耳—迷走反射与耳针降糖效应机制研究
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摘要
随着人们生活水平的日益提高，糖尿病已经成为了临床常见和好发疾病之一，针刺特别是耳针对糖尿病防治具有较好的临床疗效。然而耳针降糖效应机制的研究截至目前涉足较少，少数研究者认为耳针的作用机制可能与交感神经为主的脊髓节段性调节有关，而忽视了耳针可能激活了耳甲区迷走神经末梢与内脏神经末梢在脑干特别是“迷走复合体”的汇聚整合机制。支配外耳道和耳甲区迷走神经耳支是迷走神经分布于体表浅层的唯一分支；孤束核处存在葡萄糖和胰岛素敏感神经元，并且耳针能够影响这两种神经元的放电活动，上述事实让我们设想耳针降糖效应可能与耳-迷走神经系统反射机制有关。本研究从电生理学、形态学、生物化学三个学科角度分别观察了耳针刺激对孤束核葡萄糖和胰岛素敏感神经的放电活动影响，耳与迷走初级中枢的神经突触联系，以及电针耳穴对正常与糖尿病状态下大鼠血糖及胰岛素浓度的影响，试图从耳-迷走-胰岛素系统反射角度探讨耳针降糖效应的中枢脑干参与机制。
1电生理实验：电针耳穴对孤束核葡萄糖、胰岛素敏感神经元胞外放电活动的影响
1.1实验动物
实验用健康成年Sprague-Dawley(SD)雄性大鼠51只，清洁级，体重200～220g，中国医学科学院实验动物中心提供(合格证编号：A06-053)。
1.2实验步骤
大鼠麻醉后，动物俯卧，将大鼠头部用耳棒固定于立体定位仪头部固定器上，以玻璃微电极细胞外纪录不同刺激对孤束核(NTS)神经元的放电影响，电极内充有2％滂胺天蓝溶液(电阻为10-20MΩ)实验开始后，通过微电极操作器控制，在NTS内探查神经元放电。实验探查到NTS细胞放电后，将其随机分为三组，每组按如下顺序施加因素：
①NTS葡萄糖反应神经元的鉴别：颈动脉推注葡萄糖(浓度为15％，推注速度为0.25ml/min)，观察孤束核细胞放电频率的变化，鉴别葡萄糖反应神经元的类型(放电频率升高、降低、不变)，如细胞放电频率发生变化，则待细胞恢复稳定放电频率状态，关闭三通管葡萄糖端，打开生理盐水端开关，推注生理盐水(推注速度为0.25ml/min)，继续观察细胞放电频率变化，如果所记录的神经元放电方式对生理盐水不出现反应变化(排除血压变化影响)，而对葡萄糖有反应变化则认为是实验目标神经元，否则放弃统计(下同)。
②NTS胰岛素反应神经元的鉴别及其对葡萄糖浓度升高的反应：颈动脉推注胰岛素(浓度为6IU/ml，推注速度同前)，观察孤束核细胞放电频率的变化，鉴别胰岛素反应神经元的类型(放电频率升高、降低、不变)，如细胞放电频率发生变化，则待细胞恢复稳定放电频率状态后，关闭三通管胰岛素端，打开生理盐水端开关，推注生理盐水(推注速度同前)，作空白对照。若细胞活动对胰岛素浓度升高敏感，则继续颈动脉注射葡萄糖(浓度为15％，推注速度同前)，观察胰岛素反应神经元对葡萄糖的敏感性。
③耳甲区穴位电针刺激对葡萄糖、胰岛素反应神经元的放电活动影响：耳甲区穴位(相当于人耳穴的胰腺、肾、肝胆等点)电刺激(参数：间隔：0.4s，持续时间：0.4ms，强度：2mA)每次电针刺激维持时间为30s，观察细胞放电活动的变化情况(频率变化，放电大小、有无变化)，如果细胞放电频率发生明显变化，待细胞放电活动稳定后进一步分别监测颈动脉推注葡萄糖(浓度为15％)及胰岛素(6IU/ml)对该细胞放电活动的影响(速度同前)，如果细胞放电活动发生反应，则待细胞放电活动稳定后继续推注生理盐水作空白对照。。
上述实验过程中，神经元反应变化类型鉴别：神经元放电频率比基础放电频率(或稳定状态放电频率)升高或者降低30％以上，则称为细胞活动兴奋或细胞活动抑制。
1.3实验结果
大鼠颈动脉推注葡萄糖，共观察了110个NTS神经元放电变化情况，结果显示推注葡萄糖后57个细胞(51.82％)放电频率无变化，有41个细胞(37.27％)放电频率减少，呈抑制反应，其中有两个细胞放电活动完全被抑制。放电频率的降低率为60.36±15.1％(均值±SD，下同)，12个细胞(10.91％)放电频率增加，呈兴奋反应，放电频率的增加率为56.78±9.24％。
给大鼠颈动脉注射胰岛素，共记录81个NTS细胞放电变化情况，据统计有50个细胞(61.73％)放电频率无变化，27个细胞(33.33％)放电频率增加，上升率为47.34±11.24％，4个细胞(4.94％)放电频率减少，下降率为43.24±12.11％。其中27个兴奋反应细胞中有18个细胞对葡萄糖有抑制反应，9个细胞对葡萄糖无反应；4个对胰岛素抑制反应细胞中有2个对葡萄糖有兴奋反应，2个对葡萄糖无反应。
电针大鼠耳甲穴位，共记录69个NTS细胞放电，统计结果显示针刺前后有32个细胞(46.38％)放电频率无变化，34个细胞(49.27％)针刺过程中或停针后放电频率增加，增加率为54.79±24.21％，此外3个细胞(4.35％)停针后放电频率减少，减少率为44.65±9.13％；其中34个对耳针起兴奋反应的细胞中有18个对输注葡萄糖呈现放电抑制反应，2个对葡萄糖有兴奋反应，14个对葡萄糖无反应；同样是上述34个对耳针起兴奋反应细胞中，有8个对输注胰岛素有兴奋反应，3个对胰岛素起抑制反应，23个对胰岛素无反应。
输注葡萄糖组，所记录到的孤束核神经元起兴奋反应和抑制反应的潜伏期分别为15±6s和18±8s；输注胰岛素组，孤束核神经元起兴奋反应和抑制反应的潜伏期分别为16±7s和18±10s，二者之间无显著性差异(P＞0.05)。电针耳甲穴位，记录到的相关孤束核细胞兴奋反应的潜伏期为3±1s，相关神经元细胞起抑制反应的潜伏期为5±2s，且耳针实验中，37个反应细胞中有21个细胞呈现2-5min长时程的放电频率增加，其中有2例细胞由原来的静息状态突发为放电状态；3个抑制性反应细胞均呈长时程放电频率抑制。
2形态学实验：HRP神经示踪法探讨耳甲区神经传入在低位脑干的投射联系
2.1实验动物
实验用健康成年SD大鼠10只，雌雄不拘，清洁级，体重200～220g，中国医学科学院实验动物中心提供(合格证编号：A06-084)。
2.2实验步骤
大鼠经2％戊巴比妥钠腹腔注射麻醉(40mg/kg)后，在其耳甲部用手术刀片横向划开一条长约2mm缝，向外耳道口方向分离缝内耳甲皮肤与皮下组织，微量注射器针头向分离后的缝内注射辣根过氧化物酶(HRP：30％，40μl)，注射完毕，退针后，紧按缝口3-5min，每只大鼠两耳各注射3-5个点，注射点尽量分布于整个耳甲艇和耳甲腔。HRP注射后动物存活36-48h，动物用2％戊巴比妥钠腹腔注射再次麻醉(30mg/kg)后，动脉灌流固定，次日取延髓部(闩上下2-3cm)冰冻切片，片厚30μm，裱于挂胶载玻片上风干以待呈色反应。经TMB呈色反应后，酒精梯度脱水，二甲苯透明，树脂封片，并在显微镜下观察结果。
2.3实验结果
10只大鼠在耳甲区注射HRP后，经TMB反应呈色，除了在三叉神经脊束核发现了标记细胞外，在孤束核和迷走神经背核也发现了标记神经元或神经纤维，其中孤束核处标记细胞多呈圆形或菱形，其胞浆充满了蓝黑色的颗粒，围绕细胞核的周围，其直径大小约20μm；迷走神经背核处标记细胞多为菱形或卵圆形，细胞大小较孤束核处标记细胞大，神经元突触标记明显，并且可见阳性纤维存在。此外在腹外侧网状核以及疑核等处也发现了阳性标记细胞，细胞呈圆形或菱形，直径大小约20-30μm。
3生化实验：耳针刺激对正常和糖尿病大鼠血糖水平、胰岛素水平的影响，及耳针降糖的时间窗效应观察
3.1实验动物
健康雄性SD大鼠100只，体重150-220g，清洁级，中国医学科学院实验动物中心提供(合格证编号：A06-084)。室温(25±2)℃，维持12h/24h昼夜规律，饲料，自来水自由摄取，适应环境1周。
3.2实验步骤
3.2.1模型制作
参照《药理学实验方法》，采用速发型糖尿病动物模型制作方法：动物腹腔注射2％链脲佐菌素(STZ)60mg/kg，溶于柠檬酸-柠檬酸钠缓冲液(PH=4.2，0.1M)过滤除菌，冰浴操作，一周后尾静脉取血，测定大鼠空腹血糖(采用美国强生Ⅱ型快速血糖仪)，血糖高于15mmol/L定为DM模型制作成功。
3.2.2实验分组
正常饲养的大鼠随机分为三组：
A组高强度耳针组(刺激参数：波宽1ms，强度10mA)；
B组低强度耳针组(刺激参数：波宽0.5ms，强度2mA)；
C组低强度电针足三里组(刺激参数：波宽0.5ms，强度2mA)
造模成功的糖尿病大鼠随机分为另外三组：
D组耳针组(刺激参数：波宽0.5ms，强度2mA)
E组迷走切断耳针组(刺激参数：波宽0.5ms，强度2mA)
F组足三里组(刺激参数：波宽0.5ms，强度2mA)
3.2.3针刺处理过程
上述各组大鼠电针处理刺激前用10％的乌拉坦腹腔注射麻醉(1g/kg)，手术分离股动脉插管(吸入肝素湿润管周，下同)采血0.3-0.5ml，快速血糖仪测针刺前血糖水平，并离心取血浆待测胰岛素浓度。
A组：将自制的电针耳豆用医用胶布粘贴至大鼠耳甲区穴位(耳甲艇或耳甲腔)，正极置于耳甲腔面，负极置于耳甲背面正对正极耳豆处，正负极连接于刺激器，打开刺激器，电针30min停止，10min后采血0.3-0.5ml，快速测量血糖浓度，离心分离血浆-78℃保存待测胰岛素浓度。
B组：方法同A组，电针刺激30min停止，分别于电针停止即刻、电针停止后10min、20min、30min抽取血样各0.3ml-0.5ml，快速测量血糖浓度，分别离心分离血浆后-78℃保存待测各次血浆胰岛素浓度。
C组：参照华兴邦大鼠穴位图谱针刺大鼠后三里(负极)及中脘穴(正极)，正负极连接于刺激器，打开刺激器，电针30min停止，分别于电针停止即刻、电针停止后10min、20min、30min抽取血样各0.3ml-0.5ml，快速测量血糖浓度，分别离心分离血浆后-78℃保存待测各次血浆胰岛素浓度。
D组：耳针方法参数同B组，电针刺激30min停止，10min后采血0.5ml，快速测血糖浓度，离心分离血浆-78℃保存待测胰岛素浓度。
E组：电针前股动脉采血后，手术分离并切断大鼠右侧颈部迷走神经，余下电针处理同D组。
F组：电针处理同C组，电针刺激30min停止，10min后采血0.5ml，快速测血糖浓度，离心分离血浆-78℃保存待测胰岛素浓度。
3.2.4指标检测
血糖浓度采用强生公司One-touch-Ⅱ型快速血糖仪及血糖试纸测量，检测原理为葡萄糖氧化酶法。胰岛素浓度检测采用酶联免疫法，试剂盒由美国BPB生物公司提供，检测方法严格按照试剂盒说明说进行。
3.3实验结果
3.3.1不同强度的针刺对正常大鼠血糖水平影响
