Research Of The Auriculo-vagus Reflex And Mechanism Of Hypoglycemia Effect Induced By Electro-acupuncture At Auricular Concha Acupoints

Diabetes mellitus is becoming one of the most common diseases associated withthe improvement of peoples' lives. Acupuncture, particularly auricular acupuncture,exhibits pronounced hypoglycemia effect in treating diabetes. But the mechanism isunclear up to now. Some researchers hold that the mechanism underlying the effectsof auricular acupuncture on hypoglycemia was conclusively attributed to thesegmental innervations of sympathetic nervous systems. However, they ignored thefact that auricular acupuncture possibly participated in regulating the interactionsbetween the afferent vagal nerve from aricular concha and the visceral organs whichconverge in the brainstem especially the complex of vagus. Based on the fact that theauricular vagus branch is the only peripheral branch of the vagal nerve, and thatglucose-responsive and insulin-responsive neurons possibly exist in the nucleustractrus solitarri (NTS), furthermore, acupuncture in auricular concha acupoints canaffect the physiological activity of the above two types of neurons. So, wehypothesize that the mechanism of hypoglycemia effect induced by needling orpressing on auricular point is correlative with the auriculo-vagus nerve systems reflex.The present study investigated the effects of electrical stimulation at the conchaacupoints on the discharges of glucose-responsive and insulin-responsive neurons, thesynaptic correlation between the auricular concha and the vagal primary center, andchange of glucose and insulin concentration of normal and diabetic rats which wereapplied with electrical acupuncture in auricular acupoints. In this study we tried toelucidate the CNS mechanism of effects caused by needling stimulation at auricularconcha acupoints in the light of auriculo-vagal nerve system reflex.PartⅠElectrophysiological experent: Responses of glucose-sensitive neuronsand insulin-sensitive neurons in nucleus tractus solitarius to electricalacupuncture at auricular concha acupointsMethods To observe responses of glucose-sensitive neurons and insulin-sensitiveneurons in nucleus tractus solitarius to acupuncture stimulation at auricular conchaacupoints (representation of pancreas, liver and gall, et,al), and to investigatepreliminarily the brainstem's mechanism of hypoglycaemia effect caused by auricular acupuncture, we used extracellular single-unit recordings which were carried out innucleus tracus solitarius of rats followed intra-carotid administration of glucose(15%), insulin (6IU/ml) and acupuncture stimulation at auricular concha(interval:0．4s, duration:0．4ms intensity:2mA),respectively.Results In the group of administration of glucose (15%, 0．25ml/min) , thedischarges of totally 110 NTS neurons were recorded. Among them there were 41(37．27%) neurons responding to glucose infusion with a discharges decrease inpercent of 60．36±15．1%, while 10．91 %(12/110) neurons responding to glucoseinfusion with a discharges increase with a percentage of 56．78±9．24%. 57 (51．82%)neurons showed no response in discharges frequency during or after administration ofsugar solution via carotis. During or after intra-carotid administration of insulin(6IU/ml, 0．25ml/min) , totally 81 NTS neurons' discharges were observed . Amongthem, there were 33．33% (27/81) neurons showed increase in spike frequency with apercent of 47．34±11．24%. Meanwhile, there were 4(4．9%) neurons exhibited decreasein spike frequency with a percent of 43．24±12．11%. Among the above 27exciting-responding neurons, 18 cells showed decrease in activity to administration ofglucose via carotis, while 9 cells showed no response in spike frequency to sugarinfusion. Among the 4 neurons which were inhibitory-responsive to insulinadministration, there were 2 neurons showed contrary responsive to sugar infusion viacarotis, the others exhibited no response in frequency of discharge.The discharges of totally 69 neurons in NTS were examined during or afteradministration of electro-acupuncture at auricular concha. Among them, there were 34(49．27%)neurons showed increase in spike frequency with a percentage of 54．79±24．21%, 3 (4．35%) ones showed decease in spike frequency with a percentage of44．65±9．13%, the others (46．38%)demonstrated no response to the acupuncture atauricular concha. Glucose infusion via carotis resulted in a decrease in firing rate of18 neurons in the totally 34 neurons which showed exciting-response to auricularacupuncture, while there were 2 neurons in the 34 ones showed contrary response tothe administration of glucose, and the others showed no response to glucoseconcentration's elevation. Among the same 34 neurons, 8 neurons showed anexcitatory response to insulin infusion, 3 ones showed contrary response to theadministration of insulin, the others showed no response to insulin concentration'selevation. During or after glucose infusion via carotis, the latence ofexcitatory-response and inhibitory-response neurons recorded in NTS wererespectively 15±6 and 18±8s, and those were respectively 16±7s and 18±10s during or after administration of insulin, no significant difference between them (P>0．05) . Thelatence of neurons recorded in NTS showing excitatory-response andinhibitory-response to auricular acupuncture were respectively 3±1s and 5±2s.Furthermore, among the totally 37 recorded during auricular acupuncture, there were21 neurons exhibited 2-5 minutes' long-term increase in firing rate. 2 neurons werestimulated to waken up from silence. 