【作者】 王晓宇；

【导师】 朱兵；

【作者基本信息】 中国中医科学院 ， 中西医结合基础， 2010， 博士

【摘要】 耳针刺激耳甲区域能激活迷走神经增强副交感神经系统兴奋性。本课题组研究结果表明耳针刺激耳甲区能更好地激活孤束核和迷走神经背核神经元放电,产生耳迷走效应,调整心率和血压,并促进胃运动；同时在对糖尿病的降糖效应、高血压病的降压作用的研究中也已经取得了不少的研究成果。癫痫是一种脑部疾病,其特点是脑部有持续存在的癫痫反复发作的易感性,以及由于这种疾病引起的神经生化、认知、心理和社会后果；癫痫具体表现是反复发作的神经元异常放电所致的暂时性中枢神经系统功能失常。根据有关神经元的部位和放电扩散的范围的不同,功能失常可能分为运动、感觉、意识、行为、自主神经等不同障碍,或混合存在。治疗癫痫病的主要手段为药物控制,或是手术治疗(切除致痫灶本身或切断其传导通路)。除此之外还有迷走神经刺激(vagus nerve stimulation, VNS)疗法。VNS疗法的作用机制多数认为是通过激活迷走神经投射到NTS的通路,通过NTS与脑内的广泛联系达到去同步化脑电来完成抗癫痫作用的。在本课题组之前的研究中发现,耳针刺激耳甲区域(耳迷走神经刺激),具有与VNS相类似的神经投射规律,同样能抑制癫痫发作,具有较好的抗癫痫效应。如果耳迷走神经刺激能替代迷走神经刺激疗法,就可以解决VNS手术并发症和费用昂贵等问题,为癫痫病的治疗提供便利、经济的方法。本课题将在前期研究基础上从动物实验及人体试验两方面研究耳迷走神经刺激抑制癫痫发作的效应机制及其刺激参数等,进一步探讨耳迷走刺激抑制癫痫的作用机理,为临床应用提供理论依据。实验1耳迷走神经刺激对癫痫模型大鼠大脑皮层场电位的影响1.1实验动物健康成年雄性Sprague-Dawley大鼠14只,体重300±49g,清洁级,由中国医学科学院实验动物中心提供。1.2实验过程大鼠14只,10%乌拉坦腹腔注射麻醉(1.2-1.4g／kg)。俯卧,头部用耳棒固定于脑立体定位仪上。头顶部备皮,沿正中矢状线切开皮肤,分离皮下组织,刮除骨膜,将前后囱调至同一水平。根据大鼠脑立体定位图谱(Paxinos和Watson,1998),在距正中线右侧4-6mm,前囟前后1mm处用颅骨钻开窗后,显微镜下剥离硬脑膜,将微电极阵列(microelectrode arrays, MEA)放置在大鼠右侧大脑皮层初级躯体感觉皮层(primary somatosensory cortices, S1)区。用微电极推进器推进至皮层内0.5-1.0mm,参考电极用合金螺丝钉铆在距电极阵列3mm的颅骨上,皮层表面用37℃液体石蜡覆盖保湿。MEA另一端用headstage接于微电极放大器,放大的电信号经CerebusTM 5.0数据采集分析系统采集和分析。给予大鼠腹腔注射戊四氮(pentylenetetrazol, PTZ) 60 mg/kg造成急性癫痫模型。对大鼠进行30秒、20Hz耳迷走神经刺激,观察耳迷走刺激对癫痫模型大鼠大脑皮层场电位的变化。1.3实验结果采用电极阵列记录癫痫造模前大鼠正常皮层场电位,表现为散在的棘波,没有脑神经元异常和过度超同步化放电；造模后耳迷走神经刺激前,大鼠皮层场电位出现变化,表现为脑神经元异常和过度超同步化放电引起的高振幅的癫痫波。在耳迷走神经刺激后,大鼠癫痫发作持续时间减少。给予癫痫模型大鼠(n=14)耳迷走神经刺激后,单位时间(120sec)内发作时间从101.55±6.39秒,减少到58.5±10.25秒(P<0.01),抑制率为58.74±10.10%。