分别采用10mA和2mA的强度电针耳甲区穴，对照组为2mA的电针足三里穴位，结果显示10mA电针耳甲，血糖浓度针刺前为6.48±0.28mmol/L，针刺30min，停针后10min血糖浓度升高为7.70±0.38mmol/L，配对t检验，有显著性差异(P＜0.05)，说明高强度的电针耳穴非但不能降低血糖，而且使血糖浓度升高；2mA电针耳甲，血糖浓度由电针前7.23±0.15mmol/L下降至针后10min 6.43±0.19mmol/L，配对t检验，有显著性差异(P＜0.01)，说明低强度的电针耳穴对正常大鼠的血糖浓度具有较好的调整作用；对照组电针足三里穴，电针前血糖浓度为7.45±0.33mmol/L，停针后10min血糖浓度值为7.41±0.29mmol/L，血糖虽有下降趋势，但无统计学差异(P＞0.05)。
3.3.2低强度电针耳穴与足三里穴对正常大鼠血糖浓度的时间窗效应比较
2mA耳针刺激正常大鼠30min，针刺前大鼠血糖浓度为7.23±0.15mmol/L，耳针30min停止，停针即刻血糖浓度下降至6.65±0.14mmol/L，与针刺前相比有显著性差异(P＜0.01)；停针后10min，血糖浓度继续下降至最低值6.43±0.14mmol/L，与停针即刻浓度相比，有统计学差异(P＜0.05)；停针后20min，血糖开始慢慢上升至6.84±0.14mmol/L，与停针10min相比有显著差异(P＜0.05)，与停针即刻相比无显著差异(P＞0.05)；停针后30min，血糖恢复至电针前水平，浓度为7.17±0.14mmol/L，与电针前相比无统计学差异(P＞0.05)。
2mA电针刺激正常大鼠足三里穴30min，针前血糖浓度为7.45±0.33mmol/L，电针30min停止，停针即刻血糖浓度为7.49±0.27mmol/L，与针刺前相比无显著性差异(P＞0.05)；停针后10min，血糖浓度小幅度下降至7.41±0.29mmol/L，血糖浓度虽然下降但与停针即刻浓度相比无显著性差异(P＞0.05)；停针后20min，血糖开始缓慢上升至7.43±0.27mmol/L，但与10min前比较无显著性变化；停针后30min，血糖浓度恢复至7.44±0.25mmol/L，与电针前相比较没有统计学差异(P＞0.05)。
3.3.3不同穴位针刺对正常大鼠胰岛素浓度的影响
耳针组：2mA电针耳甲区穴位，针刺前正常大鼠血浆胰岛素浓度为44.65±2.95μmol/L，针刺后胰岛素浓度均值上升为50.06±3.88μmol/L，配对t检验，耳针前后胰岛素浓度无显著性差异(P＞0.05)；对照组：2mA针刺足三里前胰岛素浓度为：48.63±3.79μmol/L，针刺后胰岛素浓度均值也上升至51.76±2.93μmol/L，配对t检验，与针刺前比较，针刺前后胰岛素浓度亦无显著性差异(P＞0.05)。
3.3.4耳针刺激对糖尿病大鼠血糖浓度的影响
耳针组：2mA电针DM大鼠耳甲穴位，耳针前血糖浓度为17.33±0.60mmol/L，针刺后下降为16.58±0.63mmol/L，配对t检验，有显著性差异(P＜0.05)；迷切耳针组：右侧迷走神经切断后，2mA电针DM大鼠耳甲区穴位，针刺前大鼠血糖为17.57±0.80mmol/L，迷切耳针后血糖浓度升高为18.23±0.81mmol/L，配对t检验，无显著性差异(P＞0.05)；对照组：2mA电针DM大鼠足三里穴，针刺前大鼠血糖值为：17.69±0.79mmol/L，针刺后小幅度下降为17.59±0.82mmol/L，配对t检验，无显著性差异(P＞0.05)。
3.3.5耳针刺激对糖尿病大鼠胰岛素浓度的影响
耳针组：2mA电针耳甲区穴位，耳针前DM大鼠血浆胰岛素浓度为50.66±2.15μmol/L，耳针后胰岛素浓度升高为58.49±2.44μmol/L，配对t检验，针刺前后有极显著性差异(P＜0.001)；迷切耳针组：右侧迷走神经切断后，2mA电针耳甲区穴位，针刺前DM大鼠胰岛素浓度为50.53±2.06μmol/L，耳针后小幅度下降为47.86±1.34μmol/L，配对t检验，针刺前后无显著性差异(P＞0.05)；对照组：2mA针刺DM大鼠足三里前胰岛素浓度为：46.32±1.86μmol/L，针刺后升高为51.79±2.23μmol/L，配对t检验，与针刺前比较，有显著性差异(P＜0.05)。
4结论
本研究分别从电生理学、形态学、生物化学等实验角度探讨了电针耳穴降糖效应的中枢脑干参与机制。实验结果显示，不同强度电针耳穴对正常大鼠血糖浓度具有不同的效应：高强度的耳针升高血糖，而低强度的耳针则具有较好的降糖效应；与针刺足三里相比较，低强度耳针对正常大鼠的血糖调节具有时间窗效应，耳针停止后10分钟血糖下降至最低，此后血糖缓慢恢复至耳针前水平。电针耳穴与足三里穴均能升高正常大鼠血浆胰岛素水平，耳针组有极显著性意义，而足三里组与针刺前比较，胰岛素升高无显著性意义。耳针对糖尿病大鼠具有显著性降低血糖效应，这种效应可被迷走神经切断所阻断，上述实验说明耳针降糖效应可能与大鼠处于不同状态有关，耳针对血糖与血胰岛素的调节作用需要有完整的迷走神经支持。电生理学实验结果显示，孤束核处存在葡萄糖敏感和胰岛素敏感神经元，电针耳甲区穴位能够激活葡萄糖敏感和胰岛素敏感神经元，并且以葡萄糖抑制反应的细胞为主。形态学实验则运用HRP跨节段神经示踪法发现了耳甲区神经末梢与中枢脑干迷走初级中枢孤束核、迷走运动背核具有直接投射联系，这一结果颠覆了经典解剖学关于耳甲迷走分支只投射于三叉神经脊束核的传统观念，为耳针激活了孤束核葡萄糖敏感和胰岛素敏感神经元，继而升高胰岛素，降低血糖效应奠定了形态学理论基础，并为完善“耳-迷走-胰岛素系统反射”理论提供了组织学佐证。
总之，本研究从功能学证实了耳甲区穴位电刺激对不同状态的大鼠血糖和胰岛素具有不同的调节功能，耳针的效应产生可能与耳甲腔与迷走初级中枢特别是孤束核之间存在直接的纤维联系这一形态学基础有关；耳针的降糖效应可能是通过调节孤束核处的葡萄糖敏感神经元和胰岛素敏感神经元的活动，特别是调节对葡萄糖起抑制反应的神经元活动而产生的。
Diabetes mellitus is becoming one of the most common diseases associated withthe improvement of peoples' lives. Acupuncture, particularly auricular acupuncture,exhibits pronounced hypoglycemia effect in treating diabetes. But the mechanism isunclear up to now. Some researchers hold that the mechanism underlying the effectsof auricular acupuncture on hypoglycemia was conclusively attributed to thesegmental innervations of sympathetic nervous systems. However, they ignored thefact that auricular acupuncture possibly participated in regulating the interactionsbetween the afferent vagal nerve from aricular concha and the visceral organs whichconverge in the brainstem especially the complex of vagus. Based on the fact that theauricular vagus branch is the only peripheral branch of the vagal nerve, and thatglucose-responsive and insulin-responsive neurons possibly exist in the nucleustractrus solitarri (NTS), furthermore, acupuncture in auricular concha acupoints canaffect the physiological activity of the above two types of neurons. So, wehypothesize that the mechanism of hypoglycemia effect induced by needling orpressing on auricular point is correlative with the auriculo-vagus nerve systems reflex.
The present study investigated the effects of electrical stimulation at the conchaacupoints on the discharges of glucose-responsive and insulin-responsive neurons, thesynaptic correlation between the auricular concha and the vagal primary center, andchange of glucose and insulin concentration of normal and diabetic rats which wereapplied with electrical acupuncture in auricular acupoints. In this study we tried toelucidate the CNS mechanism of effects caused by needling stimulation at auricularconcha acupoints in the light of auriculo-vagal nerve system reflex.
PartⅠElectrophysiological experent: Responses of glucose-sensitive neuronsand insulin-sensitive neurons in nucleus tractus solitarius to electricalacupuncture at auricular concha acupoints
