| Parasympathetic excitation responses can be induced by the stimulation at auditory canal or auricular concha, such as auriculo-vagus-stimulation. Our previous study verifies that the stimulation of acupuncture at auricular concha might induce the excitation of nucleus of solitary tract (NTS) and alter the frequency of discharged neurons in nucleus originis dorsalis nerve vagus. Stimulation of auricular concha could regulate heart rate and blood pressure, and strengthen gastric motility. Our previous study also shows that electroacupuncture at auricular concha might induce parasympathetic excitation responses, including suppressing hypertension, suppressing hyperglycemia.The epilepsy is a chronic disease with temporal disorder function of the central nervous system caused by recurrent seizures. According to the different parts of the diseased nerve cells and the scopes of discharging with the proliferation, The seizure can be manifested as disorders in motion, feeling, intention, behavior, autonomic nerve, etc. To cure the epilepsy we can mainly use the medicine under control, or surgical operation surgical operation (cut off the nidus or its conduction passages) and vagus nerve stimulation (VNS). It is mostly considered that that the mechanism of VNS therapy is through activating projection from the vagus nerve to the NTS and the broad connection between the NTS and brain, with a result of desynchronization of EEG.In our previous study, it is found that stimulation of auricular branch of vagus nerve can also suppress the epilepsy and play a rather good effect of anti-epilepsy. If we can use the therapy of vagus nerve stimulation, we can resolve the complication of patients caused by VNS surgical operation and the high expenses and etc. and provide the treatment methods as convenient and money-saved for the people suffering epilepsy. Based on the previous study, this research will study on the effects, mechanism of effect and stimulation parameters on animal subjects and the human subjects by the stimulation to the concha auriculae of the vagus nerve to repress the epilepsy. Furthermore, we discuss the mechanism of function about how we provide the stimulation to the auricular branch of vagus nerve to repress the epilepsy, providing the corresponding theory for the clinical application.Experiments1. Effect of Transcutaneous Auriculo-vagus-stimulation on Local field potentials in cortex of epilepsy rats1.1 Animals Experiments were performed on 14 adult male Sprague-Dawley rats weighing 300±49g.1.2 Methods Anesthesia was performed by 10% urethane (1.2 g/kg, i.p.), additional sodium pentobarbital was administered as needed. Four recordings of field potentials (FPs) by microelectrode arrays (2×2,4 channels) were carried out in the right primary somatosensory cortices (AP:±1.0 mm, ML:4-6 mm, DV:0.5-1.0mm). Rats were induced by intraperitoneal injection of PTZ (pentylenetetrazol, PTZ) 60 mg/kg. Stimulation was performed by two iron slice electrodes sticked on auricular concha,with parameter as follows:Frequency of 20Hz, duration of 30 seconds, intensity of 1 mA. Field potentials was observed and recorded by CerebusTM5.0 system.1.3 Results Before PTZ, the EEG traces were horizontal relatively. After PTZ, highly synchronous, large-amplitude activity traces in field potentials occurred. Before auriculo-vagus-stimulation, the attack time of seizures in two mimutes, was 101.55±6.39 sec; after stimulation, the attack time in two mimutes was 58.5±10.25 sec. Compared with stimulation, the attack time of epilepsy decreased 58.74±10.10%(P<0.01).2. Comparation of ipsilateral and contralateral transcutaneous auriculo-vagus-stimulation on field potentials of epilepsy rats2.1 Animals Experiments were performed on 27 adult male Sprague-Dawley rats weighing 300±34g.2.2 Methods Anesthesia was performed by 10% urethane (1.2 g/kg, i.p.); additional sodium pentobarbital was administered as needed. Four recordings of field potentials (FPs) by microelectrode arrays (2×2,4 channels) were carried out in the right primary somatosensory cortices (AP:±1.0 mm, ML:4-6 mm, DV:0.5-1.0mm). Rats were induced by intraperitoneal injection of PTZ (pentylenetetrazol, PTZ) 60 mg/kg. Stimulation was performed by two iron slice electrodes sticked on auricular concha,with parameter as follows:Frequency of 20Hz, duration of 30 seconds, intensity of 1 mA. Field potentials was observed and recorded by CerebusTM 5.0 system. 