耳迷走神经刺激前后单位时间内大鼠皮层场电位中癫痫波持续时间比较,差异具有统计学意义,说明耳迷走神经刺激能明显抑制大鼠癫痫发作。实验2左右耳迷走神经刺激对癫痫模型大鼠大脑皮层场电位的影响2.1实验动物健康成年雄性Sprague-Dawley大鼠27只,体重300±34g,清洁级,由中国医学科学院实验动物中心提供。2.2实验过程实验动物用10%乌拉坦腹腔注射麻醉(1.2-1.4g/kg)。俯卧,头部用耳棒固定于脑立体定位仪上。头顶部备皮,沿正中矢状线切开皮肤,分离皮下组织,刮除骨膜,将前后囱调至同一水平。根据大鼠脑立体定位图谱(Paxinos和Watson,1998),在距正中线右侧5-6mm,前囟前后1mm处用颅骨钻开窗后,显微镜下剥离硬脑膜,将MEA放置在大鼠右侧大脑S1区。用微电极推进器推进至皮层内0.5-1.0mm,参考电极用合金螺丝钉铆在距电极阵列3mm的颅骨上,皮层表面用37℃液体石蜡覆盖保湿。MEA另一端用headstage接于微电极放大器,放大的电信号经CerebusTM5.0数据采集分析系统采集和分析。给予大鼠腹腔注射PTZ 60 mg/kg造成急性癫痫模型。将27只大鼠随机分为两组,同侧刺激组11只,对侧刺激组16只,经PTZ造模成功后,分别在左右两侧耳甲腔区域给予耳迷走神经刺激,观察进行30秒、20Hz耳迷走神经刺激后,两组癫痫模型大鼠大脑皮层场电位的变化。2.3实验结果在两侧耳甲腔给予20 Hz,强度1 mA,脉宽500μm,持续时间30s的耳迷走神经刺激均可减少癫痫模型大鼠癫痫发作持续时间。其中,在单位时间(120秒)内,对侧刺激组(n=16)刺激前后,癫痫波持续时间分别为116.00±3.21秒和88.67±6.43秒,癫痫发作减少了16.29±9.53%；同侧刺激组(n=11)刺激前后,癫痫波持续时间分别为113.83±2.47秒和61.867±16.79秒,癫痫发作减少了58.47±10.24%,两组变化相比具有显著差异性(P<0.01),同侧耳迷走神经刺激效应明显优于对侧。实验3观察不同频率(2Hz、20Hz和100Hz)耳迷走神经刺激对癫痫模型大鼠异常皮层场电位影响的差异3.1实验动物健康成年雄性Sprague-Dawley大鼠51只,体重300±29g,清洁级,由中国医学科学院实验动物中心提供。3.2实验过程手术过程中动物体温用计算机温度时间控制仪维持在37℃。10%乌拉坦腹腔注射麻醉(1.2g／kg)。俯卧,头部用耳棒固定于脑立体定位仪上。头顶部备皮,沿正中矢状线切开皮肤,分离皮下组织,刮除骨膜,将前后囱调至同一水平。根据大鼠脑立体定位图谱(Paxinos和Watson,1998),在距正中线右侧5-6mm,前囟前后1mm处用颅骨钻开窗后,显微镜下剥离硬脑膜,将MEA放置在大鼠右侧大脑皮层S1。用微电极推进器推进至皮层内0.5-1.0mm,参考电极用合金螺丝钉铆在距电极阵列3mm的颅骨上,皮层表面用37℃液体石蜡覆盖保湿。MEA另一端用headstage接于微电极放大器,放大的电信号经CerebusTM 5.0数据采集分析系统采集和分析。选择不同的刺激频率,将51只癫痫模型大鼠根据刺激持续时间不同随机分为4组：30秒组、5分钟组、10分钟组和30分钟组,观察这4组大鼠在刺激后大脑皮层场电位的变化和癫痫发作情况的差异。3.3实验结果3.3.1 2Hz频率不同刺激持续时间的耳迷走神经刺激对癫痫模型大鼠电位影响的比较给予不同刺激持续时间的2Hz频率的耳迷走神经刺激后,在单位时间内(120sec),刺激持续时间30秒组抑制率最高,与其他刺激持续时间组相比,差异具有统计学意义(P<0.01)；刺激5分钟、10分钟和30分钟的3个组之间相比较,其差异没有统计学意义(P>0.05)。