孤束核在针刺调节大鼠血压及胃肠感觉和运动中的作用
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摘要
《灵枢·海论》“夫十二经脉者,内属于府藏,外络于肢节”的记载,说明沟通脏腑与体表肢节的联系是十二经脉的重要功能。针刺通过刺激人体体表的穴位以达到治疗内脏疾病调节内脏功能的作用。现代临床研究显示针刺治疗心血管、胃肠等各种内脏疾病效果显著,并有一定的穴位特异性。
针刺对内脏功能的调节是由脊髓及脊髓上中枢协同完成的。针刺引起的体表-内脏反射是其调节心血管、胃肠等内脏功能的重要神经反射形式,可分为脊髓固有反射及脊髓上反射两种形式。体表-自主神经反射分为体表-交感和体表-副交感反射两种,由器官的自主神经支配特性和刺激部位的脊髓节段性决定,同时受刺激强度和种类的影响。孤束核(nucleus of tractus solitary, NTS)位于延髓背侧,是迷走神经感觉传入的第一级中枢,参与心血管、呼吸、胃肠等多种内脏功能的调控,也是心血管压力感受性反射、胃肠感觉的第一级感觉中继站；同时NTS与迷走神经背核、延髓尾端腹外侧核(rostral Ventrolateral Medulla, rVLM)、延髓头端腹外侧核(caudal Ventrolateral Medulla, cVLM)、疑核等中枢核团有纤维联系,通过交感和副交感神经传出通路完成对内脏功能的调节。
针刺调节内脏功能,常见单一穴位或依据传统穴位配伍的多穴调节一脏的报道。例如内关对心血管功能的影响,足三里对胃肠功能的作用。未涉及穴位选取的规律性总结。本研究根据哺乳动物皮肤,肌肉,内脏神经分布的节段性特点,结合体表刺激引起躯体—植物神经反射的前期研究报道,探讨不同节段穴位,不同刺激方式引起的内脏植物神经反射的规律,力求从动物实验总结出针刺对特定内脏功能进行交感样和副交感样调节的规律。本研究通过电生理学方法在中枢鉴别出NTS与心血管、胃肠等内脏功能及结直肠扩张(colorectal distention, CRD)刺激相关的神经元,同时记录平均动脉压(mean arterial pressure, MAP)、胃运动,选取不同神经节段的穴位进行针刺刺激,观察不同穴位对MAP和胃运动的影响,并同步观察NTS与血压调节、胃运动、CRD刺激相关神经元放电活动,研究NTS在降压、调节胃运动和抑制CRD诱导内脏痛中的作用。
1针刺对MAP的影响及NTS与血压调节相关神经元在针刺降压中的作用
1.1实验目的
研究针刺对正常麻醉大鼠MAP、静脉注射去氧肾上腺素(phenylephrine,PE),硝普钠(sodium nitroprusside, NP)分别引起升压平台期、降压平台期时MAP的影响,并同步观察NTS与升压(hypertensive related neurons)、降压反射相关神经元(hypotensive related neurons)在针刺调节血压时放电活动的变化,探讨NTS中与升压、降压反射相关神经元在介导针剌降压中的作用。
1.2实验方法
本课题所有研究的动物实验过程均遵守美国国立卫生院倡导的实验动物保护和使用指导原则(Guide for Care and Use of Laboratory Animals, NIH),实验过程中对动物的处置遵照科技部发布的《关于善待实验动物的指导性意见》规定。
实验选用健康成年Sprague-Dawley雄性大鼠81只,体重300-380g之间,用10%的乌拉坦腹腔注射麻醉(urethane,1.0-1.2g/kg体重)。手术及实验中动物体温用动物恒温系统维持在37℃左右。大鼠麻醉后进行颈动、静脉插管、气管插管,并暴露延髓后,监测动脉血压和心率,同步记录MAP及NTS神经元放电,在微电极推进器下,找到自发放电稳定的NTS神经元,经静脉推注30s左右PE(4μg/kg)、NP (NP,20ug/kg),以判断该神经元对升压反射、降压反射的反应及鉴别出与升压反射和降压反射相关神经元。对升压反射和降压反射相关神经元,待自发放电稳定、MAP'恢复至正常后,记录30s放电脉冲数作为对照值,分别电针(波宽500ms,10Hz,1mA)“耳穴心”、“内关”、“足三里”各30秒。为排除针刺穴位顺序对神经元放电结果的影响,穴位针刺顺序随机选择,观察对MAP及血压调节相关神经元放电的影响。然后在大鼠颈静脉微量注射PE(6～121μg/kg,10-15min)或者NP(60～80μg/kg,10-15min)分别造成药物性血压升高、药物性血压降低模型各15min左右,在升压平台期、降压平台期再分别电针上述穴位,并观察对动脉血压和NTS神经元放电的影响。
1.3实验结果
1.3.1NTS与血压反射相关神经元的鉴别
麻醉大鼠颈静脉注射PE,完整记录120个NTS细胞放电(细胞记录不完整者不做统计,下同),其中70个细胞(58.3%)放电频率无变化；26个细胞(21.7%)放电频率减少,呈抑制反应；24个细胞(20.0%)放电频率增加,呈兴奋反应。
麻醉大鼠颈静脉注射NP,完整记录101个NTS细胞放电,其中52个细胞(51.5%)放电频率无变化；29个细胞(28.7%)放电频率减少,呈抑制反应；20个细胞(19.8%)放电频率增加,呈兴奋反应。
13.2电针刺激对麻醉大鼠生理状态下MAP及NTS神经元放电活动的影响
电针“耳穴心”和“足三里”能够明显降低实验动物的MAP,与此同时,NTS与升压、降压反射相关神经元的放电明显增加。电针“内关”虽然也能轻度激活NTS神经元放电,但对MAP没有明显影响。统计结果表明,正常血压状态下,电针“耳穴心”使动物的MAP由100.11±3.3mmHg下降为88.47±3.75mmHg,下降幅度为11.64±0.45mmHg(P<0.001)；电针“足三里”使MAP下降了5.74±0.64mmHg(P<0.001),电针“内关”对MAP无显著影响。不同穴位组间降压效应比较显示,电针“耳穴心”、“足三里”对正常MAP的影响大于“内关”(P<0.05)。