27 Sprague-Dawley rats were divided into two groups, ipsilateral vagus-stimulation group and contralateral vagus-stimulation group.2.3 Results Ipsilateral and contralateral transcutaneous auriculo-vagus-stimulation could both suppress epileptic seizures of rats. The inhibiting rate in ipsilateral vagus-stimulation group (n=16) was 16.29±9.53% and the inhibiting rate in contralateral vagus-stimulation group (n= 16) was 16.29±9.53%(n=11). There is significant difference between the inhibiting rate in the two groups. (P<0.01).3. Effect of different frequency (2Hz,20Hz,100Hz) of Transcutaneous Auriculo-vagus-stimulation on field potentials of epilepsy rats3.1 Animals Experiments were performed on 51 adult male Sprague-Dawley rats weighing 300±29g.3.2 Methods Anesthesia was performed by 10% urethane (1.2 g/kg, i.p.); additional sodium pentobarbital was administered as needed. Four recordings of field potentials (FPs) by microelectrode arrays (2×2,4 channels) were carried out in the right primary somatosensory cortices (AP:±1.0 mm, ML:4-6 mm, DV:0.5-1.0mm). Rats were induced by intraperitoneal injection of PTZ (pentylenetetrazol, PTZ) 60 mg/kg. Stimulation was performed by two iron slice electrodes sticked on auricular concha,with parameter as follows:duration of 30 seconds, intensity of 1 mA. Field potentials was observed and recorded by CerebusTM 5.0 system.According to different durations of simulation,51 Sprague-Dawley rats were divided into four groups:30 seconds group,5 minutes group,10 minutes group and 30 minutes group.3.3 Results3.3.1 Effect of 2Hz transcutaneous auriculo-vagus-stimulation on field potentials of epilepsy rats After 2 Hz stimulation, the inhibiting rate in 30 seconds group (n=10) was 61.51±18.21%; the inhibiting rate in 5 minutes group (n=9) was 10.87±3.1%; the inhibiting rate in 10 minutes group (n=10) was 11.57±13.71%; and the inhibiting rate in 30 minutes group (n=8) was 5.17±4.84%. Compared with 5 minutes group, the inhibiting rate of epilepsy seizure decreased significantly in the others groups (P<0.01).3.3.2 Effect of 20Hz transcutaneous Auriculo-vagus-stimulation on field potentials of epilepsy ratsAfter 20 Hz stimulation, the inhibiting rate in 30 seconds group (n=14) was 58.74±10.10%, in 5 minutes group (n=8) 36.34±13.87%, in 10 minutes group (n=9) 38.53±12.85%, and in 30 minutes group (n=7) 61.36±16.14%. The post anti-epileptic effect in 30 seconds group (n=14) was 149.22±59.80 sec, in 5 minutes group (n=8) 205.00±134.37 sec, in 10 minutes group (n=9) 213.25±122.46 sec, and in 30 minutes group (n=7) 166.00±89.71 sec. All groups could suppress the epileptic seizure, but with no significant difference between them (P>0.05).3.3.3 Effect of 100Hz transcutaneous Auriculo-vagus-stimulation on field potentials of epilepsy ratsAfter 100 Hz stimulation, the inhibiting rate in 30 seconds group (n=10) was 12.58±4.33%, in 5 minutes group (n=8) 36.26±11.92%, in 10 minutes group (n=9) 32.87±14.04%, and in 30 minutes group (n=7) 50.92±17.99%. Compared with 30 seconds group, the inhibiting rate in 30 minutes group increased significantly (P<0.01) with no significant difference between 5 minutes group and 10 minutes group.The post anti-epileptic effect in 30 seconds group (n=10) was 13.54±4.37 sec, in 5 minutes group (n=8) 59.56±10.25 sec, in 10 minutes group (n=9) 55.51±10.04 sec, and in 30 minutes group (n=7) 160.20±58.26 sec. Compared with 30 seconds group, the post anti-epileptic effect increased significantly in the others groups (P<0.05).4. Effect of Different time (30sec,5min, 10min,30min) of Transcutaneous Auriculo-vagus-stimulation on field potentials of epilepsy rats 4.1 Animals Experiments were performed on 51 adult male Sprague-Dawley rats weighing 300±29g.4.2 Methods Anesthesia was performed