给予癫痫模型大鼠2Hz的耳迷走神经刺激后,不同持续时间的刺激抑制癫痫发作的效应有所不同。刺激结束后5分钟内,每隔30秒钟观察统计耳迷走神经刺激抑制癫痫发作的情况,发现：在2Hz不同刺激持续时间的耳迷走神经刺激后,30秒刺激组与其他刺激时间组相比,在刺激后30和60秒钟时间段癫痫发作率明显降低,表现出了很好的抑制发作的效应,差异具有统计学意义(P<0.01)；在刺激后90秒钟时间段,30秒刺激组与其他刺激时间组相比,同样表现出较好的抑制发作的效应,差异具有统计学意义(P<0.05)。说明,在2Hz的耳迷走刺激中,持续时间为30秒的刺激产生的抑制癫痫发作的效应优于其他刺激持续时间组。3.5.2 20Hz频率不同刺激持续时间的耳迷走神经刺激对癫痫模型大鼠电位影响的比较给予20Hz频率不同刺激持续时间的耳迷走神经刺激后,在单位时间内(120sec),刺激30秒组、刺激5分钟、10分钟组和刺激30分钟组抑制率分别为：58.74±10.10%、36.34±13.87%、38.53±12.85%和61.36±16.14%,这四组间的差异不具有统计学意义(P>0.05)。比较刺激后癫痫模型大鼠癫痫波被抑制的后效应时间,刺激30秒组、刺激5分钟、10分钟组和刺激30分钟组后效应持续时间分别为：149.22±59.80秒、205.00±134.37秒、213.25±122.46秒、166.00±89.71秒,这四组间的差异同样不具有统计学意义(P>0.05)。给予癫痫模型大鼠20Hz耳迷走刺激后,观察不同持续时间的刺激抑制癫痫发作的效应的差异。以30秒钟为单位时间,分段统计刺激结束后5分钟内十个时间段中大鼠癫痫发作率。30秒组、5分钟组、10分钟和30分钟组在各时间段中癫痫发作率下降,均表现出了明显的抑制发作的效应,在不同的时间段的组间相比中,4组间差异不具有统计学意义(P>0.05)。说明,在20Hz的耳迷走刺激中,刺激持续时间长短对癫痫发作的抑制效应的影响不明显。3.5.3 100Hz频率不同刺激持续时间耳迷走神经刺激对癫痫模型大鼠皮层电位影响的比较给予100Hz频率不同刺激持续时间的耳迷走神经刺激后,在单位时间内(120sec),刺激30秒组的抑制率为12.58±4.33%、30分钟组抑制率为50.92±17.99%,两者相比差异具有统计学意义(P<0.01)；5分钟组抑制率为36.26±11.92%、10分钟组抑制率为32.87±14.04%,分别与30秒组和30分钟组相比其抑制率差异没有统计学意义(P>0.05)。比较不同时间刺激对大鼠癫痫发作抑制的后效应的差别,刺激30分钟组与其它的三个刺激时间组相比,具有最长的后效应,差异具有统计学意义(P<0.05)；刺激30秒组和30分钟组间相比较,差异具有统计学意义(P<0.05)；刺激5分钟和10分钟组与30秒组相比,差异没有统计学意义(P>0.05)；5分钟和10分钟的两组之间相比较,其差异也没有统计学意义(P>O.05)。刺激30秒组和其他不同刺激时间组相比,具有最短的后效应。3.5.4不同频率的耳迷走神经刺激对大鼠癫痫发作影响的比较2Hz低频率刺激在30秒短时间刺激后,对大鼠癫痫发作的抑制率为61.51±18.21%,和同为低频刺激的其他时间组相比具有更明显的抑制作用,与其余各组之间比较差异具有统计学意义(P<0.01)。100Hz高频率刺激在30秒短时间刺激后,对大鼠癫痫发作的抑制率为12.58±4.33%,和同为高频刺激的其他时间组相比其抑制作用最不明显,各组之间具有差异性(P<0.01)。