针刺不同穴位引起MAP变化,同时影响正常生理性血压状态下NTS与血压反射相关神经元的放电频率,这种影响主要以兴奋性为主。在MAP正常状态下,电针大鼠不同穴位,对血压反射有兴奋或抑制性反应的神经元放电频率发生明显变化。电针大鼠“耳穴心”(n=50),25个神经元(50%)放电频率增加,(净增加率为115.2±15.25%)。电针“内关”穴(n=50),24个神经元(48%)放电频率增加(净增加率为54.24±6.89%)。电针“足三里”穴(n=47),20个神经元(42.55%)放电频率增加(净增加率为103.35±14.78%)。组间比较显示,电针“耳穴心”和“足三里”两个穴位对神经元的激活百分比增加率显著高于“内关’穴(P<0.05)。
1.3.3电针刺激对PE引起血压升高状态MAP和NTS升压反射相关神经元放电活动的影响
大鼠颈静脉注射PE(12～16μg/kg,10～15min)造成药物性升压模型,MAP由99.28±3.82mmHg上升并维持在121.66±2.91mmHg(P<0.001)。电针“耳穴心”和“足三里”能明显降低实验动物升压平台期的MAP,与此同时,NTS与升压反射相关神经元的放电明显增加。电针“内关”虽然能轻度激活NTS神经元放电,但对MAP没有明显影响。统计结果表明,在PE引起的升压平台期,电针“耳穴心”使MAP由124.17±4.43mmHg下降到118.7±3.64mmHg,下降幅度8.30±0.79mmHg(P<0.001)；电针“足三里”使MAP下降6.07±0.79mmHg(P<0.05)；电针“内关”对MAP无明显影响。组间比较显示(one-way ANOVA),电针“耳穴心”、“足三里”的降压效应优于“内关”(P<0.05)。电针同一穴位对麻醉大鼠正常状态和PE引起血压升高状态下MAP的影响无明显差异(independent/test,P>0.05).
电针不同穴位引起升压平台期MAP变化,同时对NTS与升压反射相关神经元的放电频率产生影响,这种作用主要以兴奋性为主,使神经元放电增加。对升压反射有兴奋或抑制性反应的50个神经元中,完成升压平台期记录及针刺实验的共21个。电针大鼠耳甲区穴位,9个放电频率增加,净增加率为92.94±10.42%。电针“内关”穴,9个放电频率增加,净增加率为44.66±6.27%。电针“足三里”穴,8个放电频率增加,净增加率为86.73±5.57%。组间比较显示,电针“耳穴心”和“足三里”两个穴位对神经元的激活百分比增加率显著高于“内关”穴(P<0.05)。
1.3.4电针刺激对NP引起血压降低状态MAP和NTS降压反射相关神经元放电活动的影响
大鼠颈静脉注射NP(60～80μg/kg,10～15min)造成药物性降压模型,MAP由96.56±2.24mmHg下降并维持在78.29±1.84mmHg.电针不同穴位能明显降低实验动物降压平台期的MAP,与此同时,NTS与降压反射相关神经元的放电显著增加。统计结果表明,在NP引起的降压平台期,电针“耳穴心”使MAP由79.76±1.39mmHg下降到69.32±2.48mmHg,下降幅度10.44±1.60mmHg(P<0.001)；电针“内关”使MAP下降7.93±1.20mmHg(P<0.05)；电针“足三里”使MAP下降15.63±1.76mmHg(P<0.05)；组间比较显示(one-Way ANOVA),电针上述三个穴位的降压效应无差异(P>0.05)。电针“耳穴心”、“足三里”,对麻醉大鼠生理情况MAP及NP降压状态MAP的影响无明显差异,电针“内关”对降压平台期血压的降压效应好于生理状态(independent t test,P<0.05)。
电针不同穴位引起降压平台期MAP变化,同时,对NTS与降压反射相关神经元的放电频率产生影响,这种作用主要以兴奋性为主。电针“耳穴心”、“内关”和“足三里”使NTS内与降压反射有关神经元的放电增加。对降压反射有兴奋和抑制性反应的49个神经元中,完成升压平台期记录的共19个。电针大鼠耳甲区穴位,9个放电频率增加,净增加率为138.00±18.76%。电针“内关”穴,8个神经元放电频率增加,净增加率为96.75±20.51%。电针“足三里”穴,8个神经元放电频率增加,净增加率为100.75±31.87%。组间比较显示,电针三个穴位对神经元的激活百分比无显著差异(组间比较,one-way ANOVA, P>0.05)
2针刺对胃运动及NTS胃运动相关神经元放电活动的影响
2.1实验目的
观察针刺不同穴位对正常麻醉大鼠胃内压的影响,及静脉注射不同交感和副交感神经受体业型激动剂引起胃运动迟缓和亢进状态时针刺的效应,并同步观察NTS与胃运动相关神经元在针剌调节胃运动过程中放电活动的变化,探讨NTS中与胃运动相关神经元在针刺调节胃肠功能中的作用。
2.2实验方法
实验选用健康成年Sprague-Dawley雄性大鼠50只。实验前禁食12-18小时,手术及实验中动物体温用动物恒温系统维持在37℃左右。大鼠麻醉后进行颈静脉、气管插管,胃幽门部插入水囊连接压力换能器以记录胃内压,并监测血压、心率。