by 10% urethane (1.2 g/kg, i.p.); additional sodium pentobarbital was administered as needed. Four recordings of field potentials (FPs) by microelectrode arrays (2×2,4 channels) were carried out in the right primary somatosensory cortices (AP:±1.0 mm, ML:4-6 mm, DV:0.5-1.0mm). Rats were induced by intraperitoneal injection of PTZ (pentylenetetrazol, PTZ) 60 mg/kg. Stimulation was performed by two iron slice electrodes sticked on auricular concha,with parameter as follows:duration of 30 seconds, intensity of 1 mA. Field potentials was observed and recorded by CerebusTM 5.0 system.According to different durations of simulation,51 Sprague-Dawley rats were divided into three groups:2Hz group,20Hz group and 100Hz group.4.3 Results4.3.1 Effect of 30 seconds transcutaneous Auriculo-vagus-stimulation on field potentials of epilepsy ratsAfter 30 seconds stimulation, the inhibiting rate in 2Hz group (n=9) was 61.51±18.21%, in 20Hz group (n=14) 58.74±10.10% and in 100Hz group (n=10) 12.58±4.33%. Compared with 100Hz group, the inhibiting rate of epilepsy seizure increased significantly in the others groups (P<0.01). The post anti-epileptic effect in 2Hz group (n=9) was 142.38±61.83 seconds, in 20Hz group (n=14) 149.22±59.80 seconds and in 100 Hz group (n=10) 13.50±4.37. Compared with 100Hz group, other groups possessed good inhibitory effect on epileptic seizure.4.3.2 Effect of 5 minutes transcutaneous Auriculo-vagus-stimulation on field potentials of epilepsy ratsAfter 5 minutes stimulation, the inhibiting rate in 2Hz group (n=9) was 10.87±3.1%, in 20Hz group (n=8) 36.34±13.87% and in 100Hz group (n=8) 36.26±11.92%. Compared with 2Hz group, the inhibiting rate of anti-epileptic effect was increased in the others groups (P<0.05). The post anti-epileptic effect in 2Hz group (n=9) was 19.00±4.43 sec, in 20Hz group (n=14) 205.00±134.37 sec and in 100 Hz group (n=10) 59.56±10.25 sec. Compared with 2Hz group, other groups possessed the good inhibitory effect on epileptic seizure (P<0.05). There was no significant difference between 20Hz group and 100Hz group (P>0.05).4.3.3 Effect of 10 minutes transcutaneous Auriculo-vagus-stimulation on field potentials of epilepsy ratsAfter 10 minutes stimulation, the inhibiting rate in 2Hz group (n=10) was 11.57±13.71%, in 20Hz group (n=9) 38.53±12.85% and in 100Hz group (n=9) 32.87±14.04%. Compared with 2Hz group, the inhibiting rate of anti-epileptic effect increased in the others groups (P<0.05). The post anti-epileptic effect in 2Hz group (n=10) was 16.67±11.00 sec, in 20Hz group (n=9) 213.25±122.46 sec and in 100 Hz group (n=9) 55.51±10.04 sec. Compared with 2Hz group, the others group possessed the good inhibitory effect on epileptic seizure (P<0.05).4.3.4 Effect of 30 minutes transcutaneous Auriculo-vagus-stimulation on field potentials of epilepsy ratsAfter 10 minutes stimulation, the inhibiting rate in 2Hz group (n=8) was 5.17±4.84%, in 20Hz group (n=7) 61.36±16.14% and in 100Hz group (n=7) 50.92±17.99%. Compared with 2Hz group, the inhibiting rate of epilepsy seizure was increased in the others groups (P<0.01). The time of after effect in 2Hz group (n=8) was 11.00±4.95 seconds, in 20Hz group (n=7) 166.00±89.71 seconds and in 100 Hz group (n=7) 160.20±58.26 seconds. Compared with 2Hz group, the other groups possessed the good inhibitory effect on epileptic seizure (P<0.01). There was no significant difference between 20Hz group and 100 Hz group (P>0.05).5. Effect of Transcutaneous Auriculo-vagus-stimulation on field potentials of epilepsy rats after disconnection different regions of auricle5.1 Animals Experiments were performed on 21 adult male Sprague-Dawley rats weighing 300±32g.5.2 Methods Anesthesia was performed by 10% urethane (1.2 g/kg, i.p.); additional sodium pentobarbital was administered as needed. Four recordings of field potentials (FPs) by microelectrode arrays (2×2,4 channels) were carried out in the right primary somatosensory cortices (AP:±1.0 mm, ML:4-6 mm, DV:0.5-1.0mm). Rats were induced by intraperitoneal injection of PTZ (pentylenetetrazol, PTZ) 60 mg/kg. Stimulation was performed by two