Methods To observe responses of glucose-sensitive neurons and insulin-sensitiveneurons in nucleus tractus solitarius to acupuncture stimulation at auricular conchaacupoints (representation of pancreas, liver and gall, et,al), and to investigatepreliminarily the brainstem's mechanism of hypoglycaemia effect caused by auricular acupuncture, we used extracellular single-unit recordings which were carried out innucleus tracus solitarius of rats followed intra-carotid administration of glucose(15%), insulin (6IU/ml) and acupuncture stimulation at auricular concha(interval:0．4s, duration:0．4ms intensity:2mA),respectively.
Results In the group of administration of glucose (15%, 0．25ml/min) , thedischarges of totally 110 NTS neurons were recorded. Among them there were 41(37．27%) neurons responding to glucose infusion with a discharges decrease inpercent of 60．36±15．1%, while 10．91 %(12/110) neurons responding to glucoseinfusion with a discharges increase with a percentage of 56．78±9．24%. 57 (51．82%)neurons showed no response in discharges frequency during or after administration ofsugar solution via carotis. During or after intra-carotid administration of insulin(6IU/ml, 0．25ml/min) , totally 81 NTS neurons' discharges were observed . Amongthem, there were 33．33% (27/81) neurons showed increase in spike frequency with apercent of 47．34±11．24%. Meanwhile, there were 4(4．9%) neurons exhibited decreasein spike frequency with a percent of 43．24±12．11%. Among the above 27exciting-responding neurons, 18 cells showed decrease in activity to administration ofglucose via carotis, while 9 cells showed no response in spike frequency to sugarinfusion. Among the 4 neurons which were inhibitory-responsive to insulinadministration, there were 2 neurons showed contrary responsive to sugar infusion viacarotis, the others exhibited no response in frequency of discharge.
The discharges of totally 69 neurons in NTS were examined during or afteradministration of electro-acupuncture at auricular concha. Among them, there were 34(49．27%)neurons showed increase in spike frequency with a percentage of 54．79±24．21%, 3 (4．35%) ones showed decease in spike frequency with a percentage of44．65±9．13%, the others (46．38%)demonstrated no response to the acupuncture atauricular concha. Glucose infusion via carotis resulted in a decrease in firing rate of18 neurons in the totally 34 neurons which showed exciting-response to auricularacupuncture, while there were 2 neurons in the 34 ones showed contrary response tothe administration of glucose, and the others showed no response to glucoseconcentration's elevation. Among the same 34 neurons, 8 neurons showed anexcitatory response to insulin infusion, 3 ones showed contrary response to theadministration of insulin, the others showed no response to insulin concentration'selevation. During or after glucose infusion via carotis, the latence ofexcitatory-response and inhibitory-response neurons recorded in NTS wererespectively 15±6 and 18±8s, and those were respectively 16±7s and 18±10s during or after administration of insulin, no significant difference between them (P>0．05) . Thelatence of neurons recorded in NTS showing excitatory-response andinhibitory-response to auricular acupuncture were respectively 3±1s and 5±2s.Furthermore, among the totally 37 recorded during auricular acupuncture, there were21 neurons exhibited 2-5 minutes' long-term increase in firing rate. 2 neurons werestimulated to waken up from silence. 3 ones in the 37 responsive neurons showedinhibitory-response to auricular acupuncture with long-term's decrease in firing rate.
PartⅡMorphological experiment: Research the medullary distribution ofafferent fibers and cells of origin from auricular concha using transganglionictransport of horseradish peroxidase (HRP) as a neuronal tracer.
Methods 10 adult male and female healthy Sprague-Daxley rats weighing 200-250gwere anesthetized with 30% urethane solution (1g/kg i.p.). Under general anesthesia, askin incision was made in the region of auricular concha. The skin and fasciae wereseparated to expose the auricular nerve of vagus nerve, which were traced carefully tothe region of the ear root where the auricular nerve of vagus nerve was cut. Fortymicroliters of a 30% solution of HRP (Sigma) in normal saline were subcutaneouslyinjected into the cut central end of auricular branch of vagus nerve. After survivaltimes of 36hrs to 48hrs, all animals were reanesthetized and perfused intranscardiallywith 500 ml of normal saline, followed by a phosphate buffered (PH=7．4) fixativecontaining 1．25% glutaraldehyde and 1% formaldehyde. The medulla oblongata andinterbrain were moved in each animal and transferred to phosphate buffer containing5% sucrose for 12-24h at 4℃. Serial sections (30μm) were cut through the tissues ona freezing microtome. All sections were processed for HRP activity using tetramethylbenzidine as the chromogen.