同步记录胃内压及孤束核与胃运动相关神经元,找到稳定孤束核神经元胞外放电后,记录背景放电30s,然后分别电针、手针“耳穴心”、“内关”、“中脘”、“足三里”四个穴位,同步观察对胃运动和孤束核神经元细胞外放电的影响。对针刺有反应的神经元,分为5组,分别静脉注射四种药物和生理盐水：p3体激动剂BRL37344Salt Hydrate4-8μ g/kg；β2受体激动剂盐酸克仑特罗0.05-0.1mg/kg;α：受体激动剂可乐定0.4-0.8mg/kg;拟胆碱药氨基甲酰胆碱0.2-0.4mg/kg和静脉注射0.2ml0.9%的生理盐水。上述药物引起胃蠕动迟缓或亢进的同时,NTS神经元放电活动发生兴奋或抑制的同步变化,则鉴定为孤束核与胃运动相关神经元。各组药物注射后,继续观察针刺不同穴位对胃运动和孤束核与胃运动相关神经元的影响。
2.3实验结果
2.3.1生理状态下针刺对胃运动和NTS胃运动相关神经元放电活动的影响
生理状态下,麻醉大鼠的胃运动频率为4-5次/min,波幅在50-100mmH2O。结果表明电针刺激“耳穴心”、“内关”、“足三里”都能促进麻醉大鼠胃蠕动(P<0.01),而电针“中脘”穴则明显减弱胃蠕动(P<0.001)。手针效果与电针效果一致(independent t test, P>0.05)。
针刺过程共记录133个NTS神经元胞外放电,对针刺上述4个穴位有反应的神经元104个；静脉注射p3受体激动剂、β2受体激动剂、α2受体激动剂、拟胆碱药有反应的共45个,以下分组讨论。在正常情况下,针刺对孤束核与胃运动相关神经元放电的影响以抑制为主。针刺不同穴位对孤束核与胃运动相关神经元的抑制作用无明显差异(one way ANOVA, LSD, P>0.05)
2.3.2使用β3受体激动剂后针刺对胃运动和NTS相关神经元活动的影响
静脉注射β3受体激动剂后,药物对胃运动抑制作用大约持续30-60min,胃运动蠕动波波幅下降了10-20mmH20,胃内压明显降低,幅度为65.4±7.20mmH2O(P<0.001)。药物后电针对胃运动没有影响(P>0.05)。手针刺激“耳穴心”、“足三里”使胃内压增高(P<0.05)；手针刺激“中脘”使胃内压降低(P<0.05)。与电针比较,手针“耳穴心”和“中脘”分别引起的胃运动的增强和抑制效应更为显著(P<0.05),其余穴位不同刺激无明显差异(independent t test, P>0.05)。
分别针刺4个穴位对p3受体激动剂静脉注射前后孤束核与胃运动相关神经元放电的影响无统计学差异(independent t test, P>0.05)。
2.3.2使用β2受体激动剂后针刺对胃运动和NTS相关神经元电活动的影响
静脉注射β2受体激动剂Clenbuterol(克伦布特罗)后,胃运动波幅明显减低了10-20mmH20,胃内压明显下降,幅度为51.18±9.93mmH2O(P<0.001)。电针刺激“耳穴心”使胃内压升高(P<0.05；电针刺激“内关”、“中脘”、“足三里”对胃运动无作用(P>0.05)。手针刺激“耳穴心”、“内关”、“足三里”能促进胃运动(P<0.01),手针“中脘”能抑制胃运动(P<0.05)。手针“内关”、“足三里”、“中脘”相比电针对胃运动的影响作用更明显。(independent t test, P<0.05)。
在Clenbuterol药物引起胃运动抑制状态,针刺不同穴位对药物前后孤束核与胃运动相关神经元的放电的影响无明显差异(independent t test, P>0.05)。
2.3.3使用α2受体激动剂后针刺对胃运动和NTS相关神经元放电活动的影响
静脉注射α2受体激动剂Clonidine后,胃运动的波幅降低了10-20mmH20,胃内压明显下降,幅度为79.07±24.59mmH20,持续30-60min(P<0.05)。药物后电针、手针“耳穴心”、“足三里”能促进胃运动(P<0.05),在Clonidine药物注射后引起胃运动迟缓状态下,两种刺激增强胃运动的作用无显著差异。手针“中脘”能抑制胃运动且比电针的抑制作用更显著(independent t test,P>0.05)。
针刺不同穴位对Clonidine药物静脉注射前后孤束核与胃运动相关神经元的放电的影响无明显差异(independent t test, P>0.05)
2.3.4使用拟胆碱药后针刺对胃运动和NTS相关神经元放电活动的影响
静脉注射拟胆碱药氨基甲酰胆碱后,胃运动频率明显升高至6-7次/min,胃运动波幅升高了200-300mmH2O,胃内压明显升高,幅度为121.27±29.53mmH20(P<0.01)。药物注射前后,电针和手针“耳穴心”、“内关”、“足三里”均能促进胃运动(P<0.01,P<0.05,P<0.001),电针和手针“中脘”均能抑制胃运动(P<0.01；P<0.01)。与电针相比较,在拟胆碱药引起胃运动亢进状态下,手针“耳穴心”和“中脘”的作用更显著(independent t test, P<0.05)。
针刺不同穴位对拟胆碱药静脉注射前后孤束核与胃运动相关神经元的放电的影响无明显差异(independent t test, P>0.05)。静脉注射上述四种交感和副交感受体业型激动剂对针刺调节孤束核神经元放电活动无影响。
3针刺对NTS与CRD相关神经元活动的影响与针刺治疗内脏痛的机制
3.1实验目的
给予麻醉大鼠梯度的CRD条件刺激,鉴别NTS中CRD“点燃”(ON cell)型和“熄火”(OFF cell)型神经元,观察针刺作为检验刺激对此类神经元放电活动的影响,探讨针刺在孤束核部位干预内脏痛的中枢神经系统机制。
3.2实验方法