3 ones in the 37 responsive neurons showedinhibitory-response to auricular acupuncture with long-term's decrease in firing rate.PartⅡMorphological experiment: Research the medullary distribution ofafferent fibers and cells of origin from auricular concha using transganglionictransport of horseradish peroxidase (HRP) as a neuronal tracer.Methods 10 adult male and female healthy Sprague-Daxley rats weighing 200-250gwere anesthetized with 30% urethane solution (1g/kg i.p.). Under general anesthesia, askin incision was made in the region of auricular concha. The skin and fasciae wereseparated to expose the auricular nerve of vagus nerve, which were traced carefully tothe region of the ear root where the auricular nerve of vagus nerve was cut. Fortymicroliters of a 30% solution of HRP (Sigma) in normal saline were subcutaneouslyinjected into the cut central end of auricular branch of vagus nerve. After survivaltimes of 36hrs to 48hrs, all animals were reanesthetized and perfused intranscardiallywith 500 ml of normal saline, followed by a phosphate buffered (PH=7．4) fixativecontaining 1．25% glutaraldehyde and 1% formaldehyde. The medulla oblongata andinterbrain were moved in each animal and transferred to phosphate buffer containing5% sucrose for 12-24h at 4℃. Serial sections (30μm) were cut through the tissues ona freezing microtome. All sections were processed for HRP activity using tetramethylbenzidine as the chromogen.Results HRP-labeled terminal or preterminal fibers were identified under the lightmicroscope. They often appear as brown granules of various sizes and differentshapes in medulla oblongata and interbrain. Heavily labeled neurons and fibers wereseen in NTS and DMV, as well as in the nucleus of spinal tract of trigeminal nerve,where granular labeling observed was heaviest at the same level. Labeling in themedial division of the NTS extended dorsally adjacent to the AP. In the one animal inwhich measurements were made, granular labeling extended for 2．6mm in theipsilateral NTS sparing 0．4mm of the nucleus rostrally. HRP-labeled cells in NTSwere frequently fusiform and ovoid, while those in DMV were often ovoid.Retrograde labeling of neurons was also observed in the nucleus of ambiguous and the region of reticular formation, and the labeled neurons were rounded or oviform in shape and 20×30μm in size.PartⅢBiochemical experiment: Effects of electro acupuncture on the plasma glucose and insulin concentration of normal and diabetic rats.Methods The experiment was carried out on 100 healthy male rats, 150-220g in body weight. Among the whole animals, 40 were normal rats, and the others were diabetic rats, which were induced by once intraperitoneal injection of streptozotocin (60mg.kg). The normal animals were divided into three groups in this dose-finding experiments: Group A consisted of 10 normal rats which were administered with electrostimulation at the intensity of 10 mA , wave of 1ms , applied in auricular concha points; Group B were applied in the same acupoints electrostimulation at the intensity of 2 mA , wave width of 0．5ms; Group C were administered with the same intensity as Group B of electrostimulation at the Zusanli (ST36) acupoint. Diabetic rats were divided into 3 groups: auriculo-acupuncture group (Group D) received the same electrostimulation as Group B; Vagotomy followed auriculo-acupuncture group (Group E) received the same electrostimulation with Group B after cutting left cervical vagal nerve; Group F was control group which were administered the same electrostimulation as Group C. Each group rats were received 30min's electrostimulation, then several blood samples (0．2-0．5ml each) were taken from the femoral vein respectively at the time of 0, 10, 20, 30 minutes after stopping acupuncture stimulation and placed them in Eppendorf tube containing 10 IU heparin. The plasma glucose was measured using Onetouch Ultra Brand blood glucose meter and insulin were determined in the way of immunosorbent assay, American BPB Inc. supplied with commercial kits.Results1) After 30 minutes' electrical acupuncture on the auricular concha acupoints, which was of 10 mA in intensity, 1ms in wave width, the glucose concentration responded with a hyperglycemia from average of 6．48±0．28 mmol/L before the acupuncture to 7．70±0．38 mmol/L at the point of 10mins after stopping acupuncture (P<0．05, paired t-test, same to following). While electrical acupuncture on auricular concha acupoints with 2 mA of intensity, 0．5ms of wave width induced a hypoglycemia from average of 7．23±0．15 mmol/L before the acu-stimulation to 6．43±0．14 mmol/L 10 minutes after acupuncture stop; there was statistical significance in glucose concentration before and after acu-stimulation on auricular concha (P<0．01) . In the control group, no significant difference in glucose concentration was induced by the electrical stimulation applied to need inserting in the tissue at Zusanli acupoint (7．45±0．33 mmol/L vs 7．41±0．29 mmol/L, P>0．05) .2) Lower intensity (2 mA in intensity, 1ms in wave width) of auricular acupuncture applied to normal rats showed a time-window regulation of glucose concentration. Before the auri-acupuncture, the average of blood suger level was 7．23±0．15mmol/L, after 30 minutes' administration of electrical stimulation on auricular concha, the suger level reduced to 6．65±0．14mmol/L when the stimulation came to a stop (P<0．01 vs before auricular acupuncture). 10 minutes later, glucose concentration continually decreased to a lowest value of 6．