耳—迷走反射及耳针抗癫痫的效应机制研究
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摘要
刺激外耳道或耳甲区激活迷走神经耳支引起耳-心反射、耳-肺反射,类似于副交感紧张效应的临床报道很多。本课题前期的研究结果从形态学、电生理学、生物化学等方面证实了耳甲到孤束核(nucleus of solitary tract,NTS)的纤维投射,电针耳甲还可以通过激活NTS神经元的放电来增强副交感的效应,如降低血压、降低血糖、增强胃运动等。
癫痫是一种由不同因素引起的以反复发作性大脑功能失调为表现的神经系统疾病。自美国FDA于1997年7月正式批准迷走神经刺激(vagus nervestimulation,VNS)疗法作为12岁以上药物难治性部分发作性癫痫的一种辅助疗法以来,这种治疗方法被全世界范围患者所应用。虽然其作用机制仍不是十分清楚,多数的观点认为VNS可能是通过激活迷走神经投射到NTS的通路,然后通过NTS与脑内的广泛联系达到去同步化脑电,抑制癫痫的目的。已有研究提示,癫痫的发病可能与自主神经系统功能的失调有关,表现为交感神经系统功能的亢进,副交感神经系统功能低下。VNS则可能通过刺激迷走神经增强副交感的效应而抑制癫痫。
有研究报道在颈部运用经皮迷走神经电刺激治疗癫痫取得疗效,而且经皮刺激迷走神经耳支支配的耳甲区也可以治疗癫痫,中医耳针治疗癫痫选穴也以耳甲区的穴位为主。由此,我们设想,耳针可能通过刺激迷走神经耳支发挥抑制癫痫的效应。
本研究从行为学、电生理学等角度分别观察了耳针对癫痫大鼠行为学表现,NTS神经元放电,硬膜外脑电图(electroencephalogram,EEG)的影响,以及局部冷冻NTS对耳针抗癫痫效应的影响,和应用交感兴奋剂、拮抗剂对癫痫大鼠EEG的影响,试图从耳-迷走反射的角度探讨耳针抑制癫痫的作用机制。
一动物实验
实验1耳针预处理对戊四氮致癫痫大鼠行为学表现的影响
1.1实验动物
健康成年Sprague-Dawley雄性大鼠42只,体重300±28g,由中国医学科学院实验动物中心提供,清洁级。
1.2实验过程
将动物分为3组。模型组(n=14)给予大鼠腹腔注射戊四氮(pentylenetetrazol,PTZ)60 mg/kg造成急性癫痫模型,后观察30 min大鼠行为学的表现,耳甲治疗组(n=15)和大椎治疗组(n=13)分别在造模前电针耳甲或“大椎”穴30 min,后立即腹腔注射PTZ,观察30 min大鼠行为学的表现。行为学表现根据Racine分级标准进行评分。电针刺激参数:强度,1 mA;频率,20 Hz;脉宽,500μs;刺激30 s,间隔5 min,持续30 min。
1.3实验结果
模型组大鼠腹腔注射PTZ 40s左右出现癫痫发作,开始表现为小发作,如凝视,点头运动,双前肢抬起,阵挛等,1-2 min出现大发作,表现为后肢伸直,抽搐,跌倒伴翻滚,持续约9.5 s,PTZ后30 min内大鼠有1-2次大发作,多次小发作。与模型组比较,大椎治疗组和耳甲治疗组动物大发作潜伏期(即从腹腔注射PTZ到大鼠出现第一次大发作的时间)延长(P＜0.05,P＜0.01),行为学评分减少(P＜0.05,P＜0.01)。与大椎治疗组比较,除了在首次大发作持续时间上无显著差异(P＞0.05),耳甲治疗组大发作潜伏期延长(P＜0.01),行为学评分亦减少(P＜0.01)。结果提示:针刺预处理可以抑制大鼠癫痫发作,而且电针耳甲抑制癫痫的效应优于电针“大椎”穴的效应。
实验2耳针对癫痫大鼠孤束核神经元细胞外放电和脑电图的影响
2.1实验动物
健康成年雄性Sprague-Dawley大鼠45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
手术及实验过程中动物体温用计算机温度时间控制仪维持在37℃。
大鼠麻醉后仰卧,颈部正中切口,游离左侧迷走神经干1cm,将自制C型双极银质电极刺激端无张力地钩于迷走神经干上用以刺激迷走神经,缝合伤口。然后俯卧,将大鼠头部用耳棒固定于脑立体定位仪上,根据大鼠脑立体定位图谱(Paxinos和Watson,1998),在前囟前(A:1.0 mm,L:1.0 mm)和前囟后(A:-1.0 mm,L:1.0 mm)及NTS(A:-11.3～-14.3 mm,L:0～2.3 mm,H:4～7 mm)对应颅骨表面,用颅骨钻钻孔,暴露硬脑膜。双银球电极置于前囟前、后各一小孔内硬脑膜上用以记录EEG。显微镜下分离NTS对应处小孔硬脑膜和软脑膜,暴露小脑皮层,玻璃微电极(阻抗10-20 MQ)记录NTS神经元放电。
手术完毕一小时待大鼠状态稳定后开始记录。用微推进器推进玻璃微电极寻找到自发放电的NTS神经元,上下调节推进器位置,至信噪比较大时停止推进。记录30 s背景放电,待细胞放电频率稳定后,给予耳甲刺激15 s,若细胞放电频率出现变化,确定为对耳甲刺激有反应神经元。在细胞放电频率稳定后,给予大鼠腹腔注射PTZ(40-60 mg/kg)造模,以造模后5 min内EEG出现高幅的癫痫波为造模成功。分别观察造模前后,刺激迷走神经(vagus nerve,VN)、耳甲腔、耳甲艇、耳尖、耳郭外缘中点、耳垂、“大椎”穴、“丰隆”穴前后NTS神经元放电频率和EEG的变化。每一次操作在EEG恢复稳定后重复刺激程序,一只动物每组穴位刺激最多重复3次。刺激方法包括经皮电刺激(transcutaneous electrical nerve stimulation,TENS),电针刺激(electroacupuncture,EA)或手针刺激(manual acupuncture,MA)。TENS或EA参数:强度,1 mA;频率,20 Hz;脉宽,500μs;刺激30 s,间隔5 min,持续30 min。MA采用平补平泻,均匀捻转。针刺时间,30 s。