正常雄性SD大鼠,监测心率、颈总动脉血压,记录孤束核神经元细胞外放电。大鼠经肛门在直结肠放置长度4-5cm左右的气囊,通过压力计随机给予梯度为20、40、60、80mmHg的结直肠扩张刺激(CRD),同时用电生理单细胞记录的方法记录和鉴别孤束核对CRD刺激有反应的相关神经元,参照Fields等的研究大鼠RVM内存在对伤害性热刺激引起的甩尾反射相关的“熄火”型(OFF cell)和“点燃”型(ON cell)神经元,对梯度CRD刺激产生递增的兴奋性反应的神经元,称为“点燃”型(ON cell),对梯度CRD刺激产生递减的抑制性反应的神经元,称为“熄火”型(OFF cell).鉴别出NTS中CRD相关神经元后,待放电恢复至基础水平,给予CRD刺激,NTS神经元放电发生明显"ON cell"或“OFFcell"型反应时,给予2mA、10Hz持续30s的电针不同穴位的刺激,观察针刺“耳穴心”、“内关”、“足三里”在正常及CRD条件刺激引起NTS放电变化时对孤束核与CRD "ON cell"型和"OFF cell"型神经元的影响。
3.3实验结果
3.3.1NTS神经元对梯度CRD刺激的反应
本实验在57只大鼠共记录到对CRD "ON cell"型神经元21个。在CRD20、40、60、80mmHg条件刺激时,NTS神经元放电由自发的205.52±30.12个/30s分别增加了21.10±5.23个/30s、56.79±11.93个/30s、86.17±13.38个/30s、124.27±15.00个/30s(P<0.001,P<0.01,P<0.001,P<0.001),为梯度增加反应。
另外记录到12个CRD "OFF cell"型神经元。在CRD20、40、60、80mmHg条件刺激时,NTS神经元放电由自发的196.67±42.36个/30S分别减少了25.82±13.43个/30s、72.72±14.77个/30s、112.91±22.37个/30s、146.83±29.83个/30s(P<0.05,P<0.01,P<0.01,P<0.05),为梯度抑制反应。
此外记录到2个对梯度CRD刺激产生先兴奋后抑制,3个先抑制后兴奋的神经元,在本实验中未纳入统计。
3.3.2电针对NTS与CRD相关神经元放电活动的影响
CRD刺激对NTS产生"ON cell"型反应的神经元,CRD刺激前和CRD过程中电针“耳穴心”、“内关”、“足三里”都以抑制反应为主。如CRD前电针“耳穴心”放电减少了34.73±3.37个/30s(P<0.01)；CRD刺激过程电针“耳穴心”使放电减少了45.55±4.17个/30s(P<0.001)。
对CRD刺激产生"OFF cell"型反应的神经元,在CRD刺激前和CRD刺激过程中电针上述3个穴位都以兴奋反应为主。如CRD前电针“耳穴心”使放电增加了184.26±27.35个/30s(P<0.001)；CRD刺激时电针“耳穴心”使放电增加了130.14±28.23个/30s(P<0.01)。
4结论
孤束核内存在升压、降压反射相关神经元,这些神经元可以分别被升压、降压反射兴奋和抑制,表现为反应的多样性。在生理状态下及PE引起的升压平台期,这些神经元被“耳穴心”、“足三里”激活的百分比与“内关”相比更为显著；在降压平台期,这些神经元被上述三个穴位激活的百分比无明显差异,与其针刺降压相应相一致。这说明NTS在针刺调节血压过程中发挥重要的中枢整合作用,通过神经信号交互调节(cross talk)的方式完成对血压的调节。
针刺不同穴位对胃运动的影响不同,针刺“耳穴心”及与胃肠异节段的“足三里”、“内关”能显著增强胃运动,针刺与胃肠同神经节段的“中脘”使胃运动减弱。使用交感神经不同业型神经受体激动剂后,针刺对胃运动的影响减弱；电针对胃运动的作用不如手针显著；而针刺不同穴位对胃运动的影响也发生变化,“耳穴心”、“足三里”促进胃运动的效果比“内关”明显。使用副交感神经的拟胆碱药物后,不同穴位对胃运动的作用与药物前相比无明显变化。NTS内存在与胃运动同步变化的神经元,不同植物神经受体激动剂对此类神经元的影响以兴奋为主,介导了针刺“耳穴心”、“内关”、“足三里”的胃运动增强效应和“中脘”的胃运动抑制的效应,使用植物神经受体激动剂后针刺对此类神经元的效应与生理状态下无显著差异,以抑制NTS神经元放电为主。
NTS与CRD刺激相关神经元有两类：第一类对梯度的CRD刺激产生兴奋反应,表现为神经元胞外放电梯度增加,为“点燃”型神经元(ON cell);第二类对梯度的CRD刺激产生抑制反应,表现为神经元胞外放电梯度减少,为“熄灭”型神经元(OFF cell).这两类神经元介导了针刺拮抗CRD痛刺激的效应。针刺能够使孤束核CRD"ON cell"型神经元的放电抑制,而使孤束核CRD"OFF cell"型神经元的放电增加。提示针刺可以通过交互调节(cross talk)的方式在孤束核干预内脏感觉和痛觉,是针刺镇痛的脑干核团机制所在。
The Miraculous Pivot (it is one of the earliest medical classics in China) stated."Twelve regular channels belong to the zang-fu organs internally, and connect to the extremities and joints externally." Acupuncture and Moxibustion can treat visceral diseases to modulate visceral function by stimulating the acupoins. Acupuncture and Moxibustion's treatment of kinds of visceral diseases such as cardiovascular, gastrointestinal diseases are very effective, and has certain acupoints specificity.