Results HRP-labeled terminal or preterminal fibers were identified under the lightmicroscope. They often appear as brown granules of various sizes and differentshapes in medulla oblongata and interbrain. Heavily labeled neurons and fibers wereseen in NTS and DMV, as well as in the nucleus of spinal tract of trigeminal nerve,where granular labeling observed was heaviest at the same level. Labeling in themedial division of the NTS extended dorsally adjacent to the AP. In the one animal inwhich measurements were made, granular labeling extended for 2．6mm in theipsilateral NTS sparing 0．4mm of the nucleus rostrally. HRP-labeled cells in NTSwere frequently fusiform and ovoid, while those in DMV were often ovoid.Retrograde labeling of neurons was also observed in the nucleus of ambiguous and the region of reticular formation, and the labeled neurons were rounded or oviform in shape and 20×30μm in size.
PartⅢBiochemical experiment: Effects of electro acupuncture on the plasma glucose and insulin concentration of normal and diabetic rats.
Methods The experiment was carried out on 100 healthy male rats, 150-220g in body weight. Among the whole animals, 40 were normal rats, and the others were diabetic rats, which were induced by once intraperitoneal injection of streptozotocin (60mg.kg). The normal animals were divided into three groups in this dose-finding experiments: Group A consisted of 10 normal rats which were administered with electrostimulation at the intensity of 10 mA , wave of 1ms , applied in auricular concha points; Group B were applied in the same acupoints electrostimulation at the intensity of 2 mA , wave width of 0．5ms; Group C were administered with the same intensity as Group B of electrostimulation at the Zusanli (ST36) acupoint. Diabetic rats were divided into 3 groups: auriculo-acupuncture group (Group D) received the same electrostimulation as Group B; Vagotomy followed auriculo-acupuncture group (Group E) received the same electrostimulation with Group B after cutting left cervical vagal nerve; Group F was control group which were administered the same electrostimulation as Group C. Each group rats were received 30min's electrostimulation, then several blood samples (0．2-0．5ml each) were taken from the femoral vein respectively at the time of 0, 10, 20, 30 minutes after stopping acupuncture stimulation and placed them in Eppendorf tube containing 10 IU heparin. The plasma glucose was measured using Onetouch Ultra Brand blood glucose meter and insulin were determined in the way of immunosorbent assay, American BPB Inc. supplied with commercial kits.
Results
1) After 30 minutes' electrical acupuncture on the auricular concha acupoints, which was of 10 mA in intensity, 1ms in wave width, the glucose concentration responded with a hyperglycemia from average of 6．48±0．28 mmol/L before the acupuncture to 7．70±0．38 mmol/L at the point of 10mins after stopping acupuncture (P<0．05, paired t-test, same to following). While electrical acupuncture on auricular concha acupoints with 2 mA of intensity, 0．5ms of wave width induced a hypoglycemia from average of 7．23±0．15 mmol/L before the acu-stimulation to 6．43±0．14 mmol/L 10 minutes after acupuncture stop; there was statistical significance in glucose concentration before and after acu-stimulation on auricular concha (P<0．01) . In the control group, no significant difference in glucose concentration was induced by the electrical stimulation applied to need inserting in the tissue at Zusanli acupoint (7．45±0．33 mmol/L vs 7．41±0．29 mmol/L, P>0．05) .
2) Lower intensity (2 mA in intensity, 1ms in wave width) of auricular acupuncture applied to normal rats showed a time-window regulation of glucose concentration. Before the auri-acupuncture, the average of blood suger level was 7．23±0．15mmol/L, after 30 minutes' administration of electrical stimulation on auricular concha, the suger level reduced to 6．65±0．14mmol/L when the stimulation came to a stop (P<0．01 vs before auricular acupuncture). 10 minutes later, glucose concentration continually decreased to a lowest value of 6．43±0．14mmol/L (P<0．01 vs when auri-acupuncture stopped). Then a hyperglycemia appeared as time gone on, and at the point of 20 minutes later, the glucose went up to 6．84±0．14mmol/L (P<0．05 vs 10 minutes later; but P>0．05 vs before acupuncture stimulation). The glucose concentration increased to the normal level as before the auricular electrical stimulation (P>0．05 vs before acupuncture stimulation). In the control group, above time-window regulation of glucose concentration was not induced by electrical stimulation at ST36 acupoint. Before the acupuncture stimulation at ST36, glucose concentration of the control group was 7．45±0．33mmol/L, and then was 7．49±0．27mmol/L when the 30 minutes' acupuncture came to a stop. There was no statistical significance between them., 10 minutes later the glucose slowly moved down to 7．41±0．29mmol/L (P>0．05 vs when acupuncture stopped), one more 10 minutes later the concentration went up to 7．43±0．27mmol/L, then to 7．44±0．25mmol/L at the point of 30 minutes after the acupuncture stopped. There were no differences in statistics among them.
3) However, the time-window regulation of glucose concentration of normal rats induced by the lower intensity of auricular acupuncture could not be explained absolutely by the alteration of insulin concentration, which was also induced by the auricular acupuncture as well. Before the auri-acupuncture, insulin concentration was 44．65±2．95μmol/L, then went up to 50．06±3．88μmol/L after 30 minutes' acupuncture at auricular concha, but there was no distinguished difference between them which was compared by the paired t-test(P>0．05) .In the control group, the same phenomenon was obserbed in normal rats during the electrical acupuncture stimulation was applied to the ST36 acupoint. Insulin concentration was increased to 51．76±2．93μmol/L at the point of 10 minutes after the stop of stilulation from 48．63±3．79μmol/L before stimulation, no distinguished difference was found in the above two sets of data which were compared by paired t-test analysis.
4) Acupuncture applied to auricular concha acupoint could lower plasma glucose concentrations in diabitic rats. An insulin-dependent action thus could be considered. Lowering of plasma glucose in diabetic model rats was investigated from 17．33±0．60mmol/L to 16．58±063mmolL after EA-stimilation at auricular concha acupoints (P<0．05, before auri-acupunctrure vs after auri-acupuncture). This hypoglycemia activity was blocked by vagotomy (17．57±0．80mmol/L vs 18．23±0．81mmol/L, P>0．05) . In the control group, EA-stimulation at the ST36 acupoint elicited a slender reduction in plasma glucose concentration (from 17．69±0．79mmol/L to 17．59±0．82mmol/L), but no distinguished difference was found between two sets of data analysised by the paired t-test.
5) An insulin-dependent action could be explained the hypoglycemia activity elicited by acupuncuture. After the EA-stimulation at auricular concha, plasma insulin in diabetic rats were raised from 50．66±2．15μmol/L to 58．49±2．44μmol/L (P<0．001) .Blockade of auricular acupuncture-induced increase of plasma insulin was seen in the diabetic rats which were received vagotomy, and no significantly difference was found before and after the electrical acupuncture at auricular concha followed vagotomy (50．53±2．06μmol/L vs 47．86±1．34μmol/L, P>0．05) . In the control group, EA-stimulation at ST36 acupoint also raised plasma insulin concentration from 46．32±1．86μmol/L up to 51．79±2．23μmol/L (P<0．05) . So we could possibly attribute the hypoglycemia effect induced by acupuncture to the insulin-dependence.
Conclusion
The present study including three parts of experiments were carried out to investigate the relationship between the mechanism of hypoglycemia induced by electrical acupuncture at auricular acupoints and Auriculo-vagal nerve system reflex. The experiment results showed that different intensities of acupuncture at concha points casused different effects on the glucose concentration alteration in normal rats. Lower density of electrical acupuncture at concha acupoints had a pronounced time-window regulation of plasma glucose, compared with the control group, in which electrical stimulation was applied to a needle inserting into the tissue at ST36 acupoint. Acupuncture either in auricular concha or in ST36 acupoints could raise plasma insulin in diabetic rats. In the group of auricular acupuncture this raise had significant difference, while in the group of ST36 acupuncture, the raise was slender. The effect of pronounced reduction in plasma glucose concentration could be elicited by electrical auri-acupuncture, which could be blocked by vagotomy. There existed glucose-responsive neurons and insulin-responsive neurons in NTS. Electrical acupuncture stimulation at auricular concha acupoints could affect the activity of these neurons, especially could irritate the neurons' firing which showed inhibitory response to glucose administration. Directly neural projections in the NTS and DMV from the terminal of auricular vagus branch in the region of auricular concha were examined by using the HRP transganglionic tracing. The morphological results gave a rational explaination of the electrophysiolodical outcome, and supplied with morphology evidence for auriculo-vagal nerve system reflex theory. This study showed that the hypoglycemia effects of auricular acupuncture on different conditions of rats were distinct. Lowing of plasma glucose induced by auri-acupuncture should be supported by intact vagal nerve.