2.3实验结果
2.3.1造模前后孤束核神经元放电频率和脑电图的同步变化
造模前后共观察到31个细胞。造模后26个细胞放电频率降低(83.87%),3个细胞放电频率不变(9.68%),2个细胞放电频率增多(6.45%)。经统计学处理:NTS细胞放电频率造模前为9.48±0.76个/秒,造模后为4.69±0.36个/秒。其中26个放电频率降低的细胞,其放电频率的变化和脑电图的变化存在同步性,即在细胞放电频率降低时,EEG出现高幅癫痫波,当细胞放电频率增加时,EEG癫痫波消失或振幅降低。说明当NTS细胞放电频率减少时,动物癫痫发作,NTS细胞放电频率增多时,癫痫的发作消失或减少。这种同步变化在造模后1-3分钟之内开始出现,呈节律性变化,按EEG每出现一次癫痫波记为一次癫痫发作,最初发作3次/分,2秒/次,约7-10分钟内持续增多,在约15分钟后,呈基本有规律的变化,平均发作约6.21±0.48次/分,持续时间4.32±0.23秒/次,每分钟发作总的持续时间为26.83±2.25秒。
2.3.2 TENS对NTS放电频率影响的统计结果
TENS(n=31)后,放电频率从平均4.53±0.35个/秒增加到7.50±0.63个/秒,增加了2.97±0.18个/秒(P＜0.01);刺激耳甲腔(n=29)后,放电频率从4.66±0.41个/秒增加到7.64±0.72个/秒,增加了2.98±0.14个/秒(P＜0.01);刺激耳甲艇(n=32)后,放电频率从4.71±0.38个/秒增加到7.61±0.62个/秒,增加了2.90±0.15个/秒(P＜0.01);刺激耳垂(n=28)后,放电频率从4.69±0.48个/秒增加到5.72±0.56个/秒,增加了1.03±0.08个/秒(P＜0.01);刺激耳郭外缘中点(n=26)后,放电频率从4.72±0.34个/秒增加到5.40±0.42个/秒,增加了0.68±0.04个/秒(P＜0.05);刺激耳尖(n=25)后,放电频率从4.59±0.31个/秒增加到5.42±0.53个/秒,增加了0.83±0.05个/秒(P＜0.05);刺激“大椎”(n=28)后,放电频率从4.67±0.34个/秒增加到5.23±0.28个/秒,增加了0.56±0.06个/秒(P＜0.05);刺激“丰隆”(n=20)后,放电频率从4.82±0.49个/秒增加到5.08±0.38个/秒,增加了0.26±0.03个/秒(P＞0.05)。结果:除了“丰隆”穴,TENS其他部位前后NTS放电频率比较,差异有统计学意义。
2.3.3 EA对NTS放电频率影响的统计结果
VNS(n=26)后,放电频率从4.63±0.39个/秒增加到8.25±0.43个/秒,增加了3.62±0.25个/秒(P＜0.01);刺激耳甲腔(n=25)后,放电频率从4.59±0.54个/秒增加到8.18±0.61个/秒,增加了3.59±0.34个/秒(P＜0.01);刺激耳甲艇(n=27)后,放电频率从4.58±0.46个/秒增加到8.19±0.62个/秒,增加了3.61±0.14个/秒(P＜0.01);刺激耳垂(n=24)后,放电频率从4.73±0.45个/秒增加到5.85±0.64个/秒,增加了1.12±0.09个/秒(P＜0.01);刺激耳郭外缘中点(n=22)后,放电频率从4.65±0.24个/秒增加到5.51±0.59个/秒,增加了0.86±0.07个/秒(P＜0.01);刺激耳尖(n=25)后,放电频率从4.59±0.48个/秒增加到5.60±0.54个/秒,增加了1.01±0.09个/秒(P＜0.01);刺激“大椎”(n=20)后,放电频率从4.65±0.34个/秒增加到5.31±0.32个/秒,增加了0.66±0.07个/秒(P＜0.05);刺激“丰隆”(n=21)后,放电频率从4.56±0.49个/秒增加到4.91±0.55个/秒,增加了0.35±0.05个/秒(P＞0.05)。结果,除了“丰隆”穴,电针其他部位前后NTS放电频率比较,差异有统计学意义。
2.3.4 MA对NTS放电频率影响的统计结果
刺激耳甲腔(n=28)后,放电频率从4.68±0.46个/秒增加到8.52±0.74个/秒,增加了3.84±0.35个/秒(P＜0.01);刺激耳甲艇(n=32)后,放电频率从4.72±0.57个/秒增加到8.53±0.75个/秒,增加了3.81±0.45个/秒(P＜0.01);刺激耳垂(n=33)后,放电频率从4.61±0.43个/秒增加到6.00±0.52个/秒,增加了1.39±0.17个/秒(P＜0.01);刺激耳郭外缘中点(n=29)后,放电频率从4.65±0.58个/秒增加到5.63±0.55个/秒,增加了0.98±0.12个/秒(P＜0.01);刺激耳尖(n=26)后,放电频率从4.61±0.35个/秒增加到5.87±0.43个/秒,增加了1.21±0.15个/秒(P＜0.01);刺激“大椎”(n=25)后,放电频率从4.62±0.59个/秒增加到5.33±0.37个/秒,增加了0.91±0.12个/秒(P＜0.01);刺激“丰隆”(n=20)后,放电频率从4.65±0.30个/秒增加到5.15±0.59个/秒,增加了0.50±0.06个/秒(P＜0.05)。结果,手针每个部位前后NTS放电频率比较,差异都有统计学意义。