43±0．14mmol/L (P<0．01 vs when auri-acupuncture stopped). Then a hyperglycemia appeared as time gone on, and at the point of 20 minutes later, the glucose went up to 6．84±0．14mmol/L (P<0．05 vs 10 minutes later; but P>0．05 vs before acupuncture stimulation). The glucose concentration increased to the normal level as before the auricular electrical stimulation (P>0．05 vs before acupuncture stimulation). In the control group, above time-window regulation of glucose concentration was not induced by electrical stimulation at ST36 acupoint. Before the acupuncture stimulation at ST36, glucose concentration of the control group was 7．45±0．33mmol/L, and then was 7．49±0．27mmol/L when the 30 minutes' acupuncture came to a stop. There was no statistical significance between them., 10 minutes later the glucose slowly moved down to 7．41±0．29mmol/L (P>0．05 vs when acupuncture stopped), one more 10 minutes later the concentration went up to 7．43±0．27mmol/L, then to 7．44±0．25mmol/L at the point of 30 minutes after the acupuncture stopped. There were no differences in statistics among them.3) However, the time-window regulation of glucose concentration of normal rats induced by the lower intensity of auricular acupuncture could not be explained absolutely by the alteration of insulin concentration, which was also induced by the auricular acupuncture as well. Before the auri-acupuncture, insulin concentration was 44．65±2．95μmol/L, then went up to 50．06±3．88μmol/L after 30 minutes' acupuncture at auricular concha, but there was no distinguished difference between them which was compared by the paired t-test(P>0．05) .In the control group, the same phenomenon was obserbed in normal rats during the electrical acupuncture stimulation was applied to the ST36 acupoint. Insulin concentration was increased to 51．76±2．93μmol/L at the point of 10 minutes after the stop of stilulation from 48．63±3．79μmol/L before stimulation, no distinguished difference was found in the above two sets of data which were compared by paired t-test analysis. 4) Acupuncture applied to auricular concha acupoint could lower plasma glucose concentrations in diabitic rats. An insulin-dependent action thus could be considered. Lowering of plasma glucose in diabetic model rats was investigated from 17．33±0．60mmol/L to 16．58±063mmolL after EA-stimilation at auricular concha acupoints (P<0．05, before auri-acupunctrure vs after auri-acupuncture). This hypoglycemia activity was blocked by vagotomy (17．57±0．80mmol/L vs 18．23±0．81mmol/L, P>0．05) . In the control group, EA-stimulation at the ST36 acupoint elicited a slender reduction in plasma glucose concentration (from 17．69±0．79mmol/L to 17．59±0．82mmol/L), but no distinguished difference was found between two sets of data analysised by the paired t-test.5) An insulin-dependent action could be explained the hypoglycemia activity elicited by acupuncuture. After the EA-stimulation at auricular concha, plasma insulin in diabetic rats were raised from 50．66±2．15μmol/L to 58．49±2．44μmol/L (P<0．001) .Blockade of auricular acupuncture-induced increase of plasma insulin was seen in the diabetic rats which were received vagotomy, and no significantly difference was found before and after the electrical acupuncture at auricular concha followed vagotomy (50．53±2．06μmol/L vs 47．86±1．34μmol/L, P>0．05) . In the control group, EA-stimulation at ST36 acupoint also raised plasma insulin concentration from 46．32±1．86μmol/L up to 51．79±2．23μmol/L (P<0．05) . So we could possibly attribute the hypoglycemia effect induced by acupuncture to the insulin-dependence.ConclusionThe present study including three parts of experiments were carried out to investigate the relationship between the mechanism of hypoglycemia induced by electrical acupuncture at auricular acupoints and Auriculo-vagal nerve system reflex. The experiment results showed that different intensities of acupuncture at concha points casused different effects on the glucose concentration alteration in normal rats. Lower density of electrical acupuncture at concha acupoints had a pronounced time-window regulation of plasma glucose, compared with the control group, in which electrical stimulation was applied to a needle inserting into the tissue at ST36 acupoint. Acupuncture either in auricular concha or in ST36 acupoints could raise plasma insulin in diabetic rats. In the group of auricular acupuncture this raise had significant difference, while in the group of ST36 acupuncture, the raise was slender. The effect of pronounced reduction in plasma glucose concentration could be elicited by electrical auri-acupuncture, which could be blocked by vagotomy. There existed glucose-responsive neurons and insulin-responsive neurons in NTS. Electrical acupuncture stimulation at auricular concha acupoints could affect the activity of these neurons, especially could irritate the neurons' firing which showed inhibitory response to glucose administration. Directly neural projections in the NTS and DMV from the terminal of auricular vagus branch in the region of auricular concha were examined by using the HRP transganglionic tracing. The morphological results gave a rational explaination of the electrophysiolodical outcome, and supplied with morphology evidence for auriculo-vagal nerve system reflex theory. This study showed that the hypoglycemia effects of auricular acupuncture on different conditions of rats were distinct. Lowing of plasma glucose induced by auri-acupuncture should be supported by intact vagal nerve.