20Hz的刺激在30秒、5分钟、10分钟、30分钟刺激持续时间下均有良好的抑制作用,其抑制率分别为：58.74±10.10%、36.34±13.87%、38.53±12.85%、61.36±16.14%,四组数据间的差异不具有统计学意义(P>0.05)。实验4观察不同刺激持续时间(30秒、5分钟、10分钟和30分钟)耳迷走神经刺激对癫痫模型大鼠异常皮层场电位影响的差异4.1实验动物健康成年雄性Sprague-Dawley大鼠51只,体重300±29g,清洁级,由中国医学科学院实验动物中心提供。4.2实验过程手术过程中动物体温用计算机温度时间控制仪维持在37℃。10%乌拉坦腹腔注射麻醉(1.2g／kg)。俯卧,头部用耳棒固定于脑立体定位仪上。头顶部备皮,沿正中矢状线切开皮肤,分离皮下组织,刮除骨膜,将前后囱调至同一水平。根据大鼠脑立体定位图谱(Paxinos和Watson,1998),在距正中线右侧5-6mm,前囟前后1mm处用颅骨钻开窗后,显微镜下剥离硬脑膜,将MEA放置在大鼠右侧大脑皮层S1。用微电极推进器推进至皮层内0.5-1.0mm,参考电极用合金螺丝钉铆在距电极阵列3mm的颅骨上,皮层表面用37℃液体石蜡覆盖保湿。MEA另一端用headstage接于微电极放大器,放大的电信号经CerebusTM5.0数据采集分析系统采集和分析。选择不同的刺激频率,将51只癫痫模型大鼠根据刺激频率不同随机分为3组：2Hz、20Hz和100Hz,观察这3组大鼠在刺激后大脑皮层场电位的变化和癫痫发作情况的差异。4.3实验结果4.3.1刺激持续时间为30秒条件下,不同频率的耳迷走神经刺激对癫痫模型大鼠场电位影响的比较给予癫痫模型大鼠30秒不同刺激频率耳迷走神经刺激后,在单位时间内(120sec),2Hz刺激组、20Hz刺激组中对癫痫发作的抑制率为61.51±18.21%、58.744±10.10%,与100Hz组的抑制率12.58±4.33%相比,2Hz和20Hz组抑制率更高,差异具有统计学意义(P<0.01)。2Hz组、20Hz组中癫痫发作抑制的后效应持续时间为142.38±61.83秒、149.22±59.80秒,与100Hz组的抑制后效应时间13.50±4.37秒相比,2Hz和20Hz组具有更好的抑制癫痫发作的后效应,差异具有统计学意义(P＜0.01)。给予癫痫模型大鼠30秒不同刺激频率耳迷走神经刺激后,观察不同刺激对癫痫发作抑制效应的差异性。以30秒钟为单位时间,分段统计刺激结束后5分钟内十个时间段中大鼠癫痫发作率：刺激后120秒内,2Hz和20Hz组发作率下降,100Hz组刺激后发作率变化不大,其差异具有显著的统计学意义(P<0.01)；在刺激后120秒至210秒时间段,2Hz和20Hz组癫痫发作率低于100Hz组,差异具有统计学意义(P<0.05)；在刺激后240秒至300秒时间段,2Hz和20Hz组中单位时间癫痫发作率升高,和100Hz组相比差异不具有统计学意义(P>0.05)。4.3.2刺激持续时间为5分钟条件下,不同频率的耳迷走神经刺激对癫痫模型大鼠场电位影响的比较给予癫痫模型大鼠5分钟不同刺激频率耳迷走神经刺激后,在单位时间内(120sec),20Hz组、100Hz组对癫痫发作的抑制率分别为36.34±13.87%、36.26±11.92%,与2Hz组的抑制率10.87±3.1%相比,差异具有统计学意义(P<0.05)。