2.3.5不同针刺方法不同部位刺激对NTS神经元放电频率影响的比较
同一刺激部位,不同方法刺激前后NTS神经元放电频率的差值比较:MA与TENS、EA相比,差异有统计学意义(P＜0.05),EA与TENS相比,差异有统计学意义(P＜0.05),说明增加NTS神经元放电频率的效应比较,MA较TENS、EA显著,EA较TENS显著。同一刺激方法,不同部位刺激前后NTS神经元放电频率的差值比较:VNS、耳甲腔、耳甲艇分别与其他部位相比,差异有统计学意义(P＜0.01),VNS、耳甲腔、耳甲艇两两相比,差异没有统计学意义(P＞0.05),说明刺激VNS、耳甲腔、耳甲艇增加NTS神经元放电频率的效应较刺激其他部位的效应显著,而且与VNS相比无显著差异。
2.3.6不同针刺方法不同部位刺激对EEG影响的比较
同一刺激部位,不同方法刺激前后大鼠癫痫发作持续时间的差值比较:MA与TENS、EA相比,差异有统计学意义(P＜0.05),EA与TENS相比,差异有统计学意义(P＜0.05),说明抑制癫痫的效应比较,MA优于EA和TENS,EA优于TENS。同一刺激方法,不同部位刺激前后大鼠癫痫发作持续时间的差值比较:VNS、耳甲腔、耳甲艇分别与其他部位相比,差异有统计学意义(P＜0.01),VNS、耳甲腔、耳甲艇三者两两比较,差异没有统计学意义(P＞0.05),说明刺激VNS、耳甲腔、耳甲艇抑制癫痫的效应优于刺激其他部位的效应。
实验3耳针对癫痫大鼠行为学表现、孤束核场电位、皮层场电位的影响
3.1实验动物:健康成年雄性Sprague-Dawley大鼠124只,体重298±31g,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
3.2.1单电极记录耳针对癫痫大鼠皮层场电位的影响(急性实验)
手术过程中动物体温用计算机温度时间控制仪维持在37℃。大鼠12只,麻醉后俯卧。根据大鼠脑立体定位图谱,暴露初级体感皮层(SI)(A:-3～-6 mm;L:4～6 mm:H:1.5 mm),用单根直径为50μ的金属微电极记录癫痫大鼠SI场电位(FPs),分别观察5个部位耳针(包括耳甲腔、耳甲艇、耳垂、耳郭外缘中点、耳尖)30 s前后癫痫大鼠SI FPs的变化。电针刺激参数:强度,1 mA;频率,20 Hz;脉宽,500μs;刺激30 s。
3.2.2单电极记录耳针对癫痫大鼠行为学表现、皮层场电位影响(慢性实验)
大鼠50只,根据外耳5个刺激部位分为5组,每组10只。手术前部分同3.2.1,将金属微电极推进至SI后,牙托水泥固定电极,动物清醒并恢复一周后,分别观察5个部位耳针30 min后癫痫大鼠行为学表现和SI FPs的变化。电针刺激参数:强度,1 mA;频率,20 Hz;脉宽,500μs;刺激30 s,间隔5 min,持续30 min。
3.2.3微电极阵列记录耳针对癫痫大鼠孤束核场电位、皮层场电位的影响(急性实验)
手术过程中动物体温用计算机温度时间控制仪维持在37℃。大鼠12只,根据大鼠脑立体定位图谱,用微电极推进器将两组微电极阵列(MEA)推进至NTS(电极类型:2×2)和SI(电极类型:2×8),分别记录NTS FPs和SI FPs,分别观察5个部位耳针30 s前后NTS FPs和SI FPs的变化。电针刺激参数同3.2.1。
3.2.4微阵记录耳针对癫痫大鼠行为学表现、皮层场电位影响(慢性实验)
大鼠50只,根据外耳5个刺激部位分为5组,每组10只。手术前部分同3.2.3,将MEA推进至SI(电极类型:2×8)后,牙托水泥固定电极,动物清醒并恢复一周后,分别观察5个部位耳针30 min后大鼠行为学表现和SI FPs的变化。电针刺激参数同3.2.2。
3.3实验结果
3.3.1单电极记录耳针对癫痫大鼠皮层场电位的影响(急性实验)
电针耳甲腔(n=14)后,发作持续时间从26.15±2.31秒减少到5.07±0.64秒,减少了21.08±2.06秒(P＜0.01);电针耳甲艇(n=13)后,发作持续时间从25.98±2.17秒减少到4.97±0.43秒,减少了21.01±1.97秒(P＜0.01);电针耳垂(n=13)后,发作持续时间从26.29±2.24秒减少到19.28±1.53秒,减少了7.01±0.82秒(P＜0.01);电针耳郭外缘中点(n=11)后,发作持续时间从26.38±2.37秒减少到22.16±1.98秒,减少了4.02±0.35秒(P＜0.01);电针耳尖(n=10)后,发作持续时间从26.41±2.23秒减少到19.78±1.62秒,减少了6.63±0.47秒(P＜0.01)。每个部位电针前后大鼠癫痫发作总的持续时间比较,差异有统计学意义,说明耳针能明显抑制大鼠癫痫发作。
3.3.2单电极记录耳针对癫痫大鼠行为学积分、皮层场电位的影响(慢性实验)
耳针后行为学积分比较:电针耳甲腔、电针耳甲艇组分别与其它组相比,差异有统计学意义(P＜0.01);电针耳甲腔与电针耳甲艇比较,差异没有统计学意义(P＞0.05)。耳针后FPs记录的癫痫发作持续时间比较:电针耳甲腔、电针耳甲艇分别与其它组相比,差异有统计学意义(P＜0.01);电针耳甲腔与电针耳甲艇比较,差异没有统计学意义(P＞0.05)。说明电针耳甲抑制癫痫的效应优于电针其它耳部的效应。