1电生理实验：电针耳穴对孤束核葡萄糖、胰岛素敏感神经元胞外放电活动的影响
1.1实验动物
实验用健康成年Sprague-Dawley(SD)雄性大鼠51只，清洁级，体重200～220g，中国医学科学院实验动物中心提供(合格证编号：A06-053)。
1.2实验步骤
大鼠麻醉后，动物俯卧，将大鼠头部用耳棒固定于立体定位仪头部固定器上，以玻璃微电极细胞外纪录不同刺激对孤束核(NTS)神经元的放电影响，电极内充有2％滂胺天蓝溶液(电阻为10-20MΩ)实验开始后，通过微电极操作器控制，在NTS内探查神经元放电。实验探查到NTS细胞放电后，将其随机分为三组，每组按如下顺序施加因素：
①NTS葡萄糖反应神经元的鉴别：颈动脉推注葡萄糖(浓度为15％，推注速度为0.25ml/min)，观察孤束核细胞放电频率的变化，鉴别葡萄糖反应神经元的类型(放电频率升高、降低、不变)，如细胞放电频率发生变化，则待细胞恢复稳定放电频率状态，关闭三通管葡萄糖端，打开生理盐水端开关，推注生理盐水(推注速度为0.25ml/min)，继续观察细胞放电频率变化，如果所记录的神经元放电方式对生理盐水不出现反应变化(排除血压变化影响)，而对葡萄糖有反应变化则认为是实验目标神经元，否则放弃统计(下同)。
②NTS胰岛素反应神经元的鉴别及其对葡萄糖浓度升高的反应：颈动脉推注胰岛素(浓度为6IU/ml，推注速度同前)，观察孤束核细胞放电频率的变化，鉴别胰岛素反应神经元的类型(放电频率升高、降低、不变)，如细胞放电频率发生变化，则待细胞恢复稳定放电频率状态后，关闭三通管胰岛素端，打开生理盐水端开关，推注生理盐水(推注速度同前)，作空白对照。若细胞活动对胰岛素浓度升高敏感，则继续颈动脉注射葡萄糖(浓度为15％，推注速度同前)，观察胰岛素反应神经元对葡萄糖的敏感性。
③耳甲区穴位电针刺激对葡萄糖、胰岛素反应神经元的放电活动影响：耳甲区穴位(相当于人耳穴的胰腺、肾、肝胆等点)电刺激(参数：间隔：0.4s，持续时间：0.4ms，强度：2mA)每次电针刺激维持时间为30s，观察细胞放电活动的变化情况(频率变化，放电大小、有无变化)，如果细胞放电频率发生明显变化，待细胞放电活动稳定后进一步分别监测颈动脉推注葡萄糖(浓度为15％)及胰岛素(6IU/ml)对该细胞放电活动的影响(速度同前)，如果细胞放电活动发生反应，则待细胞放电活动稳定后继续推注生理盐水作空白对照。。
上述实验过程中，神经元反应变化类型鉴别：神经元放电频率比基础放电频率(或稳定状态放电频率)升高或者降低30％以上，则称为细胞活动兴奋或细胞活动抑制。
1.3实验结果
大鼠颈动脉推注葡萄糖，共观察了110个NTS神经元放电变化情况，结果显示推注葡萄糖后57个细胞(51.82％)放电频率无变化，有41个细胞(37.27％)放电频率减少，呈抑制反应，其中有两个细胞放电活动完全被抑制。放电频率的降低率为60.36±15.1％(均值±SD，下同)，12个细胞(10.91％)放电频率增加，呈兴奋反应，放电频率的增加率为56.78±9.24％。
给大鼠颈动脉注射胰岛素，共记录81个NTS细胞放电变化情况，据统计有50个细胞(61.73％)放电频率无变化，27个细胞(33.33％)放电频率增加，上升率为47.34±11.24％，4个细胞(4.94％)放电频率减少，下降率为43.24±12.11％。其中27个兴奋反应细胞中有18个细胞对葡萄糖有抑制反应，9个细胞对葡萄糖无反应；4个对胰岛素抑制反应细胞中有2个对葡萄糖有兴奋反应，2个对葡萄糖无反应。
电针大鼠耳甲穴位，共记录69个NTS细胞放电，统计结果显示针刺前后有32个细胞(46.38％)放电频率无变化，34个细胞(49.27％)针刺过程中或停针后放电频率增加，增加率为54.79±24.21％，此外3个细胞(4.35％)停针后放电频率减少，减少率为44.65±9.13％；其中34个对耳针起兴奋反应的细胞中有18个对输注葡萄糖呈现放电抑制反应，2个对葡萄糖有兴奋反应，14个对葡萄糖无反应；同样是上述34个对耳针起兴奋反应细胞中，有8个对输注胰岛素有兴奋反应，3个对胰岛素起抑制反应，23个对胰岛素无反应。
输注葡萄糖组，所记录到的孤束核神经元起兴奋反应和抑制反应的潜伏期分别为15±6s和18±8s；输注胰岛素组，孤束核神经元起兴奋反应和抑制反应的潜伏期分别为16±7s和18±10s，二者之间无显著性差异(P＞0.05)。电针耳甲穴位，记录到的相关孤束核细胞兴奋反应的潜伏期为3±1s，相关神经元细胞起抑制反应的潜伏期为5±2s，且耳针实验中，37个反应细胞中有21个细胞呈现2-5min长时程的放电频率增加，其中有2例细胞由原来的静息状态突发为放电状态；3个抑制性反应细胞均呈长时程放电频率抑制。
2形态学实验：HRP神经示踪法探讨耳甲区神经传入在低位脑干的投射联系
2.1实验动物
实验用健康成年SD大鼠10只，雌雄不拘，清洁级，体重200～220g，中国医学科学院实验动物中心提供(合格证编号：A06-084)。
2.2实验步骤
大鼠经2％戊巴比妥钠腹腔注射麻醉(40mg/kg)后，在其耳甲部用手术刀片横向划开一条长约2mm缝，向外耳道口方向分离缝内耳甲皮肤与皮下组织，微量注射器针头向分离后的缝内注射辣根过氧化物酶(HRP：30％，40μl)，注射完毕，退针后，紧按缝口3-5min，每只大鼠两耳各注射3-5个点，注射点尽量分布于整个耳甲艇和耳甲腔。HRP注射后动物存活36-48h，动物用2％戊巴比妥钠腹腔注射再次麻醉(30mg/kg)后，动脉灌流固定，次日取延髓部(闩上下2-3cm)冰冻切片，片厚30μm，裱于挂胶载玻片上风干以待呈色反应。经TMB呈色反应后，酒精梯度脱水，二甲苯透明，树脂封片，并在显微镜下观察结果。
2.3实验结果
10只大鼠在耳甲区注射HRP后，经TMB反应呈色，除了在三叉神经脊束核发现了标记细胞外，在孤束核和迷走神经背核也发现了标记神经元或神经纤维，其中孤束核处标记细胞多呈圆形或菱形，其胞浆充满了蓝黑色的颗粒，围绕细胞核的周围，其直径大小约20μm；迷走神经背核处标记细胞多为菱形或卵圆形，细胞大小较孤束核处标记细胞大，神经元突触标记明显，并且可见阳性纤维存在。此外在腹外侧网状核以及疑核等处也发现了阳性标记细胞，细胞呈圆形或菱形，直径大小约20-30μm。
3生化实验：耳针刺激对正常和糖尿病大鼠血糖水平、胰岛素水平的影响，及耳针降糖的时间窗效应观察
3.1实验动物
健康雄性SD大鼠100只，体重150-220g，清洁级，中国医学科学院实验动物中心提供(合格证编号：A06-084)。室温(25±2)℃，维持12h/24h昼夜规律，饲料，自来水自由摄取，适应环境1周。
3.2实验步骤
3.2.1模型制作
参照《药理学实验方法》，采用速发型糖尿病动物模型制作方法：动物腹腔注射2％链脲佐菌素(STZ)60mg/kg，溶于柠檬酸-柠檬酸钠缓冲液(PH=4.2，0.1M)过滤除菌，冰浴操作，一周后尾静脉取血，测定大鼠空腹血糖(采用美国强生Ⅱ型快速血糖仪)，血糖高于15mmol/L定为DM模型制作成功。
3.2.2实验分组
正常饲养的大鼠随机分为三组：
A组高强度耳针组(刺激参数：波宽1ms，强度10mA)；
B组低强度耳针组(刺激参数：波宽0.5ms，强度2mA)；
C组低强度电针足三里组(刺激参数：波宽0.5ms，强度2mA)
造模成功的糖尿病大鼠随机分为另外三组：
D组耳针组(刺激参数：波宽0.5ms，强度2mA)
E组迷走切断耳针组(刺激参数：波宽0.5ms，强度2mA)
F组足三里组(刺激参数：波宽0.5ms，强度2mA)
3.2.3针刺处理过程
上述各组大鼠电针处理刺激前用10％的乌拉坦腹腔注射麻醉(1g/kg)，手术分离股动脉插管(吸入肝素湿润管周，下同)采血0.3-0.5ml，快速血糖仪测针刺前血糖水平，并离心取血浆待测胰岛素浓度。
A组：将自制的电针耳豆用医用胶布粘贴至大鼠耳甲区穴位(耳甲艇或耳甲腔)，正极置于耳甲腔面，负极置于耳甲背面正对正极耳豆处，正负极连接于刺激器，打开刺激器，电针30min停止，10min后采血0.3-0.5ml，快速测量血糖浓度，离心分离血浆-78℃保存待测胰岛素浓度。
B组：方法同A组，电针刺激30min停止，分别于电针停止即刻、电针停止后10min、20min、30min抽取血样各0.3ml-0.5ml，快速测量血糖浓度，分别离心分离血浆后-78℃保存待测各次血浆胰岛素浓度。
C组：参照华兴邦大鼠穴位图谱针刺大鼠后三里(负极)及中脘穴(正极)，正负极连接于刺激器，打开刺激器，电针30min停止，分别于电针停止即刻、电针停止后10min、20min、30min抽取血样各0.3ml-0.5ml，快速测量血糖浓度，分别离心分离血浆后-78℃保存待测各次血浆胰岛素浓度。
D组：耳针方法参数同B组，电针刺激30min停止，10min后采血0.5ml，快速测血糖浓度，离心分离血浆-78℃保存待测胰岛素浓度。
E组：电针前股动脉采血后，手术分离并切断大鼠右侧颈部迷走神经，余下电针处理同D组。
F组：电针处理同C组，电针刺激30min停止，10min后采血0.5ml，快速测血糖浓度，离心分离血浆-78℃保存待测胰岛素浓度。
3.2.4指标检测
血糖浓度采用强生公司One-touch-Ⅱ型快速血糖仪及血糖试纸测量，检测原理为葡萄糖氧化酶法。胰岛素浓度检测采用酶联免疫法，试剂盒由美国BPB生物公司提供，检测方法严格按照试剂盒说明说进行。
3.3实验结果
3.3.1不同强度的针刺对正常大鼠血糖水平影响
分别采用10mA和2mA的强度电针耳甲区穴，对照组为2mA的电针足三里穴位，结果显示10mA电针耳甲，血糖浓度针刺前为6.48±0.28mmol/L，针刺30min，停针后10min血糖浓度升高为7.70±0.38mmol/L，配对t检验，有显著性差异(P＜0.05)，说明高强度的电针耳穴非但不能降低血糖，而且使血糖浓度升高；2mA电针耳甲，血糖浓度由电针前7.23±0.15mmol/L下降至针后10min 6.43±0.19mmol/L，配对t检验，有显著性差异(P＜0.01)，说明低强度的电针耳穴对正常大鼠的血糖浓度具有较好的调整作用；对照组电针足三里穴，电针前血糖浓度为7.45±0.33mmol/L，停针后10min血糖浓度值为7.41±0.29mmol/L，血糖虽有下降趋势，但无统计学差异(P＞0.05)。
3.3.2低强度电针耳穴与足三里穴对正常大鼠血糖浓度的时间窗效应比较