20Hz组、100Hz组对癫痫发作抑制的后效应持续时间分别为205.00±134.37秒、59.56±10.25秒,与2Hz组的抑制后效应持续时间19.00±4.43秒相比,20Hz和100Hz的刺激具有更好的抑制癫痫发作的后效应,差异具有统计学意义(P<0.01)。给予癫痫模型大鼠5分钟不同刺激频率的耳迷走神经刺激后,各组抑制癫痫发作的效应不尽相同。以30秒钟为单位时间,分段统计刺激结束后5分钟内十个时间段中大鼠癫痫发作率。在5分钟刺激持续时间条件下,刺激后90秒内,20Hz组较2Hz组发作率下降,对癫痫发作的抑制作用较强,差异具有统计学意义(P<0.05)。在其余时间段中,2Hz、20Hz和100Hz组发作率相比没有统计学意义(P>0.05)。4.3.3刺激持续时间为10分钟条件下,不同频率的耳迷走神经刺激对癫痫模型大鼠场电位影响的比较给予癫痫模型大鼠10分钟不同刺激频率耳迷走神经刺激后,在单位时间内(120sec),20Hz组、100Hz组对癫痫发作的抑制率为38.53±12.85%、32.87±14.04%,与2Hz组的抑制率11.57±13.71%相比,差异具有统计学意义(P<0.05)。20Hz组、100Hz组对癫痫发作抑制的后效应持续时间为213.25±122.46秒、55.51±10.04秒,与2Hz组的后效应持续时间16.67±11.00秒相比,20Hz和100Hz组具有更好的抑制癫痫发作的后效应,差异具有显著统计学意义(P<0.01)。给予癫痫模型大鼠10分钟不同刺激频率耳迷走神经刺激后,不同频率刺激抑制癫痫发作的效应有所不同。以30秒钟为单位时间,分段统计刺激结束后5分钟内十个时间段中大鼠癫痫发作率,在10分钟刺激持续时间条件下,刺激后30秒内,20Hz组较2Hz组发作率下降,对癫痫发作的抑制作用较强,差异具有统计学意义(P<0.01)；刺激后30至60秒内,20Hz组较2Hz组发作率下降,对癫痫发作的抑制作用较强,差异具有统计学意义(P<0.05)。在其余时间段中,2Hz、20Hz和100Hz组发作率比较高,组间相比没有统计学意义(P>0.05)。4.3.4刺激持续时间为30分钟条件下,不同频率的耳迷走神经刺激对癫痫模型大鼠场电位影响的比较给予癫痫模型大鼠30分钟不同刺激频率耳迷走神经刺激后,在单位时间内(120sec),20Hz组、100Hz组对癫痫发作的抑制率为61.36±16.14%、50.92±17.99%,与2Hz组的抑制率5.17±4.84%相比,差异具有统计学意义(P<0.01)。20Hz组、100Hz组对癫痫发作抑制的后效应持续时间为166.00±89.71秒、160.20±58.26秒,与2Hz组的后效应持续时间11.00±4.95秒相比,20Hz和100Hz组具有更好的抑制癫痫发作的后效应,差异具有统计学意义(P<0.01)。给予癫痫模型大鼠10分钟不同刺激频率耳迷走神经刺激后,不同频率抑制癫痫发作的效应有所不同。以30秒钟为单位时间,分段统计刺激结束后5分钟内十个时间段中大鼠癫痫发作率,在30分钟刺激持续时间条件下,刺激后60秒内,20Hz组较2Hz组发作率下降,对癫痫发作的抑制作用较强,差异具有统计学意义(P<0.05)；刺激后90至180秒,100Hz组较2Hz组发作率下降,对癫痫发作的抑制作用较强,差异具有统计学意义(P<0.05)。在其余时间段中,2Hz、20Hz和100Hz组发作率相比没有统计学意义(P>0.05)。4.3.5不同刺激持续时间的耳迷走神经刺激对大鼠癫痫发作影响的比较30秒刺激后,100Hz组对大鼠癫痫发作的抑制率为12.58±4.33%,和同为短期刺激的其他组相比抑制作用较不明显,差异具有统计学意义(P<0.01)。