3.3.3微电极阵列记录耳针对癫痫大鼠孤束核场电位、皮层场电位的影响(急性实验)
电针耳甲腔(n=12)后,发作持续时间从25.94±2.12秒减少到4.91±0.37秒,减少了21.03±1.96秒(P＜0.01);电针耳甲艇(n=13)后,发作持续时间从26.29±2.40秒减少到5.02±0.38秒,减少了21.27±2.01秒(P＜0.01);电针耳垂(n=10)后,发作持续时间从26.42±3.01秒减少到19.20±2.02秒,减少了7.22±0.69秒(P＜0.01);电针耳郭外缘中点(n=10)后,发作持续时间从26.61±3.12秒减少到21.60±2.05秒,减少了5.01±0.36秒(P＜0.01);电针耳尖(n=10)后,发作持续时间从25.74±2.23秒减少到18.41±1.62秒,减少了6.93±0.52秒(P＜0.01)。每个部位电针前后大鼠癫痫发作总的持续时间比较,差异有统计学意义(P＜0.01),说明耳针能明显抑制大鼠癫痫发作。
而且MEA记录显示癫痫大鼠NTS FPs与SI FPs电位振幅的变化同步。
3.3.4微电极阵列记录耳针对癫痫大鼠行为学积分、皮层场电位的影响(慢性实验)
耳针后行为学积分比较:电针耳甲腔组、电针耳甲艇组分别与其它组相比,差异有统计学意义(P＜0.01);电针耳甲腔组与电针耳甲艇比较,差异没有统计学意义(P＞0.05)。耳针后FPs记录的癫痫发作持续时间比较:电针耳甲腔组、电针耳甲艇组分别与其它组相比,差异有统计学意义(P＜0.01);电针耳甲腔组与电针耳甲艇组比较,差异没有统计学意义(P＞0.05)。说明电针耳甲抑制癫痫的效应优于电针其它耳部的效应。
实验4物理冷冻孤束核对耳针抗癫痫效应的影响
4.1实验动物
健康成年雄性Sprague-Dawley大鼠10只,体重300±30g,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
手术及实验过程中动物体温用计算机温度时间控制仪维持在37℃。双银球电极记录硬脑膜外EEG。沿大鼠项部正中切开皮肤,充分暴露闩部,并保持闩部水平。将U型管固定于微推进器,用微推器将U型管底部轻轻接触于孤束核在延髓投影区,即闩部区域。
将冷冻至-8℃的防冻液用注射器匀速推进通过U型管,局部冷冻孤束核。观察冷冻孤束核对癫痫大鼠EEG的影响,以及对耳针(包括耳甲腔、耳甲艇、耳垂、耳郭外缘中点、耳尖)抑制癫痫EEG效应的影响。
4.3结果
造模前,孤束核冷冻前、冷冻时、及冷冻后EEG无明显变化。腹腔注射PTZ造模后,同一刺激部位电针前后发作持续时间的差值比较:冷冻NTS中与冷冻NTS前比较,差异有统计学意义(P＜0.01);撤除冷冻NTS 5 min后与冷冻NTS前比较,差异无统计学意义(P＞0.05)。结果表明冷冻NTS时,耳针抑制癫痫的效应减弱,撤除冷冻5min后,耳针抑制癫痫的效应恢复。说明NTS的功能受损削弱了耳针抗癫痫的效应。
实验5静脉注射盐酸肾上腺素,心得安对癫痫大鼠脑电图的影响
5.1实验动物
健康成年雄性Sprague-Dawley大鼠10只,体重300±30g,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
手术及实验过程中动物体温用计算机温度时间控制仪维持在37℃。双银球电极记录硬膜外EEG.。股静脉插管,以备静脉用药。造模成功后,给予大鼠股静脉注射盐酸肾上腺素或心得安,观察药物对癫痫大鼠EEG的影响。
5.3结果
癫痫发作较少时静脉注射肾上腺素,可以加剧癫痫的发作;当癫痫发作频繁时静脉注射心得安,可以抑制癫痫的发作。
二结论
本项研究从行为学和电生理的角度探讨了耳针治疗癫痫的效应机制。实验结果表明:癫痫的发病表现为行为学发作和脑电图的高幅癫痫波,同时可能伴有自主神经系统功能的失衡。针刺特别是耳针能明显改善癫痫大鼠的行为学表现,这种作用可能通过激活NTS的活动来增强副交感的功能从而去同步化脑电,最终抑制癫痫发作。刺激耳甲的急性抗癫痫效应与VNS的急性抗癫痫效应相比无显著性差异,且明显优于刺激其它部位的效应,可能与耳甲部分布有迷走神经耳支相关。这项研究从行为学和电生理的角度探讨了耳针治疗癫痫的效应机制,不仅为耳针治疗癫痫提供了理论依据,也为耳-迷走理论提供更丰富的依据。
同时本研究发现癫痫大鼠NTS的放电频率与EEG癫痫波存在着时间上的同步变化的关系,物理冷冻NTS损削弱了耳针抑制癫痫脑电图的效应,说明NTS可能参与癫痫的发生和发展。这一结果为解释癫痫的发病机制和VNS治疗癫痫的作用机制提供了理论依据。
本部分实验还进行了经皮电刺激(TENS)、电针(EA)、手针(MA)三种刺激方法治疗癫痫大鼠的效应的比较,发现MA效果优于EA和TENS,EA效果优于TENS,说明不同刺激方法所产生的效应不同,这项研究结果为临床针灸治疗癫痫选用适宜的方法提供了一定的实验依据。
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 experiments
1 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.