2mA耳针刺激正常大鼠30min，针刺前大鼠血糖浓度为7.23±0.15mmol/L，耳针30min停止，停针即刻血糖浓度下降至6.65±0.14mmol/L，与针刺前相比有显著性差异(P＜0.01)；停针后10min，血糖浓度继续下降至最低值6.43±0.14mmol/L，与停针即刻浓度相比，有统计学差异(P＜0.05)；停针后20min，血糖开始慢慢上升至6.84±0.14mmol/L，与停针10min相比有显著差异(P＜0.05)，与停针即刻相比无显著差异(P＞0.05)；停针后30min，血糖恢复至电针前水平，浓度为7.17±0.14mmol/L，与电针前相比无统计学差异(P＞0.05)。
2mA电针刺激正常大鼠足三里穴30min，针前血糖浓度为7.45±0.33mmol/L，电针30min停止，停针即刻血糖浓度为7.49±0.27mmol/L，与针刺前相比无显著性差异(P＞0.05)；停针后10min，血糖浓度小幅度下降至7.41±0.29mmol/L，血糖浓度虽然下降但与停针即刻浓度相比无显著性差异(P＞0.05)；停针后20min，血糖开始缓慢上升至7.43±0.27mmol/L，但与10min前比较无显著性变化；停针后30min，血糖浓度恢复至7.44±0.25mmol/L，与电针前相比较没有统计学差异(P＞0.05)。
3.3.3不同穴位针刺对正常大鼠胰岛素浓度的影响
耳针组：2mA电针耳甲区穴位，针刺前正常大鼠血浆胰岛素浓度为44.65±2.95μmol/L，针刺后胰岛素浓度均值上升为50.06±3.88μmol/L，配对t检验，耳针前后胰岛素浓度无显著性差异(P＞0.05)；对照组：2mA针刺足三里前胰岛素浓度为：48.63±3.79μmol/L，针刺后胰岛素浓度均值也上升至51.76±2.93μmol/L，配对t检验，与针刺前比较，针刺前后胰岛素浓度亦无显著性差异(P＞0.05)。
3.3.4耳针刺激对糖尿病大鼠血糖浓度的影响
耳针组：2mA电针DM大鼠耳甲穴位，耳针前血糖浓度为17.33±0.60mmol/L，针刺后下降为16.58±0.63mmol/L，配对t检验，有显著性差异(P＜0.05)；迷切耳针组：右侧迷走神经切断后，2mA电针DM大鼠耳甲区穴位，针刺前大鼠血糖为17.57±0.80mmol/L，迷切耳针后血糖浓度升高为18.23±0.81mmol/L，配对t检验，无显著性差异(P＞0.05)；对照组：2mA电针DM大鼠足三里穴，针刺前大鼠血糖值为：17.69±0.79mmol/L，针刺后小幅度下降为17.59±0.82mmol/L，配对t检验，无显著性差异(P＞0.05)。
3.3.5耳针刺激对糖尿病大鼠胰岛素浓度的影响
耳针组：2mA电针耳甲区穴位，耳针前DM大鼠血浆胰岛素浓度为50.66±2.15μmol/L，耳针后胰岛素浓度升高为58.49±2.44μmol/L，配对t检验，针刺前后有极显著性差异(P＜0.001)；迷切耳针组：右侧迷走神经切断后，2mA电针耳甲区穴位，针刺前DM大鼠胰岛素浓度为50.53±2.06μmol/L，耳针后小幅度下降为47.86±1.34μmol/L，配对t检验，针刺前后无显著性差异(P＞0.05)；对照组：2mA针刺DM大鼠足三里前胰岛素浓度为：46.32±1.86μmol/L，针刺后升高为51.79±2.23μmol/L，配对t检验，与针刺前比较，有显著性差异(P＜0.05)。
4结论
本研究分别从电生理学、形态学、生物化学等实验角度探讨了电针耳穴降糖效应的中枢脑干参与机制。实验结果显示，不同强度电针耳穴对正常大鼠血糖浓度具有不同的效应：高强度的耳针升高血糖，而低强度的耳针则具有较好的降糖效应；与针刺足三里相比较，低强度耳针对正常大鼠的血糖调节具有时间窗效应，耳针停止后10分钟血糖下降至最低，此后血糖缓慢恢复至耳针前水平。电针耳穴与足三里穴均能升高正常大鼠血浆胰岛素水平，耳针组有极显著性意义，而足三里组与针刺前比较，胰岛素升高无显著性意义。耳针对糖尿病大鼠具有显著性降低血糖效应，这种效应可被迷走神经切断所阻断，上述实验说明耳针降糖效应可能与大鼠处于不同状态有关，耳针对血糖与血胰岛素的调节作用需要有完整的迷走神经支持。电生理学实验结果显示，孤束核处存在葡萄糖敏感和胰岛素敏感神经元，电针耳甲区穴位能够激活葡萄糖敏感和胰岛素敏感神经元，并且以葡萄糖抑制反应的细胞为主。形态学实验则运用HRP跨节段神经示踪法发现了耳甲区神经末梢与中枢脑干迷走初级中枢孤束核、迷走运动背核具有直接投射联系，这一结果颠覆了经典解剖学关于耳甲迷走分支只投射于三叉神经脊束核的传统观念，为耳针激活了孤束核葡萄糖敏感和胰岛素敏感神经元，继而升高胰岛素，降低血糖效应奠定了形态学理论基础，并为完善“耳-迷走-胰岛素系统反射”理论提供了组织学佐证。
总之，本研究从功能学证实了耳甲区穴位电刺激对不同状态的大鼠血糖和胰岛素具有不同的调节功能，耳针的效应产生可能与耳甲腔与迷走初级中枢特别是孤束核之间存在直接的纤维联系这一形态学基础有关；耳针的降糖效应可能是通过调节孤束核处的葡萄糖敏感神经元和胰岛素敏感神经元的活动，特别是调节对葡萄糖起抑制反应的神经元活动而产生的。
Diabetes mellitus is becoming one of the most common diseases associated withthe improvement of peoples' lives. Acupuncture, particularly auricular acupuncture,exhibits pronounced hypoglycemia effect in treating diabetes. But the mechanism isunclear up to now. Some researchers hold that the mechanism underlying the effectsof auricular acupuncture on hypoglycemia was conclusively attributed to thesegmental innervations of sympathetic nervous systems. However, they ignored thefact that auricular acupuncture possibly participated in regulating the interactionsbetween the afferent vagal nerve from aricular concha and the visceral organs whichconverge in the brainstem especially the complex of vagus. Based on the fact that theauricular vagus branch is the only peripheral branch of the vagal nerve, and thatglucose-responsive and insulin-responsive neurons possibly exist in the nucleustractrus solitarri (NTS), furthermore, acupuncture in auricular concha acupoints canaffect the physiological activity of the above two types of neurons. So, wehypothesize that the mechanism of hypoglycemia effect induced by needling orpressing on auricular point is correlative with the auriculo-vagus nerve systems reflex.
The present study investigated the effects of electrical stimulation at the conchaacupoints on the discharges of glucose-responsive and insulin-responsive neurons, thesynaptic correlation between the auricular concha and the vagal primary center, andchange of glucose and insulin concentration of normal and diabetic rats which wereapplied with electrical acupuncture in auricular acupoints. In this study we tried toelucidate the CNS mechanism of effects caused by needling stimulation at auricularconcha acupoints in the light of auriculo-vagal nerve system reflex.
PartⅠElectrophysiological experent: Responses of glucose-sensitive neuronsand insulin-sensitive neurons in nucleus tractus solitarius to electricalacupuncture at auricular concha acupoints
Methods To observe responses of glucose-sensitive neurons and insulin-sensitiveneurons in nucleus tractus solitarius to acupuncture stimulation at auricular conchaacupoints (representation of pancreas, liver and gall, et,al), and to investigatepreliminarily the brainstem's mechanism of hypoglycaemia effect caused by auricular acupuncture, we used extracellular single-unit recordings which were carried out innucleus tracus solitarius of rats followed intra-carotid administration of glucose(15%), insulin (6IU/ml) and acupuncture stimulation at auricular concha(interval:0．4s, duration:0．4ms intensity:2mA),respectively.
