76 mmH2O which was 8.31±1.87% in percentage (PO.001). There was no statistical significance among the effects produced by acupuncture at ipsi- auricular concha, contra-lateral auricular concha, ST36 and P6 stimulations on GP (P>0.05), but all of them had different effect on GP with acupuncture at S25, which had inverse effect on GP(PO.OOl).1.2 Results on atropine-treated ratsAfter intravenous administration of atropine in 34 rats the discharges in 22 NTS neurons responding to acupuncture at ipsi-lateral auricular concha and contra-lateral auricular concha were increased by 3.58±0.82 spikes/sec and 3.44±0.76 spikes/sec, corresponding to the percentages of 61.82±12.04% (PO.OOl) and 75.24±19.26% (PO.001) respectively. The discharges in 20 NTS neurons responding to acupuncture at ST36 points and 24 NTS neurons responding to acupuncture at S25 points were increased by 1.51 ±0.43 spikes/sec and 1.40±0.43 spikes/sec, equal to a percentage of 26.03±6.44%(P<0.01) and 26.26±6.52% (P<0.01) separately; in 24 neurons responding to acupuncture at P6 points the discharges were increased by 1.40±0.44 spikes/sec , a percentage of 28.10±6.18% (PO.01). Compare effect of acupuncture at auricular concha and body points on the NTS extracellular discharges after atropine, there was no statistical significance between ipsi- and contra-lateral auricular (P>0.05) stimulation, but there did exist differences between both auricular and other body points (P<0.05), by and large, indicating a better exciting effect of auricular than of body points after atropine injection. Compare the excitation effect of the same stimulation site pre- and post-atropine, there was no statistical difference all the points (P>0.05). There was no statistical difference in the excitatory effects induced by every acupoint stimulated before and after administration of atropine.After intravenous administration of atropine, the HR was recorded in some animals. After acupuncture at ipsi-lateral auricular concha in 30 rats, the increment of HR was 2.80 ± 2.05 beats/sec, a percentage of 0.65 ± 0.51% (P>0.05); acupuncture at contra-lateral auricular concha on 32 rats, the increment of HR was 3.75 ± 2.31 beats/sec, a percentage of 0.79 ± 0.51% (P>0.05); acupuncture at ST36 on 31 rats, the increment of HR was 4.65 ± 1.07 beats/sec, a percentage of 1.07 ± 0.24% (?<0.001); acupuncture at S25 on 27 rats, the increment of HR was 3.11 ±1.76 beats/sec, a percentage of 0.69 ± 0.40% (P>0.05); acupuncture at P6 on 30 rats, the increment of HR was 3.20 ± 1.40 beats/sec, a percentage of 0.73 ±0.31% (P<0.05). Compare the effect of acupuncture at auricular concha and body points on HR, there was no statistical significance among all the groups (P>0.05). Compare the influence of the same stimulation site pre- and post-atropine, there existed apparent statistical difference in both auricular stimulation groups (PO.01) for contrary responsive direction; ST36 and S25 had similar effect pre- and post-atropine injection (P>0.05); acupuncture at P6 had a small increase effect on HR after atropine CP<0.05).After intravenous administration of atropine, the MAP was recorded on some animals. Acupuncture at ipsi-lateral auricular concha on 29 rats, the reduction of MAP was 3.84 ± 0.90 mmHg, a percentage of 4.80 ± 1.11% (PO.001); acupuncture at contra-lateral auricular concha on 29 rats, the reduction of MAP was 3.28 ± 0.91 mmHg (PO.01), a percentage of 4.23 ± 1.17%; acupuncture at ST36 on 27 rats, the reduction of MAP was 1.69 ± 0.80 mmHg, a percentage of 2.07 ± 1.00%(PO.05); acupuncture at S25 on 27 rats, the reduction of MAP was 1.75 ±0.97 mmHg, a percentage of 5.83 ± 1.08 (PO.001); acupuncture at P6 on 27 rats, the reduction of MAP was 1.75 ± 0.97mmHg, a percentage of 2.21 ± 1.33% (P>0.05). Compare the depressor effect of acupuncture at auricular concha and body points after atropine, there was no statistical difference on depressor effect among all the points except S20, which still had better depressor effect than the other two points (PO.05). Compare the depressor effect of the same stimulation site pre- and post-atropine, there existed apparent statistical difference in both auricular stimulation groups (PO.001) and S20 points (PO.05).After intravenous administration of atropine, the GP was recorded on some animals. Acupuncture at ipsi-lateral auricular concha on 14 rats, the increment of GP was 1.01 ± 1.21 mmH20, a percentage of 0.52 ± 1.89% (P>0.05); acupuncture at contra-lateral auricular concha on 15 rats, the increment of GP was 2.78 ± 1.75 mmH20, a percentage of 4.72 ± 1.75% (P>0.05); acupuncture at ST36 on 18 rats, the increment of GP was 2.09 ± 1.01 mmH20, a percentage of 1.51 ± 2.30% (P>0.01); acupuncture at S2s on 18 rats, the increment of GP was 0.00 ± 1.05 mmH20, a percentage of 1.09 ± 1.07% (P>0.05); acupuncture at P6 on 18 rats, the increment of GP was 0.50 ± 2.01 mmH20 , a percentage of 0.24 ± 2.87% (P>0.05). There was nostatistical significance among all the groups after atropine (P>0.05). There existed statistical difference in all groups (PO.05) pre- and post- atropine injection.2 Morphological experiment: research the direct projection from auricular concha to NTS by neuronal tracing technology and immunohistochemistry methodsThe male healthy Sprague-Dawley rats weighing 200-250 g were anesthetized with 10% urethane (1.0~1.2gkg"' i.p.). 20% PBS solution of biocytin and 4% PBS solution of Pseudorabies Virus (PrV) were subcutaneously injected into rats' auricular concha and external auditory canal opening from 3-4 separate points. The medulla oblongata and interbrain were removed rapidly under anesthesia with 10% urethane 72 hours after injection of biocytin and PrV and cut into 20|i slices. The brain slices were detected by immunohistochemistry methods.In addition, the Fos-protein expressed in NTS of the rats was also determined with immunohistochemistry methods after the stimulation of electroacupuncture at auricular concha for 30 minutes. The results are as follows.2.1 Result after injection of biocytin as a neuronal pathway tracerSparsely labeled fibers were found from rostral to obex in NTS. And comparatively more labeled fibers were exhibited in the nucleus of spinal tract of trigeminal nerve, postrema and reticular formation.2.2 Result after injection of PrV as a neuronal pathway tracerThe labeled neurons appeared in NTS, DMV, the nucleus of spinal tract of trigeminal nerve and the nucleus of ambiguous. In NTS the labeled neurons mainly scattered from rostral to obex.2.3 c-fos protooncogene expression in oblongata after electro-acupuncture stimulation on auricular concha of ratsFos protein stained neurons were spread along whole NTS, but more labeled neurons appeared from rostral to the opening base of the four ventricle. Quite more expressions were found in DMV, and densely labeled neurons existed in the bilateral nucleus of spinal tract of trigeminal nerve and reticular formation of medulla oblongata.
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