Conclusion
The 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.
癫痫是一种由不同因素引起的以反复发作性大脑功能失调为表现的神经系统疾病。自美国FDA于1997年7月正式批准迷走神经刺激(vagus nervestimulation,VNS)疗法作为12岁以上药物难治性部分发作性癫痫的一种辅助疗法以来,这种治疗方法被全世界范围患者所应用。虽然其作用机制仍不是十分清楚,多数的观点认为VNS可能是通过激活迷走神经投射到NTS的通路,然后通过NTS与脑内的广泛联系达到去同步化脑电,抑制癫痫的目的。已有研究提示,癫痫的发病可能与自主神经系统功能的失调有关,表现为交感神经系统功能的亢进,副交感神经系统功能低下。VNS则可能通过刺激迷走神经增强副交感的效应而抑制癫痫。
有研究报道在颈部运用经皮迷走神经电刺激治疗癫痫取得疗效,而且经皮刺激迷走神经耳支支配的耳甲区也可以治疗癫痫,中医耳针治疗癫痫选穴也以耳甲区的穴位为主。由此,我们设想,耳针可能通过刺激迷走神经耳支发挥抑制癫痫的效应。
本研究从行为学、电生理学等角度分别观察了耳针对癫痫大鼠行为学表现,NTS神经元放电,硬膜外脑电图(electroencephalogram,EEG)的影响,以及局部冷冻NTS对耳针抗癫痫效应的影响,和应用交感兴奋剂、拮抗剂对癫痫大鼠EEG的影响,试图从耳-迷走反射的角度探讨耳针抑制癫痫的作用机制。
一动物实验
实验1耳针预处理对戊四氮致癫痫大鼠行为学表现的影响
1.1实验动物
健康成年Sprague-Dawley雄性大鼠42只,体重300±28g,由中国医学科学院实验动物中心提供,清洁级。
1.2实验过程
将动物分为3组。模型组(n=14)给予大鼠腹腔注射戊四氮(pentylenetetrazol,PTZ)60 mg/kg造成急性癫痫模型,后观察30 min大鼠行为学的表现,耳甲治疗组(n=15)和大椎治疗组(n=13)分别在造模前电针耳甲或“大椎”穴30 min,后立即腹腔注射PTZ,观察30 min大鼠行为学的表现。行为学表现根据Racine分级标准进行评分。电针刺激参数:强度,1 mA;频率,20 Hz;脉宽,500μs;刺激30 s,间隔5 min,持续30 min。
1.3实验结果
模型组大鼠腹腔注射PTZ 40s左右出现癫痫发作,开始表现为小发作,如凝视,点头运动,双前肢抬起,阵挛等,1-2 min出现大发作,表现为后肢伸直,抽搐,跌倒伴翻滚,持续约9.5 s,PTZ后30 min内大鼠有1-2次大发作,多次小发作。与模型组比较,大椎治疗组和耳甲治疗组动物大发作潜伏期(即从腹腔注射PTZ到大鼠出现第一次大发作的时间)延长(P＜0.05,P＜0.01),行为学评分减少(P＜0.05,P＜0.01)。与大椎治疗组比较,除了在首次大发作持续时间上无显著差异(P＞0.05),耳甲治疗组大发作潜伏期延长(P＜0.01),行为学评分亦减少(P＜0.01)。结果提示:针刺预处理可以抑制大鼠癫痫发作,而且电针耳甲抑制癫痫的效应优于电针“大椎”穴的效应。
实验2耳针对癫痫大鼠孤束核神经元细胞外放电和脑电图的影响
2.1实验动物
健康成年雄性Sprague-Dawley大鼠45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
手术及实验过程中动物体温用计算机温度时间控制仪维持在37℃。
大鼠麻醉后仰卧,颈部正中切口,游离左侧迷走神经干1cm,将自制C型双极银质电极刺激端无张力地钩于迷走神经干上用以刺激迷走神经,缝合伤口。然后俯卧,将大鼠头部用耳棒固定于脑立体定位仪上,根据大鼠脑立体定位图谱(Paxinos和Watson,1998),在前囟前(A:1.0 mm,L:1.0 mm)和前囟后(A:-1.0 mm,L:1.0 mm)及NTS(A:-11.3～-14.3 mm,L:0～2.3 mm,H:4～7 mm)对应颅骨表面,用颅骨钻钻孔,暴露硬脑膜。双银球电极置于前囟前、后各一小孔内硬脑膜上用以记录EEG。显微镜下分离NTS对应处小孔硬脑膜和软脑膜,暴露小脑皮层,玻璃微电极(阻抗10-20 MQ)记录NTS神经元放电。
手术完毕一小时待大鼠状态稳定后开始记录。用微推进器推进玻璃微电极寻找到自发放电的NTS神经元,上下调节推进器位置,至信噪比较大时停止推进。记录30 s背景放电,待细胞放电频率稳定后,给予耳甲刺激15 s,若细胞放电频率出现变化,确定为对耳甲刺激有反应神经元。在细胞放电频率稳定后,给予大鼠腹腔注射PTZ(40-60 mg/kg)造模,以造模后5 min内EEG出现高幅的癫痫波为造模成功。分别观察造模前后,刺激迷走神经(vagus nerve,VN)、耳甲腔、耳甲艇、耳尖、耳郭外缘中点、耳垂、“大椎”穴、“丰隆”穴前后NTS神经元放电频率和EEG的变化。每一次操作在EEG恢复稳定后重复刺激程序,一只动物每组穴位刺激最多重复3次。刺激方法包括经皮电刺激(transcutaneous electrical nerve stimulation,TENS),电针刺激(electroacupuncture,EA)或手针刺激(manual acupuncture,MA)。TENS或EA参数:强度,1 mA;频率,20 Hz;脉宽,500μs;刺激30 s,间隔5 min,持续30 min。MA采用平补平泻,均匀捻转。针刺时间,30 s。