Results In the group of administration of glucose (15%, 0．25ml/min) , thedischarges of totally 110 NTS neurons were recorded. Among them there were 41(37．27%) neurons responding to glucose infusion with a discharges decrease inpercent of 60．36±15．1%, while 10．91 %(12/110) neurons responding to glucoseinfusion with a discharges increase with a percentage of 56．78±9．24%. 57 (51．82%)neurons showed no response in discharges frequency during or after administration ofsugar solution via carotis. During or after intra-carotid administration of insulin(6IU/ml, 0．25ml/min) , totally 81 NTS neurons' discharges were observed . Amongthem, there were 33．33% (27/81) neurons showed increase in spike frequency with apercent of 47．34±11．24%. Meanwhile, there were 4(4．9%) neurons exhibited decreasein spike frequency with a percent of 43．24±12．11%. Among the above 27exciting-responding neurons, 18 cells showed decrease in activity to administration ofglucose via carotis, while 9 cells showed no response in spike frequency to sugarinfusion. Among the 4 neurons which were inhibitory-responsive to insulinadministration, there were 2 neurons showed contrary responsive to sugar infusion viacarotis, the others exhibited no response in frequency of discharge.
The discharges of totally 69 neurons in NTS were examined during or afteradministration of electro-acupuncture at auricular concha. Among them, there were 34(49．27%)neurons showed increase in spike frequency with a percentage of 54．79±24．21%, 3 (4．35%) ones showed decease in spike frequency with a percentage of44．65±9．13%, the others (46．38%)demonstrated no response to the acupuncture atauricular concha. Glucose infusion via carotis resulted in a decrease in firing rate of18 neurons in the totally 34 neurons which showed exciting-response to auricularacupuncture, while there were 2 neurons in the 34 ones showed contrary response tothe administration of glucose, and the others showed no response to glucoseconcentration's elevation. Among the same 34 neurons, 8 neurons showed anexcitatory response to insulin infusion, 3 ones showed contrary response to theadministration of insulin, the others showed no response to insulin concentration'selevation. During or after glucose infusion via carotis, the latence ofexcitatory-response and inhibitory-response neurons recorded in NTS wererespectively 15±6 and 18±8s, and those were respectively 16±7s and 18±10s during or after administration of insulin, no significant difference between them (P>0．05) . Thelatence of neurons recorded in NTS showing excitatory-response andinhibitory-response to auricular acupuncture were respectively 3±1s and 5±2s.Furthermore, among the totally 37 recorded during auricular acupuncture, there were21 neurons exhibited 2-5 minutes' long-term increase in firing rate. 2 neurons werestimulated to waken up from silence. 3 ones in the 37 responsive neurons showedinhibitory-response to auricular acupuncture with long-term's decrease in firing rate.
PartⅡMorphological experiment: Research the medullary distribution ofafferent fibers and cells of origin from auricular concha using transganglionictransport of horseradish peroxidase (HRP) as a neuronal tracer.
Methods 10 adult male and female healthy Sprague-Daxley rats weighing 200-250gwere anesthetized with 30% urethane solution (1g/kg i.p.). Under general anesthesia, askin incision was made in the region of auricular concha. The skin and fasciae wereseparated to expose the auricular nerve of vagus nerve, which were traced carefully tothe region of the ear root where the auricular nerve of vagus nerve was cut. Fortymicroliters of a 30% solution of HRP (Sigma) in normal saline were subcutaneouslyinjected into the cut central end of auricular branch of vagus nerve. After survivaltimes of 36hrs to 48hrs, all animals were reanesthetized and perfused intranscardiallywith 500 ml of normal saline, followed by a phosphate buffered (PH=7．4) fixativecontaining 1．25% glutaraldehyde and 1% formaldehyde. The medulla oblongata andinterbrain were moved in each animal and transferred to phosphate buffer containing5% sucrose for 12-24h at 4℃. Serial sections (30μm) were cut through the tissues ona freezing microtome. All sections were processed for HRP activity using tetramethylbenzidine as the chromogen.
