| There were numerous clinic reports about parasympathetic excitation responses induced by stimulation of auditory canal or auricular concha such as auriculo-cardiac-reflex and ear-cough-reflex.Our previous study confirmed the exist of the primary projection of the auricular branch of vagus to the nucleus of solitary tract(NTS),and also showed that electroacupuncture on auricular concha might induce parasympathetic excitation responses including suppressing hypertension, suppressing hyperglycemia,strengthening gastric motility by increasing the firing rates of the NTS.Epilepsy is a disorder of the brain characterized by recurrent seizures.On July 16, 1997,the U.S.Food and Drug Administration(FDA)approved the use of vagus nerve stimulation(VNS)as an adjunctive treatment for refractory partial-onset seizures in adults and adolescents aged＞12 years.VNS has been approved for use in reducing seizures in patients who have mostly partial or generalized seizures in more and more countries.Yet the mechanism by which VNS might exert an antiepileptic effect is unknown.Putative antiseizure mechanisms are mostly considered as that mediated by activated vagal afferent activities to the NTS,and probably include altered activities in the reticular activating system,the central autonomic network,the limbic system, and the diffuse noradrenergic projection system.Several reports found an imbalance within and between sympathetic and parasympathetic activities which was frequently associated with sympathetic over-activity and parasympathetic low-activity,so the antiseizure effect of VNS may be induced by activating the parasympathetic activity. Transcutaneus vagus nerve stimulations(TVNS)on the body surface projection of vagus nerve and the auricular branch of vagus nerve were both confirmed to be medicative to suppress seizure.The frequently used ear acupoints to treat epilepsy were mainly situated at auricular concha,which was innervated by the auricular branch of vagus nerve.Therefore,we presumed that the antiseizure effect of auriculo-acupuncture was mediated by stimulating the auricular branch of vagus nerve. The present study investigated the effects of stimulation at auricular concha on the epileptiform behaviors,firing rates of the NTS,epidural electroencephalogram(EEG), field potentials of the NTS and the cortex of the epilepsy rats,and explored the effect on the epidural EEG of the epilepsy rats induced by cooling the NTS or vena femoralis injection of excitomotory or antagon of sympathesis,aiming at explore the antiseizure mechanism of auriculo-acupuncture on the basis of the auriculo-vagus-reflex theory.Animal experiments1 Effect of auriculo-acupuncture pretreatment on the behaviors of PTZ-induced epilepsy rats.Animals Experiments were performed on 42 adult male Sprague-Dawley rats weighing 300±28g.Methods Acute seizures were induced by intraperitoneal injection of PTZ (pentylenetetrazol,PTZ)60 mg/kg.The animals were divided into three groups:PTZ group(n=14),auricular eoncha group(n=15),and Dazhui group(n=13).The latter two groups were pretreated with stimulation at auricular concha or "Dazhui" point respectively for 30 min before intraperitoneal injection of PTZ.Behaviors of the rats had been observed and marked according to the standard of Racine' s for 30 min immediately after the intraperitoneal injection of PTZ.Results Epileptic behaviors were suppressed by acupuncture pretreatment.Compared with the PTZ group,epileptic behaviors of the auricular concha group and the Dazhui group were suppressed in terms of duration of the first grand mal and marks of epileptic behaviors.Compared with the Dazhui group,epileptic behaviors of auricular concha group were more suppressed(p＜0.05)in terms of the duration of the first grand mal and marks of epileptic behaviors.2 Effect of auriculo-acupuncture on activities of neurons in the NTS and epidural EEG of epilepsy rats. Animals Experiments were performed on 45 adult male Sprague-Dawley rats weighing 300±34g.Methods Anesthesia was initiated by 10%urethane(1.2 g/kg,i p),additional sodium pentobarbital was administered as needed.Exposed left vagus nerve was then placed over the cuff electrode for VNS.Three small holes were drilled by burr drill,one in the right side of frontal region(AP:1.0 mm,ML:1.0 mm),one in the right side of parietal region(AP:-1.0mm,ML:1.0 mm),the other in the left side of the occipital region(AP:-11.3～-14.3mm,ML:0～2.3mm).For the bipolar recording of epidural EEG,two silver globe electrodes were placed over the dura in the former two holes respectively.Extracellular single-unit recordings in the site of the NTS(AP:-11.3～-14.3mm;ML:0～2.3mm;DV:4～7mm)were recorded by glass microelectrodes.Transcutaneous electrical nerve stimulation(TENS),electroacupuncture(EA), manual acupuncture(MA)were used respectively to stimulate vagus nerve,five points of the pinna such as cavity of auricular concha,cymba of auricular concha, auricular lobule,middle point of the exterior margin of the pinna,or apex conchae auris,"Du 14 "point,and" ST 40 "point.Results Totally 31 cells of the NTS were recorded.Before PTZ,EEG traces were horizontal relatively.When PTZ were administered with no stimulation provided,the firing rates of 26 cells declined(26/31,83.87%),simultaneously highly synchronous, large-amplitude activity in epidural