2.3实验结果
2.3.1造模前后孤束核神经元放电频率和脑电图的同步变化
造模前后共观察到31个细胞。造模后26个细胞放电频率降低(83.87%),3个细胞放电频率不变(9.68%),2个细胞放电频率增多(6.45%)。经统计学处理:NTS细胞放电频率造模前为9.48±0.76个/秒,造模后为4.69±0.36个/秒。其中26个放电频率降低的细胞,其放电频率的变化和脑电图的变化存在同步性,即在细胞放电频率降低时,EEG出现高幅癫痫波,当细胞放电频率增加时,EEG癫痫波消失或振幅降低。说明当NTS细胞放电频率减少时,动物癫痫发作,NTS细胞放电频率增多时,癫痫的发作消失或减少。这种同步变化在造模后1-3分钟之内开始出现,呈节律性变化,按EEG每出现一次癫痫波记为一次癫痫发作,最初发作3次/分,2秒/次,约7-10分钟内持续增多,在约15分钟后,呈基本有规律的变化,平均发作约6.21±0.48次/分,持续时间4.32±0.23秒/次,每分钟发作总的持续时间为26.83±2.25秒。
2.3.2 TENS对NTS放电频率影响的统计结果
TENS(n=31)后,放电频率从平均4.53±0.35个/秒增加到7.50±0.63个/秒,增加了2.97±0.18个/秒(P＜0.01);刺激耳甲腔(n=29)后,放电频率从4.66±0.41个/秒增加到7.64±0.72个/秒,增加了2.98±0.14个/秒(P＜0.01);刺激耳甲艇(n=32)后,放电频率从4.71±0.38个/秒增加到7.61±0.62个/秒,增加了2.90±0.15个/秒(P＜0.01);刺激耳垂(n=28)后,放电频率从4.69±0.48个/秒增加到5.72±0.56个/秒,增加了1.03±0.08个/秒(P＜0.01);刺激耳郭外缘中点(n=26)后,放电频率从4.72±0.34个/秒增加到5.40±0.42个/秒,增加了0.68±0.04个/秒(P＜0.05);刺激耳尖(n=25)后,放电频率从4.59±0.31个/秒增加到5.42±0.53个/秒,增加了0.83±0.05个/秒(P＜0.05);刺激“大椎”(n=28)后,放电频率从4.67±0.34个/秒增加到5.23±0.28个/秒,增加了0.56±0.06个/秒(P＜0.05);刺激“丰隆”(n=20)后,放电频率从4.82±0.49个/秒增加到5.08±0.38个/秒,增加了0.26±0.03个/秒(P＞0.05)。结果:除了“丰隆”穴,TENS其他部位前后NTS放电频率比较,差异有统计学意义。
2.3.3 EA对NTS放电频率影响的统计结果
VNS(n=26)后,放电频率从4.63±0.39个/秒增加到8.25±0.43个/秒,增加了3.62±0.25个/秒(P＜0.01);刺激耳甲腔(n=25)后,放电频率从4.59±0.54个/秒增加到8.18±0.61个/秒,增加了3.59±0.34个/秒(P＜0.01);刺激耳甲艇(n=27)后,放电频率从4.58±0.46个/秒增加到8.19±0.62个/秒,增加了3.61±0.14个/秒(P＜0.01);刺激耳垂(n=24)后,放电频率从4.73±0.45个/秒增加到5.85±0.64个/秒,增加了1.12±0.09个/秒(P＜0.01);刺激耳郭外缘中点(n=22)后,放电频率从4.65±0.24个/秒增加到5.51±0.59个/秒,增加了0.86±0.07个/秒(P＜0.01);刺激耳尖(n=25)后,放电频率从4.59±0.48个/秒增加到5.60±0.54个/秒,增加了1.01±0.09个/秒(P＜0.01);刺激“大椎”(n=20)后,放电频率从4.65±0.34个/秒增加到5.31±0.32个/秒,增加了0.66±0.07个/秒(P＜0.05);刺激“丰隆”(n=21)后,放电频率从4.56±0.49个/秒增加到4.91±0.55个/秒,增加了0.35±0.05个/秒(P＞0.05)。结果,除了“丰隆”穴,电针其他部位前后NTS放电频率比较,差异有统计学意义。
2.3.4 MA对NTS放电频率影响的统计结果
刺激耳甲腔(n=28)后,放电频率从4.68±0.46个/秒增加到8.52±0.74个/秒,增加了3.84±0.35个/秒(P＜0.01);刺激耳甲艇(n=32)后,放电频率从4.72±0.57个/秒增加到8.53±0.75个/秒,增加了3.81±0.45个/秒(P＜0.01);刺激耳垂(n=33)后,放电频率从4.61±0.43个/秒增加到6.00±0.52个/秒,增加了1.39±0.17个/秒(P＜0.01);刺激耳郭外缘中点(n=29)后,放电频率从4.65±0.58个/秒增加到5.63±0.55个/秒,增加了0.98±0.12个/秒(P＜0.01);刺激耳尖(n=26)后,放电频率从4.61±0.35个/秒增加到5.87±0.43个/秒,增加了1.21±0.15个/秒(P＜0.01);刺激“大椎”(n=25)后,放电频率从4.62±0.59个/秒增加到5.33±0.37个/秒,增加了0.91±0.12个/秒(P＜0.01);刺激“丰隆”(n=20)后,放电频率从4.65±0.30个/秒增加到5.15±0.59个/秒,增加了0.50±0.06个/秒(P＜0.05)。结果,手针每个部位前后NTS放电频率比较,差异都有统计学意义。