Acupuncture modulating visceral diseases through coordination of spinal and supraspinal centers. Acupuncture-induced somato-viscera reflexes have two patterns of propriospinal reflex and supraspinal reflex. Somato-autonomic nervous reflexes are divided into two kinds:the somato-sympathetic and the somato-parasympathetic reflex, dominated by the autonomic nervous characteristics of the organs and the spinal cord segments of the stimulated parts, as well as the impact of stimulus intensity and type. The medullary dorsal nucleus of solitary tract (NTS) is the first level of the central vagus nerve afferent involved in the regulation of cardiovascular, respiratory, gastrointestinal and other visceral functions of cardiovascular baroreceptor reflex, the relay station of gastrointestinal senses. NTS has fibric connections with the dorsal nucleus of the vagus nerve, the rostral Ventralateral Medulla (rVLM,), the caudal Ventrolateral Medulla (cVLM), nucleus ambiguous (NA) and other central nuclei of the fiber contact, to complete the regulation of visceral functions through the sympathetic and parasympathetic efferent pathways.
Acupuncture regulating visceral function was commonly reported using single point or multi-points according to the traditional acupuncture compatibility. For example, the impact of Neiguan (PC6) on cardiovascular function, Zusanli (ST36) on gastrointestinal function, yet the summary of the regularity of acupuncture points selected is not involved in. In this study, according to the segmental characteristics of mammalian skin, muscle, and splanchnic nerve distribution, combined with a preliminary study of surface stimulation of the body, we tried to explore the law of the visceral autonomic reflex caused by the different segments of acupuncture points, the different stimulation, and strive to summarize the sympathetic and parasympathetic-like regulation of the law of acupuncture on the specific function of internal organs from animal experiments. This study identified the central NTS neurons that related to the cardiovascular, gastrointestinal and colorectal distention (CRD) by electrophysiological methods, simultaneously recording the blood pressure, gastric motility, to stimulate by selecting the different acupoints of the body segments, and to observe the NTS-related neuronal activity, to study the role of NTS related neurons in the effects of acupuncture reducing MAP, modulating gastric motility and inhibiting CRD-induced visceral pain.