2Hz低频刺激在5分钟、10分钟和30分钟刺激后,对大鼠癫痫发作的抑制率为10.87±3.1%、11.57±13.71%、5.17±4.84%,其抑制作用最不明显,和20Hz和100Hz各组相比具有显著的差异性(P<0.01)。20Hz刺激在30秒、5分钟、10分钟、30分钟刺激持续时间下均表现出良好的抑制作用,其抑制率分别为：58.74±10.10%、36.344±13.87%、38.53±12.85%、61.36±16.14%,四组数据间的差异不具有统计学意义(P＞0.05)。100Hz高频率刺激在30秒刺激后,大鼠癫痫发作抑制的后效应持续时间为13.544±4.37秒,和同为短期刺激的其他时间组相比抑制作用不明显,差异具有统计学意义(P<0.01)。2Hz低频率刺激在5分钟、10分钟、30分钟刺激后,大鼠癫痫发作抑制的后效应持续时间为19.00±4.43秒、16.67±11.00秒、11.00±4.95秒,和其他刺激频率组相比其抑制作用最不明显,差异具有统计学意义(P<0.01)；20Hz的刺激在30秒、5分钟、10分钟、30分钟刺激持续时间下均有良好的抑制作用,刺激后大鼠癫痫发作抑制的后效应持续时间分别为：149.22±59.80秒、205.00±134.37秒、213.25±122.46秒、166.00±89.71秒,四组数据间的差异不具有统计学意义(P>0.05)。不同持续时间的耳迷走神经刺激,对大鼠癫痫发作的抑制效应的影响差别不大,高频率的短期刺激和低频频率的长期刺激,对大鼠癫痫发作的抑制效应均不明显。实验5切断大鼠耳廓不同部位,观察耳迷走神经刺激对癫痫模型大鼠异常皮层场电位影响的差异及切断部位的神经分布情况5.1实验动物健康成年雄性Sprague-Dawley大鼠27只,体重300±32g,清洁级,由中国医学科学院实验动物中心提供。5.2实验过程手术过程中动物体温用计算机温度时间控制仪维持在37℃。10%乌拉坦腹腔注射麻醉(1.2g／kg)。俯卧,头部用耳棒固定于脑立体定位仪上。头顶部备皮,沿正中矢状线切开皮肤,分离皮下组织,刮除骨膜,将前后囱调至同一水平。根据大鼠脑立体定位图谱(Paxinos和Watson,1998),在距正中线右侧5-6mm,前囟前后1mm处用颅骨钻开窗后,显微镜下剥离硬脑膜,将MEA放置在大鼠右侧大脑皮层S1。用微电极推进器推进至皮层内0.5-1.0mm,参考电极用合金螺丝钉铆在距电极阵列3mm的颅骨上,皮层表面用37℃液体石蜡覆盖保湿。MEA另一端用headstage接于微电极放大器,放大的电信号经CerebusTM5.0数据采集分析系统采集和分析。将21只大鼠随机分为两组,其中11只在记录癫痫模型大鼠大脑皮层场电位的过程中,沿大鼠耳根部,(将右侧外耳和躯体连接的部位分为长度相等的5份),切断外耳与躯体连接部位的后5／2处,另11只切断外耳与躯体连接部位的前5／2处,使用频率20 Hz,强度1mA,脉宽500μm,持续时间30秒为刺激参数,在右侧耳甲腔区域给予耳迷走神经刺激,观察切断外耳不同部位后耳迷走神经刺激对癫痫模型大鼠大脑皮层场电位的影响。5.3结果在切断大鼠耳根后2／5前,右侧耳迷走神经刺激可以减少大鼠癫痫发作持续时间。其中,在单位时间(120秒)内,耳迷走神经刺激前后(n=11),发作持续时间分别为118.29±4.3秒和78.4±7.66秒,两者相比其差别具有统计学意义(P<0.01),抑制率为31.26±3.07%。在切断大鼠耳根后2／5后,右侧耳迷走神经刺激减少大鼠癫痫发作的效应消失。