2.3.5不同针刺方法不同部位刺激对NTS神经元放电频率影响的比较
同一刺激部位,不同方法刺激前后NTS神经元放电频率的差值比较:MA与TENS、EA相比,差异有统计学意义(P＜0.05),EA与TENS相比,差异有统计学意义(P＜0.05),说明增加NTS神经元放电频率的效应比较,MA较TENS、EA显著,EA较TENS显著。同一刺激方法,不同部位刺激前后NTS神经元放电频率的差值比较:VNS、耳甲腔、耳甲艇分别与其他部位相比,差异有统计学意义(P＜0.01),VNS、耳甲腔、耳甲艇两两相比,差异没有统计学意义(P＞0.05),说明刺激VNS、耳甲腔、耳甲艇增加NTS神经元放电频率的效应较刺激其他部位的效应显著,而且与VNS相比无显著差异。
2.3.6不同针刺方法不同部位刺激对EEG影响的比较
同一刺激部位,不同方法刺激前后大鼠癫痫发作持续时间的差值比较:MA与TENS、EA相比,差异有统计学意义(P＜0.05),EA与TENS相比,差异有统计学意义(P＜0.05),说明抑制癫痫的效应比较,MA优于EA和TENS,EA优于TENS。同一刺激方法,不同部位刺激前后大鼠癫痫发作持续时间的差值比较:VNS、耳甲腔、耳甲艇分别与其他部位相比,差异有统计学意义(P＜0.01),VNS、耳甲腔、耳甲艇三者两两比较,差异没有统计学意义(P＞0.05),说明刺激VNS、耳甲腔、耳甲艇抑制癫痫的效应优于刺激其他部位的效应。
实验3耳针对癫痫大鼠行为学表现、孤束核场电位、皮层场电位的影响
3.1实验动物:健康成年雄性Sprague-Dawley大鼠124只,体重298±31g,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
3.2.1单电极记录耳针对癫痫大鼠皮层场电位的影响(急性实验)
手术过程中动物体温用计算机温度时间控制仪维持在37℃。大鼠12只,麻醉后俯卧。根据大鼠脑立体定位图谱,暴露初级体感皮层(SI)(A:-3～-6 mm;L:4～6 mm:H:1.5 mm),用单根直径为50μ的金属微电极记录癫痫大鼠SI场电位(FPs),分别观察5个部位耳针(包括耳甲腔、耳甲艇、耳垂、耳郭外缘中点、耳尖)30 s前后癫痫大鼠SI FPs的变化。电针刺激参数:强度,1 mA;频率,20 Hz;脉宽,500μs;刺激30 s。
3.2.2单电极记录耳针对癫痫大鼠行为学表现、皮层场电位影响(慢性实验)
大鼠50只,根据外耳5个刺激部位分为5组,每组10只。手术前部分同3.2.1,将金属微电极推进至SI后,牙托水泥固定电极,动物清醒并恢复一周后,分别观察5个部位耳针30 min后癫痫大鼠行为学表现和SI FPs的变化。电针刺激参数:强度,1 mA;频率,20 Hz;脉宽,500μs;刺激30 s,间隔5 min,持续30 min。
3.2.3微电极阵列记录耳针对癫痫大鼠孤束核场电位、皮层场电位的影响(急性实验)
手术过程中动物体温用计算机温度时间控制仪维持在37℃。大鼠12只,根据大鼠脑立体定位图谱,用微电极推进器将两组微电极阵列(MEA)推进至NTS(电极类型:2×2)和SI(电极类型:2×8),分别记录NTS FPs和SI FPs,分别观察5个部位耳针30 s前后NTS FPs和SI FPs的变化。电针刺激参数同3.2.1。
3.2.4微阵记录耳针对癫痫大鼠行为学表现、皮层场电位影响(慢性实验)
大鼠50只,根据外耳5个刺激部位分为5组,每组10只。手术前部分同3.2.3,将MEA推进至SI(电极类型:2×8)后,牙托水泥固定电极,动物清醒并恢复一周后,分别观察5个部位耳针30 min后大鼠行为学表现和SI FPs的变化。电针刺激参数同3.2.2。
3.3实验结果
3.3.1单电极记录耳针对癫痫大鼠皮层场电位的影响(急性实验)
电针耳甲腔(n=14)后,发作持续时间从26.15±2.31秒减少到5.07±0.64秒,减少了21.08±2.06秒(P＜0.01);电针耳甲艇(n=13)后,发作持续时间从25.98±2.17秒减少到4.97±0.43秒,减少了21.01±1.97秒(P＜0.01);电针耳垂(n=13)后,发作持续时间从26.29±2.24秒减少到19.28±1.53秒,减少了7.01±0.82秒(P＜0.01);电针耳郭外缘中点(n=11)后,发作持续时间从26.38±2.37秒减少到22.16±1.98秒,减少了4.02±0.35秒(P＜0.01);电针耳尖(n=10)后,发作持续时间从26.41±2.23秒减少到19.78±1.62秒,减少了6.63±0.47秒(P＜0.01)。每个部位电针前后大鼠癫痫发作总的持续时间比较,差异有统计学意义,说明耳针能明显抑制大鼠癫痫发作。
3.3.2单电极记录耳针对癫痫大鼠行为学积分、皮层场电位的影响(慢性实验)
耳针后行为学积分比较:电针耳甲腔、电针耳甲艇组分别与其它组相比,差异有统计学意义(P＜0.01);电针耳甲腔与电针耳甲艇比较,差异没有统计学意义(P＞0.05)。耳针后FPs记录的癫痫发作持续时间比较:电针耳甲腔、电针耳甲艇分别与其它组相比,差异有统计学意义(P＜0.01);电针耳甲腔与电针耳甲艇比较,差异没有统计学意义(P＞0.05)。说明电针耳甲抑制癫痫的效应优于电针其它耳部的效应。
3.3.3微电极阵列记录耳针对癫痫大鼠孤束核场电位、皮层场电位的影响(急性实验)