Results HRP-labeled terminal or preterminal fibers were identified under the lightmicroscope. They often appear as brown granules of various sizes and differentshapes in medulla oblongata and interbrain. Heavily labeled neurons and fibers wereseen in NTS and DMV, as well as in the nucleus of spinal tract of trigeminal nerve,where granular labeling observed was heaviest at the same level. Labeling in themedial division of the NTS extended dorsally adjacent to the AP. In the one animal inwhich measurements were made, granular labeling extended for 2．6mm in theipsilateral NTS sparing 0．4mm of the nucleus rostrally. HRP-labeled cells in NTSwere frequently fusiform and ovoid, while those in DMV were often ovoid.Retrograde labeling of neurons was also observed in the nucleus of ambiguous and the region of reticular formation, and the labeled neurons were rounded or oviform in shape and 20×30μm in size.
PartⅢBiochemical experiment: Effects of electro acupuncture on the plasma glucose and insulin concentration of normal and diabetic rats.
Methods The experiment was carried out on 100 healthy male rats, 150-220g in body weight. Among the whole animals, 40 were normal rats, and the others were diabetic rats, which were induced by once intraperitoneal injection of streptozotocin (60mg.kg). The normal animals were divided into three groups in this dose-finding experiments: Group A consisted of 10 normal rats which were administered with electrostimulation at the intensity of 10 mA , wave of 1ms , applied in auricular concha points; Group B were applied in the same acupoints electrostimulation at the intensity of 2 mA , wave width of 0．5ms; Group C were administered with the same intensity as Group B of electrostimulation at the Zusanli (ST36) acupoint. Diabetic rats were divided into 3 groups: auriculo-acupuncture group (Group D) received the same electrostimulation as Group B; Vagotomy followed auriculo-acupuncture group (Group E) received the same electrostimulation with Group B after cutting left cervical vagal nerve; Group F was control group which were administered the same electrostimulation as Group C. Each group rats were received 30min's electrostimulation, then several blood samples (0．2-0．5ml each) were taken from the femoral vein respectively at the time of 0, 10, 20, 30 minutes after stopping acupuncture stimulation and placed them in Eppendorf tube containing 10 IU heparin. The plasma glucose was measured using Onetouch Ultra Brand blood glucose meter and insulin were determined in the way of immunosorbent assay, American BPB Inc. supplied with commercial kits.
Results
1) After 30 minutes' electrical acupuncture on the auricular concha acupoints, which was of 10 mA in intensity, 1ms in wave width, the glucose concentration responded with a hyperglycemia from average of 6．48±0．28 mmol/L before the acupuncture to 7．70±0．38 mmol/L at the point of 10mins after stopping acupuncture (P<0．05, paired t-test, same to following). While electrical acupuncture on auricular concha acupoints with 2 mA of intensity, 0．5ms of wave width induced a hypoglycemia from average of 7．23±0．15 mmol/L before the acu-stimulation to 6．43±0．14 mmol/L 10 minutes after acupuncture stop; there was statistical significance in glucose concentration before and after acu-stimulation on auricular concha (P<0．01) . In the control group, no significant difference in glucose concentration was induced by the electrical stimulation applied to need inserting in the tissue at Zusanli acupoint (7．45±0．33 mmol/L vs 7．41±0．29 mmol/L, P>0．05) .
2) Lower intensity (2 mA in intensity, 1ms in wave width) of auricular acupuncture applied to normal rats showed a time-window regulation of glucose concentration. Before the auri-acupuncture, the average of blood suger level was 7．23±0．15mmol/L, after 30 minutes' administration of electrical stimulation on auricular concha, the suger level reduced to 6．65±0．14mmol/L when the stimulation came to a stop (P<0．01 vs before auricular acupuncture). 10 minutes later, glucose concentration continually decreased to a lowest value of 6．43±0．14mmol/L (P<0．01 vs when auri-acupuncture stopped). Then a hyperglycemia appeared as time gone on, and at the point of 20 minutes later, the glucose went up to 6．84±0．14mmol/L (P<0．05 vs 10 minutes later; but P>0．05 vs before acupuncture stimulation). The glucose concentration increased to the normal level as before the auricular electrical stimulation (P>0．05 vs before acupuncture stimulation). In the control group, above time-window regulation of glucose concentration was not induced by electrical stimulation at ST36 acupoint. Before the acupuncture stimulation at ST36, glucose concentration of the control group was 7．45±0．33mmol/L, and then was 7．49±0．27mmol/L when the 30 minutes' acupuncture came to a stop. There was no statistical significance between them., 10 minutes later the glucose slowly moved down to 7．41±0．29mmol/L (P>0．05 vs when acupuncture stopped), one more 10 minutes later the concentration went up to 7．43±0．27mmol/L, then to 7．44±0．25mmol/L at the point of 30 minutes after the acupuncture stopped. There were no differences in statistics among them.
3) However, the time-window regulation of glucose concentration of normal rats induced by the lower intensity of auricular acupuncture could not be explained absolutely by the alteration of insulin concentration, which was also induced by the auricular acupuncture as well. Before the auri-acupuncture, insulin concentration was 44．65±2．95μmol/L, then went up to 50．06±3．88μmol/L after 30 minutes' acupuncture at auricular concha, but there was no distinguished difference between them which was compared by the paired t-test(P>0．05) .In the control group, the same phenomenon was obserbed in normal rats during the electrical acupuncture stimulation was applied to the ST36 acupoint. Insulin concentration was increased to 51．76±2．93μmol/L at the point of 10 minutes after the stop of stilulation from 48．63±3．79μmol/L before stimulation, no distinguished difference was found in the above two sets of data which were compared by paired t-test analysis.
4) Acupuncture applied to auricular concha acupoint could lower plasma glucose concentrations in diabitic rats. An insulin-dependent action thus could be considered. Lowering of plasma glucose in diabetic model rats was investigated from 17．33±0．60mmol/L to 16．58±063mmolL after EA-stimilation at auricular concha acupoints (P<0．05, before auri-acupunctrure vs after auri-acupuncture). This hypoglycemia activity was blocked by vagotomy (17．57±0．80mmol/L vs 18．23±0．81mmol/L, P>0．05) . In the control group, EA-stimulation at the ST36 acupoint elicited a slender reduction in plasma glucose concentration (from 17．69±0．79mmol/L to 17．59±0．82mmol/L), but no distinguished difference was found between two sets of data analysised by the paired t-test.
5) An insulin-dependent action could be explained the hypoglycemia activity elicited by acupuncuture. After the EA-stimulation at auricular concha, plasma insulin in diabetic rats were raised from 50．66±2．15μmol/L to 58．49±2．44μmol/L (P<0．001) .Blockade of auricular acupuncture-induced increase of plasma insulin was seen in the diabetic rats which were received vagotomy, and no significantly difference was found before and after the electrical acupuncture at auricular concha followed vagotomy (50．53±2．06μmol/L vs 47．86±1．34μmol/L, P>0．05) . In the control group, EA-stimulation at ST36 acupoint also raised plasma insulin concentration from 46．32±1．86μmol/L up to 51．79±2．23μmol/L (P<0．05) . So we could possibly attribute the hypoglycemia effect induced by acupuncture to the insulin-dependence.
Conclusion
The present study including three parts of experiments were carried out to investigate the relationship between the mechanism of hypoglycemia induced by electrical acupuncture at auricular acupoints and Auriculo-vagal nerve system reflex. The experiment results showed that different intensities of acupuncture at concha points casused different effects on the glucose concentration alteration in normal rats. Lower density of electrical acupuncture at concha acupoints had a pronounced time-window regulation of plasma glucose, compared with the control group, in which electrical stimulation was applied to a needle inserting into the tissue at ST36 acupoint. Acupuncture either in auricular concha or in ST36 acupoints could raise plasma insulin in diabetic rats. In the group of auricular acupuncture this raise had significant difference, while in the group of ST36 acupuncture, the raise was slender. The effect of pronounced reduction in plasma glucose concentration could be elicited by electrical auri-acupuncture, which could be blocked by vagotomy. There existed glucose-responsive neurons and insulin-responsive neurons in NTS. Electrical acupuncture stimulation at auricular concha acupoints could affect the activity of these neurons, especially could irritate the neurons' firing which showed inhibitory response to glucose administration. Directly neural projections in the NTS and DMV from the terminal of auricular vagus branch in the region of auricular concha were examined by using the HRP transganglionic tracing. The morphological results gave a rational explaination of the electrophysiolodical outcome, and supplied with morphology evidence for auriculo-vagal nerve system reflex theory. This study showed that the hypoglycemia effects of auricular acupuncture on different conditions of rats were distinct. Lowing of plasma glucose induced by auri-acupuncture should be supported by intact vagal nerve.
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