在切断大鼠耳根后2／5后,在单位时间(120秒)内,右侧耳迷走神经刺激前后(n=11),发作持续时间分别为116.44±6.35秒和113.76±3.28秒,前后相比没有统计学意义(P>0.05),抑制率为2.15±1.10%。切断前后抑制率相比其差别具有统计学意义(P<0.01)。在切断大鼠耳根前2／5前,右侧耳迷走神经刺激可以减少大鼠癫痫发作持续时间。其中,在单位时间(120秒)内,耳迷走神经刺激刺激前后(n=10),发作持续时间分别为111.6±2.74秒和52.7±12.02秒,两者相比其差别具有统计学意义(P<0.01),抑制率为51.72±9.85%。在切断大鼠耳根前2／5后,在单位时间(120秒)内,耳迷走神经刺激前后(n=10),发作持续时间分别为110.38±3.38秒和66.625±7.26秒,抑制率为38.87±7.64%。切断前后抑制率相比其差别不具有统计学意义(P>0.05)。从耳后开口,解剖观察切割耳廓后2／5处的神经分布情况,可以明显观察到该区域有两支较大的神经干走行。6.人体迷走体感诱发电位的测量6.1研究对象选取研究所内职工及学生,询问其病史和健康状况,确认没有神经疾患史、心理疾患史及心血管系统疾患史、没有服用能影响中枢神经系统功能的药物、听力正常的实验者共8人,年龄24-34岁,2男6女,均自述为右利手。6.2试验过程采用NeuroScan公司生产的64通道脑电图及诱发电位工作站记录受试者在接受耳迷走神经刺激时全程的脑电图,由提供耳迷走神经刺激的刺激器外触发同步在脑电图上标记刺激信号,根据标记到的信号时间点对刺激前后的脑电图数据进行分段、叠加和平均,呈现出与耳迷走神经刺激相关的迷走体感诱发电位。6.3结果在给予右侧耳迷走神经刺激的过程中,在双极导联C4-F4间得到一组相对稳定的诱发电位,其中包含一个出现在刺激后2.4ms的负偏差(1.3微伏),并持续2ms。在双极导联Fz-F4间得到电位变化也是一个正的偏差(小于1微伏),出现在刺激后1.7ms处；一个负向的成分随后出现在2.6ms处；这个诱发电位在3.9ms处以一个正向的偏差作为结束。同时在对侧C3-F3之间没有记录到明显的诱发电位。这个诱发电位的呈现在两个不同个体的样本中表现出成分明显,波形稳定,潜伏期固定,同时分布相同的特性。在耳朵的其他部位的刺激,没有出现相同电位变化。二结论本项研究主要从电生理的角度探讨了耳针刺激耳甲区域(耳迷走神经刺激)治疗癫痫的效应机制。实验结果表明：癫痫的发病表现脑电图的高幅癫痫波,耳迷走神经刺激能抑制癫痫大鼠发作时异常的场电位变化。这种作用可能是通过激活NTS的活动来增强副交感的功能从而去同步化脑电来完成的。同侧耳迷走神经刺激的效应明显优于对侧,也是符合耳部神经与NTS之间的联系,以及NTS在神经解剖学上的投射规律的。同时切断相应的神经后,耳迷走神经刺激的效应消失,说明耳迷走神经刺激的抑制癫痫效应是由神经介导来完成的。同时本研究发现在不同的刺激频率下,20Hz刺激相对于其他频率刺激来说,在各个不同的刺激持续时间中都能对大鼠癫痫发作起到最好的抑制作用。不同持续时间的耳迷走神经刺激,对大鼠癫痫发作的抑制效应的区别不是特别明显。以上结果为耳迷走神经刺激治疗癫痫在临床应用提供理论依据。本研究对人体迷走体感诱发电位的测量进行了摸索。在数名受试者进行耳迷走神经刺激过程中,采集到了一组相对稳定的诱发电位。为下一步在人体进行相关研究提供了一种新的方法和思路。更多还原

【Abstract】 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.更多还原