EEG traces occurred.Every time the more the firing rate declined,the larger-amplified activity in epidural EEG traces occurred,and they happened synchronously.1-3 min after PTZ,this synchronous changes happened, then after 15 min,the synchronous changes were regular relatively.We looked one large-amplitude activity in epidural EEG traces as one seizure,the average seizure duration was 6.21±0.48s,the average number of seizures per minute was 4.32±0.23, and the total seizure duration per minute was 26.83±2.25s.After 30 seconds' TENS of auriculo-points,firing rates of the NTS neurons increased. After TENS(n=31),the firing rates increased by 2.97±0.18 spikes/sec(P＜0.01); after TENS at cavity of auricular concha(n=29),the firing rates increased by 2.98±0.14 spikes/sec(P＜0.01);after TENS at cymba of auricular concha(n=32),the firing rates increased by 2.90±0.15 spikes/sec(P＜0.01);after TENS at auricular lobule (n=28),the firing rates increased by 1.03±0.08 spikes/sec(P＜0.01);after TENS at middle point of the exterior margin of the pinna(n=26),the firing rates increased by 0.68±0.04 spikes/sec(P＜0.05);after TENS at apex conchae auris,(n=25),the firing rates increased by 0.83±0.05 spikes/sec(P＜0.05);after TENS at " Du 14 " ponit (n=28),the firing rates increased by 0.56±0.06 spikes/sec(P＜0.05);after TENS at "ST 40" ponit(n=20),the firing rates increased by 0.26±0.03 spikes/sec(P＞0.05), which indicated that there were statistical difference between the firing rates before and after stimulation,except that induced by TENS at "ST 40 "ponit.After 30 seconds' EA of auriculo-points,firing rates of the NTS neurons increased. After VNS(n=26),the firing rates increased by 3.62±0.25 spikes/sec(P＜0.01);after EA at cavity of auricular concha(n=25),the firing rates increased by 3.59±0.34spikes/sec(P＜0.01);after EA at cymba of auricular concha(n=27),the firing rates increased by 3.61±0.14 spikes/sec(P＜0.01);after EA at auricular lobule, (n=24),the firing rates increased by 1.12±0.09spikes/sec(P＜0.01);after EA at middle point of the exterior margin of the pinna(n=22),the firing rates increased by 0.86±0.07 spikes/sec(P＜0.01);after EA at apex conchae auris(n=25),the firing rates increased by 1.01±0.09 spikes/sec(P＜0.01);after EA at "Du 14" ponit(n=20), the firing rates increased by 0.66±0.07 spikes/sec(P＜0.05);after EA at " ST 40 " ponit(n=21),the firing rates increased by 0.35±0.05 spikes/sec(P＞0.05),which indicated that there were statistical difference between the firing rates before and after stimulation,except that induced by EA at " ST 40 "ponit.After 30 seconds' MA of auriculo-points,firing rates of the NTS neurons increased. After MA at cavity of auricular concha(n=28),the firing rate increased by 3.84±0.35 spikes/sec(P＜0.01);after MA at cymba of auricular concha(n=32),the firing rate increased by 3.81±0.45 spikes/sec(P＜0.01);after MA at auricular lobule(n=33),the firing rate increased by 1.39±0.17spikes/sec(P＜0.01);after MA at middle point of the exterior margin of the pinna(n=29),the firing rate increased by 0.98±0.12 spikes/sec(P＜0.01);after MA at apex conchae auris(n=26),the firing rate increased by 1.21±0.15 spikes/sec(P＜0.01);after MA at " Du 14 " ponit(n=25),the firing rate increased by 0.91±0.12 spikes/sec(P＜0.01);after MA at "ST 40 "ponit(n=20), the firing rate increased by 0.50±0.06 spikes/sec(P＜0.05),which indicated that there were statistical difference between the firing rates before and after stimulation by MA.When stimulated at the same one point,the effet of increasing firing rates by MA was better than that by TENS or EA(P＜0.05),the effet of increasing firing rates by EA was better than that by TENS(P＜0.05).When stimulated by using the same method, the effet of increasing firing rates by stimulating at VNS,cavity of auricular concha or cymba of auricular concha,was better than that at any other point(P＜0.01).When stimulated at the same point,the effet of supressing epileptic EEG by MA was better than that by TENS or EA(P＜0.05),the effet of supressing epileptic EEG by EA was better than that by TENS(P＜0.05).When stimulated by using the same method,the effet of supressing epileptic EEG by VNS,or stimulating at cavity of auricular concha,cymba of auricular concha was better than that by stimulating at any other point(P＜0.01).3 Effect of auriculo-acupuncture on the behaviors and field potentials of epilepsy rats.Animals Experiments were performed on 124 adult male Sprague-Dawley rats weighing 298±31 g..Methods Four recordings were carried out respectively including acute single electrode recordings of field potentials(FPs)in the primary somatosensory cortices (SI),chronic single electrode recordings of FPs in the SI cortices,acute microelectrode arrays recordings of FPs in both of the NTS and the SI cortices, chronic microelectrode arrays recordings of FPs in the SI cortices,to observe the effect of electrostimulation at any of the five points of the pinna on the behaviors and field potentials of epilepsy rats. Results Our results showed that after electrostimulation at the five points of the pinna, epileptic behaviors were suppressed(P＜0.05).And the FPs of the NTS was synchronous with that of the SI cortices in the acute microelectrode arrays recording.In the experiment of acute single electrode recordings in SI cortices,epileptic discharges in the