1The effect of acupuncture on MAP and the role of NTS blood pressure regulation related neurons in the acupuncture reducing blood pressure
1.1Objective
To observe the effects of acupuncture on MAP in three conditions of anaesthized rats:physiological, pressor reflex, depressore reflex caused by i.v. PE, NP and the firing changes of the NTS pressor reflex related neurons, depressor reflex related neurons, to demonstrate the role of NTS blood pressure regulation related neurons in acupuncture reducing MAP.
1.2Methods
All the experimental process of animals in this study was in accordance with '"Guide for Care and Use of Laboratory Animals, NIH", the disposal of animals was in accordance with "Guidance to treat experimental animals", released by the Ministry of Science and Technology.
Eighty one healthy adult Sprague-Dawley male rats were used, weighing300-380g, anesthetized with10%urethane intraperitoneal injection of anesthetic (urethane,1.0～1.2g/kg body weight). During Surgery and experiments, animals thermostat system was used to maintain animal body temperature around37℃. Simultaneously NTS neurons and arterial blood pressure were recorded, looking for stabile spontaneous discharges of the NTS neurons under the control of microelectrode thruster. To identify the neurons'responses to the pressor reflex, depressor reflex reaction, intravenous injection of phenylephrine (PE)4μg/kg, sodium nitroprusside (NP)20ug/kg were administered. The discharges of NTS neurons responded to blood pressure reflex were recorded,30s discharges pulse number were recorded as control, then respectively EA of "Auricular acupoint"(AA),"Neiguan"(PC6)","Zusanli"(ST36) each for30seconds. To exclude the impact of acupuncture order on neuronal firing results, the acupuncture order randomly selected to observe the impact of acupuncture on the MAP and the discharge of NTS neurons. Microinjection of PE in the rat jugular vein caused drug-induced high blood pressure, or microinjection of NP in the rat jugular vein caused drug-induced low blood pressure. During the step-up platform, step-down platform, we observed the impact of EA on arterial blood pressure and the discharge of NTS neurons.
1.3Results
1.3.1The identification of the NTS blood pressure reflex related neurons
After jugular vein injection of PE in normal anesthetized rats, a complete of120NTS neuron discharges were recorded (the remaining cells were not completely recorded, and did not do statistic analysis), in which the discharge frequency of70cells (58.3%) did not change; the discharge frequency of26cells (21.7%) reduced, exhibitng inhibited response; the discharge frequency of24cells (20.0%) increased, exhibitng excited reaction.
After jugular vein injection of NP in normal anesthetized rats, a complete of101NTS neuron discharges were recorded, in which the discharge frequency of52cells (51.5%) did not change; the discharge frequency of29cells reduced (28.7%), showing inhibited responses;20cells (19.8%) increased, showing excited reaction.
Among them,50cells responded to the pressor reflex, in which31cells responded to depressor reflex. In24Pressor reflex excitated cells,9had inhibited reaction to NP,7no reaction,8excited; In26pressor reflex inhibited cells,13excited,1no response,12inhibited.
1.3.2The effects of electro acupuncture (EA) on MAP and NTS neurons' discharges in anesthetized rats under physiological conditions.
EA of A A and ST36can significantly reduce the MAP of the experimental animals, at the same time, discharges in the NTS pressor reflex neurons increased significantly. EA of PC6can also mildly activate NTS neural discharges, but almost no effect on the MAP. The statistical results showed that during the state of normal blood pressure, EA of AA reduced MAP of the animals from100.11±3.3mmHg to88.47±3.75mmHg, decreased by11.64±0.45mmHg (P<0.001); EA of ST36decreased normal MAP for5.74±0.64mmHg (P<0.001),EA of PC6had no significant effect on the normal MAP. The comparison of the antihypertensive effects of different acupoint groups, EA of AA, ST36is greater than PC6under normal MAP condition (P<0.05).
MAP changed during acupuncture at different acupoints, at the same time, the NTS pressor reflex related neurons'discharge activities were excited. EA of AA, PC6and ST36increased NTS pressor reflex related neural discharges. When MAP restored to normal state, EA of AA increased discharge frequencies of25neurons of pressor reflex excitatory or inhibitory response of50neurons (net increase in the rate was115.2±15.25%). EA of PC6,24neurons discharge frequency increased (net increase in the rate of54.24±6.89%). EA of ST36,20neurons'firing rate increased (net increase was103.35±14.78%). The results of the comparison between groups showed that the EA of AA and ST36two points compared to PC6, the neuron activation percentage increased even more significantly (P<0.05).