电针耳甲腔(n=12)后,发作持续时间从25.94±2.12秒减少到4.91±0.37秒,减少了21.03±1.96秒(P＜0.01);电针耳甲艇(n=13)后,发作持续时间从26.29±2.40秒减少到5.02±0.38秒,减少了21.27±2.01秒(P＜0.01);电针耳垂(n=10)后,发作持续时间从26.42±3.01秒减少到19.20±2.02秒,减少了7.22±0.69秒(P＜0.01);电针耳郭外缘中点(n=10)后,发作持续时间从26.61±3.12秒减少到21.60±2.05秒,减少了5.01±0.36秒(P＜0.01);电针耳尖(n=10)后,发作持续时间从25.74±2.23秒减少到18.41±1.62秒,减少了6.93±0.52秒(P＜0.01)。每个部位电针前后大鼠癫痫发作总的持续时间比较,差异有统计学意义(P＜0.01),说明耳针能明显抑制大鼠癫痫发作。
而且MEA记录显示癫痫大鼠NTS FPs与SI FPs电位振幅的变化同步。
3.3.4微电极阵列记录耳针对癫痫大鼠行为学积分、皮层场电位的影响(慢性实验)
耳针后行为学积分比较:电针耳甲腔组、电针耳甲艇组分别与其它组相比,差异有统计学意义(P＜0.01);电针耳甲腔组与电针耳甲艇比较,差异没有统计学意义(P＞0.05)。耳针后FPs记录的癫痫发作持续时间比较:电针耳甲腔组、电针耳甲艇组分别与其它组相比,差异有统计学意义(P＜0.01);电针耳甲腔组与电针耳甲艇组比较,差异没有统计学意义(P＞0.05)。说明电针耳甲抑制癫痫的效应优于电针其它耳部的效应。
实验4物理冷冻孤束核对耳针抗癫痫效应的影响
4.1实验动物
健康成年雄性Sprague-Dawley大鼠10只,体重300±30g,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
手术及实验过程中动物体温用计算机温度时间控制仪维持在37℃。双银球电极记录硬脑膜外EEG。沿大鼠项部正中切开皮肤,充分暴露闩部,并保持闩部水平。将U型管固定于微推进器,用微推器将U型管底部轻轻接触于孤束核在延髓投影区,即闩部区域。
将冷冻至-8℃的防冻液用注射器匀速推进通过U型管,局部冷冻孤束核。观察冷冻孤束核对癫痫大鼠EEG的影响,以及对耳针(包括耳甲腔、耳甲艇、耳垂、耳郭外缘中点、耳尖)抑制癫痫EEG效应的影响。
4.3结果
造模前,孤束核冷冻前、冷冻时、及冷冻后EEG无明显变化。腹腔注射PTZ造模后,同一刺激部位电针前后发作持续时间的差值比较:冷冻NTS中与冷冻NTS前比较,差异有统计学意义(P＜0.01);撤除冷冻NTS 5 min后与冷冻NTS前比较,差异无统计学意义(P＞0.05)。结果表明冷冻NTS时,耳针抑制癫痫的效应减弱,撤除冷冻5min后,耳针抑制癫痫的效应恢复。说明NTS的功能受损削弱了耳针抗癫痫的效应。
实验5静脉注射盐酸肾上腺素,心得安对癫痫大鼠脑电图的影响
5.1实验动物
健康成年雄性Sprague-Dawley大鼠10只,体重300±30g,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
手术及实验过程中动物体温用计算机温度时间控制仪维持在37℃。双银球电极记录硬膜外EEG.。股静脉插管,以备静脉用药。造模成功后,给予大鼠股静脉注射盐酸肾上腺素或心得安,观察药物对癫痫大鼠EEG的影响。
5.3结果
癫痫发作较少时静脉注射肾上腺素,可以加剧癫痫的发作;当癫痫发作频繁时静脉注射心得安,可以抑制癫痫的发作。
二结论
本项研究从行为学和电生理的角度探讨了耳针治疗癫痫的效应机制。实验结果表明:癫痫的发病表现为行为学发作和脑电图的高幅癫痫波,同时可能伴有自主神经系统功能的失衡。针刺特别是耳针能明显改善癫痫大鼠的行为学表现,这种作用可能通过激活NTS的活动来增强副交感的功能从而去同步化脑电,最终抑制癫痫发作。刺激耳甲的急性抗癫痫效应与VNS的急性抗癫痫效应相比无显著性差异,且明显优于刺激其它部位的效应,可能与耳甲部分布有迷走神经耳支相关。这项研究从行为学和电生理的角度探讨了耳针治疗癫痫的效应机制,不仅为耳针治疗癫痫提供了理论依据,也为耳-迷走理论提供更丰富的依据。
同时本研究发现癫痫大鼠NTS的放电频率与EEG癫痫波存在着时间上的同步变化的关系,物理冷冻NTS损削弱了耳针抑制癫痫脑电图的效应,说明NTS可能参与癫痫的发生和发展。这一结果为解释癫痫的发病机制和VNS治疗癫痫的作用机制提供了理论依据。
本部分实验还进行了经皮电刺激(TENS)、电针(EA)、手针(MA)三种刺激方法治疗癫痫大鼠的效应的比较,发现MA效果优于EA和TENS,EA效果优于TENS,说明不同刺激方法所产生的效应不同,这项研究结果为临床针灸治疗癫痫选用适宜的方法提供了一定的实验依据。
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 experiments
1 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.
Conclusion
The 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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