field potential traces were suppressed after electrostimulation at the five points of the pinna(P＜0.01),The antiseizure effect induced by stimulation at the cavity of auricular concha or the cymba of auricular concha was better than that induced by stimulation at any other position(P＜0.01).In the experiment of chronic single electrode recordings in the SI cortices,epileptic behaviors and epileptic discharges in the field potential traces were more suppressed by stimulation at the cavity of auricular concha or the cymba of auricular concha than that induced by stimulation at any other positions(P＜0.01),and there was no significant difference between the antiseizure effect of stimulation at the cavity of auricular concha and the cymba of auricular concha(P＞0.05).In the experiment of acute microelectrode arrays recordings in the SI cortices, epileptic discharges in the field potential traces were suppressed after electrostimulation at the five points of the pinna(P＜0.01),The antiseizure effect induced by stimulation at the cavity of auricular concha or the cymba of auricular concha was better than that induced by stimulation at other position(P＜0.01).In the experiment of chronic microelectrode arrays recordings in the SI cortices, epileptic behaviors and epileptic discharges in the field potential traces were more suppressed by stimulation at the cavity of auricular concha or the cymba of auricular concha than that induced by stimulation at any other positions(P＜0.01),there was no significant difference between the antiseizure effect of stimulation at the cavity of auricular concha and the cymba of auricular concha(P＞0.05).4 Effect of cooling the NTS on antiseizure effect of auriculo-acupuncture.Animals Experiments were performed on 10 adult male Sprague-Dawley rats weighing 300±30g. Methods Two silver globe electrodes were placed over dura of the frontal region and the parietal region respectively for the bipolar recording of epidural EEG.U-shaped glass tube was placed on the region of the obex.- 8℃ethylene glycol was injected through the U-shaped glass tube to cool the NTS.We have observed the effect of cooling the NTS on antiseizure effect of auriculo-acupuncture at the five points of the pinna respectively.Results Before PTZ,the EEG traces were horizontal relatively,and cooling the NTS had no obvious effect on the EEG traces.After PTZ,highly synchronous, large-amplitude activity in epidural EEG traces occurred.Compared with that before cooling the NTS,the antiseizure effect of the auriculo-acupuncture was impaired significantly when cooling the NTS(P＜0.01),5 min after removing cooling,the antiseizure effect of the auriculo-acupuncture recovered to the level before cooling,(P＞0.05).5 Effect of Adrenaline Hydrochloride and Propranolol on epidural EEG of epilepsy rats.Animals Experiments were performed on 10 adult male Sprague-Dawley rats weighing 300±30g.Methods Two silver globe electrodes were placed over dura of the frontal region and the parietal region for the bipolar recording of epidural EEG.Adrenaline Hydrochloride or Propranolol was injected respectively by vena femoralis injection to observe the effect of excitomotory or antagon of sympathesis on the EEG of the epilepsy rats.Results After PTZ,vena femoralis injection of Adrenaline Hydrochloride could aggravate seizures,vena femoralis injection of Propranolol could suppress seizures.ConclusionThe present study has exlpored the mechanism of the antiseizure effect induced by auriculo-acupuncture.The results demonstrated that epilepsy was accompanied with seizures and highly synchronous,large-amplitude activity in epidural EEG traces,epileptic EEG could be significantly suppressed by auriculo-acupuncture through increasing the firing rates of the NTS to activate the parasympathetic activity.The antiseizure effect induced by stimulation at the cavity of auricular concha or the cymba of auricular concha was better than that induced by stimulation at any other position,there was no significant difference between the acute antiseizure effect of stimulation at auricular concha and VNS,which indicated that the antiseizure effect of stimulation at auricular concha perhaps was mediated by stimulation of the auricular branch of vagus nerve.Our result also provided more evidence to explain the antiseizure effect of auriculo-acupuncture, and to found the auriculo-vagus-reflex theory.Our study had showed that the variation of firing rates of the NTS was synchronous with the variation of epileptic EEG.The antiseizure effect induced by auriculo-acupuncture could be weakened if the activity of the NTS was impaired. Perhaps our result was the first to confirm in vivo that the NTS had participated in the development of epilepsy,which gave a prompt to the research on the pathogenesis of epilepsy and the antiseizure mechanism of VNS.Our study also observed the antiseizure effects of three different treating methods induced by TENS,EA or MA.The antiseizure effect induced by MA was better than that induced by TENS or EA.The antiseizure effect induced by EA was better than that induced by TENS.The result indicated that different curative effects occurred when treated by different methods,which gave us experimental evidence to select a better therapy method in clinic.
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