1.3.3The effects of EA on MAP and NTS neurons discharges in PE caused MAP increased state rats
In the rats'carotid, intravenous injection of PE (12～16μg/kg,10～15min) caused drug-induced step-up model, the MAP increased and maintained from normal99.28±3.82mmHg to121.66±2.91mmHg. EA of AA and ST36can significantly reduce the step-up platform of MAP in experimental animals. At the same time, the discharges of NTS pressor reflex related neurons increased significantly. EA of PC6also can mildly activate NTS neural discharges, but almost no significant effect on the MAP. The statistical results showed that during the PE pressor plateau, EA of AA reduced step-up platform of MAP from124.17±4.43mmHg to118.7±3.64mmHg, decreased by8.30±0.79mmHg (P<0.001); EA of ST36decreased the step-up platform of the MAP by6.07±0.79mmHg (P<0.05); EA of PC6had no significant effect on step-up platform of MAP. Between the depressor effect of EA of AA and ST36to PC6, the former two acupoints had better pressor effects (one way ANOVA, P>0.05).
EA of A A, PC6, ST36on the MAP states of normal anesthetized rats and PE caused high blood pressure state, had no significant difference (independent t test, P>0.05).
EA caused MAP changes during the step-up platform, at the same time, EA excited the discharged of NTS pressor related neurons. EA of AA, PC6and ST36increased the NTS pressor reflex related neurons'discharges. Of the50NTS pressor reflex excitatory and inhibitory neurons, a total of21NTS discharges were recorded during the step-up platform. EA of AA, nine discharge frequencies increased, the net increase rate was92.94±10.42%,7had no change in discharge frequency, five discharge frequency decreased. EA of PC6increased nine neurons firing rate, a net increasing rate was44.66±6.27%, seven discharge frequencies did not change,5discharge frequencies reduced. EA of ST36, eight neuronal firing frequencies increased, the net increasing rate was86.73±5.57%, eight discharge frequencies did not change, five discharge frequencies were reduced. The results of the comparison between groups showed that the EA of AA and ST36excited the neuronal discharges more significantly compared to PC6(P<0.05).
1.3.4The effects of EA on MAP and NTS neurons discharges of rats in the NP caused MAP reduced state
In rat jugular vein injection of the NP (60～80μg/kg, and10～15min) caused drug-induced step-down MAP model, the MAP reduced from the normal96.56±2.24mmHg to78.29±1.84mmHg. EA of AA, PC6and ST36can significantly reduce the step-down platform of MAP in experimental animals. At the same time, neurons in the NTS step-down reflex related neurons discharges increased significantly. Statistical results showed that the EA of AA in NP caused step-down platform of MAP dropped from79.76±1.39mmHg to69.32±2.48mmHg, decreased by10.44±1.60mmHg (P<0.001); In NP caused step-down platform of MAP, MAP decreased by7.93±1.20mmHg by EA of PC6(P<0.05);15.63±1.76mmHg by ST36decreased by (P<0.05); Comparison between groups showed that (one way ANOVA), the depressor effect of EA at these three points had no significant difference (P>0.05).
EA of AA and ST36can reduce the MAP during physiological and NP induced depressor conditions and there was no significant difference between these two states, EA of PC6can reduce the MAP in the NP induced depressor conditions, which was better than in the physiological state.(Independent t test, P<0.05).
EA caused the changes of the MAP during the step-down platform, at the same time, EA excited the discharge frequencies of neurons in the NTS. EA of AA, PC6and ST36can increase the discharges of NTS depressor reflex related neurons. In the49excitatory and inhibitory response neurons,19NTS discharges were recorded during the step-down platform. During EA of AA, nine discharge frequencies increased, the net increase in the rate was138.00±18.76%, seven discharge frequencies did not change,3discharge frequencies reduced. EA of PC6, eight neuronal firing frequencies increased, the net increase rate was96.75±20.51%, and seven had no change in the discharge frequencies, and four reduced discharge frequencies. During EA of ST36, eight neurons increased the discharge frequencies, the net increase rate was100.75±31.87%, eight firing rate did not change,3discharge frequencies reduced. The results of the comparison between groups showed that EA of these three acupoints on the activation percentage had no significant difference (between the two groups, One-way ANOVA, P>0.05).
2The role of NTS gastric motility related neurons in acupuncture adjusting gastrointestinal function
2.1Objective
To investigate the effect of acupuncture of different acupoints on physiological gastric pressure and the inhibited or excited conditions caused by i.v different sympathetic and parasympathetic subtype receptor agonists, and synchronizely on the firing rates of NTS gatric motility related neurons, to discuss the role of NTS gastric motility related neurons in acupuncture regulating gastrointestinal functions.
2.2Methods
Fifty Sprague-Dawley male rats were used, weighing300～380g, anesthized with10%urethane intraperitoneal injection (urethane,1.0～1.2g/kg body weight). Fasted for18-20hours before the experiment, animal body temperature during surgical and experimental periods maintained at around37℃by an animal thermostatic system.
Simultaneous recording intragastric pressure and NTS gastric motility related neurons, the stabile discharges of NTS were recorded for background30s, and then EA, manual acupuncture (MA) of AA, PC6, CV12and ST36. The acupuncture responses of neurons divided into5groups were injected intravenously with four drugs:β3agonist, BRL37344the Salt Hydrate4-8μg/kg; β2receptor agonist, Clenbuterol Hydrochloride0.05-0.1mg/kg; α2receptor agonist, Clonidine Hydrochloride0.4-0.8mg/kg; Cholinomimetics, carbamyl-B-methylcholine chloridecrystal0.2-0.4mg/kg and the fifth group of intravenous0.2ml0.9%saline. Of these drugs to slow or facilitate gastric motility, at the same time, the NTS neuron discharge activities had excitatory or inhibitory changes, and then they were identified as NTS gastric motility related neurons. Continue to observe the different effects of acupuncture points on the gastric motility and the NTS gastric motility related neurons after drug injection.