iron slice electrodes sticked on auricular concha,with parameter as follows:Frequency of 20Hz, duration of 30 seconds, intensity of 1 mA. Field potentials was observed and recorded by CerebusTM 5.0 system. According to disconnection different regions of right auricle,21 Sprague-Dawley rats were divided into two groups, the front disconnection group and the back disconnection group.5.3 ResultsBefore the disconnection different regions of right auricle, the inhibiting rate in the front disconnection group (n=10) was 51.72±9.85% and the rate in back disconnection group was 36.26±3.07%(n=11).There was no significant difference between the front group and the back group (P>0.05). After the disconnection, the inhibiting rate in the front disconnection group (n=10) was 38.87±7.64% and the rate in back disconnection group was 2.15±1.10%(n= 11).Compared with the front disconnection group, the inhibiting rate of epilepsy in the back group was impaired significantly (P<0.01).6. Measure of Vagus Sensory Evoked Potential6.1 Subject of research:A group of 8 healthy subjects (6f,2m; age range 24-34years, all self-reported right-handers)6.2 Process of experiment:The work station with 64 channels for the electroencephalograms and the inducting electric potential (NeuroScanTM) was used to record the EEG of the subjects in the whole process of the stimulation to the auricular branch of vagus nerve. The stimulator providing stimulation to the auricular branch of vagus nerve was used to trigger it externally and mark the stimulation signals on the EEG synchronously. According to the time point of signals marked, the data of EEG before or after the stimulation was divided,folded and analyzed, providing and presenting the appeared induction electric potential by somatic sensation of the vagus nerve concerned with the auricular branch of vagus nerve. 6.3 Result:In the process of stimulation to the auricular branch of vagus nerve, we can get a stable inducting Evoked potential (EP). This EP consisted of a positive deflection of about 1.3μV occurring about 2.4ms after stimulation and a lasting about 2ms measured between electrodes C4 and F4. We can also get the electric potential variety between the electrodes Fz and F4. No Clear EP was measured in the recording of C3 vs F3.The two dissimilarity individual samples presented by the inducting EP can show the feature of an obvious composition with stable wave form and fixed incubation period and same distribution. The same EP could not be acquired by presenting the stimulation on other parts in the ear.ConclusionThis research mainly investigated the mechanism of transcutaneous auriculo-vagus-stimulation for the treatment of epilepsy by using electrophysiological techniques. it was showed that stimulation at the auricular branch of vagus nerve of the rat can suppress epileptic seizures. This kind of function can strengthen the function of parasympathetic activities by activating the activities of the NTS and thus alter the desynchronization process of the brain electricity, producing the effect of repressing the epilepsy. The effect of the auricular branch of vagus nerve on the same side is much better than that on the other side which is consistent with the projecting laws of the NTS in neurotomy. After cutting off the homologous nerve, the anti-epileptic effect of stimulation at the auricular branch of vagus nerve disappeared. It perhaps can be explained that simulation of the auricular branch of vagus nerve can suppress the epilepsy through the nerve media. This research finds that among all the stimulations at different frequencies, the stimulation at 20 Hz can optimally repress the epilepsy of rat compared with the stimulations in other frequencies. As to the different duration time of the stimulation to the auricular branch of vagus nerve, the influenced effect to repressing the epilepsy of rat is not so obvious. The result mentioned above can provide the theory to support the clinical application as to stimulate the auricular branch of vagus nerve to cure epilepsy.
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