2.3Results
2.3.1The effects of acupuncture on gastric motility and the NTS gastrointestinal motility related neurons under physiological conditions
The results showed that EA of AA, PC6, ST36stimulation can promote gastric motility (P<0.01), EA of CV12significantly reduced the gastric motor function (P0.001). MA had the same results with EA (Independent t test, P>0.05).
Recorded a total of133NTS neuron extracellular discharges,29had no response to MA,104Neurons responded;45neurons responded to intravenous injection of one of the four drugs.
Acupuncture inhibited neurons in the NTS gastric motility related neurons in normal conditions. Acupuncture of the four acupoints inhibiting effects on the NTS gastric motility related neurons had no significant difference (P>0.05, ONE WAYANNOVA of LSD).
2.3.2The effects of β3adrenergic agonist on acupuncture regulating gastric motility and NTS related neurons activities
After intravenous injection of β3adrenergic agonist, EA of AA, PC6and ST36no longer appeared to promote gastric motility, the inhibitory effect of EA of CV12on gastric motility disappeared (P>0.05). MA of AA, ST36increased gastric pressure (P<0.05). MA of CV12decreased the intragastric pressure (P<0.05). MA of PC6in promoting gastric motility disappeared. MA of AA enhanced gastric motility more significantly than EA of AA. MA of CV12had better inhibitory effect on gastric motility than EA. The exciting effect of EA and MA on rest of the acupoints had no significant difference (independent t test, P>0.05).
After the β3agonist BRL37344drugs intravenously, the acupuncture effects on NTS related neurons were still inhibiting.
2.3.3The effects of β2adrenergic agonist on acupuncture regulation of gastric motility and NTS neurons discharge activities
After intravenous drug administration, EA of AA increased intragastric pressure (P<0.05), EA of PC6, CV12and ST36had no effect on gastric motility (P>0.05). MA of AA, PC6and ST36can promote gastric motility (P<0.01), MA of CV12can inhibit gastric motility (P<0.05). MA and EA of AA had no significant difference in the enhancement of gastric motility, MA in the abdominal had more significant inhibitory effect on gastric motility than EA of CV12. MA of PC6and ST36had better gastric motility enhancement than EA of PC6and ST36.(Independent t test, P <0.05).
Acupuncture of the four points on the NTS gastric motility related neurons discharges before and after Clenbuterol drugs had no significant difference (independent t-test).
2.3.4The effects of α2receptor agonist on acupuncture in regulating gastric motility and NTS neurons discharge activities
After intravenous drug administration, EA of A A and ST36can promote gastric motility (P<0.05), EA of PC6, CV12had no significant effect on gastric motility (P>0.05). MA of AA, ST36can promote gastric motility, PC6had no effect on gastric motility (P>0.05). MA and EA had no significant difference on the gastric motility on AA, PC6and ST36after Clonidine injection, MA in the abdomen caused inhibitory effect on gastric motility was more significant than EA in the abdomen (independent t test, P>0.05).
Before and after the intravenous injection of Clonidine,acupuncture at different acupoints reduced NTS gastric motility related neurons (P<0.01, P<0.05).
The impact of acupuncture four acupoints on NTS gastric motility related neurons discharges before and after Clonidine drug injection had no significant difference (independent t test, P>0.05).
2.3.5The effects of Cholinomimetics on acupuncture regulating gastric motility and NTS neurons discharge activities
Before and after the intravenous injection of Cholinomimetics, EA of AA, PC6, ST36can promote gastric motility (P<0.01, P<0.05), EA of CV12can inhibit gastric motility (P<0.01). MA of AA, PC6, ST36obviously can promote gastric motility (P <0.001), MA of CV12in the abdomen significantly inhibited gastric motility (P <0.01). MA and EA of ST36, PC6had no difference in gastric motility enhancement.
Acupuncture at different acupoints inhibited NTS gastric motility related neurons (P<0.01, P<0.05). There was no difference in the effects of acupuncture on NTS gastric motility related neuron discharges (independent t test).
Of the four intravenously injected autonomic agonist drugs, none of them had effects on acupuncture regulating NTS neurons (P<0.001).
3The effects of NTS colorectal distention (CRD) related "ON cell","OFF cell" neurons and their roles in the acupuncture treatment of visceral pain
3.1Objective
To identify NTS CRD related "ON cell" or “OFF cell” neurons by graded CRD in anaesthized rats, to observe the effects of acupuncture on these NTS CRD related neurons, to discover the central nervous mechanism of acupuncture interfering vescral pain in the NTS.
3.2Methods
In normal male SD rats, heart rate, blood pressure of the carotid artery, NTS neuronal discharges were monitored. A4cm ballon was placed in rectum connected to a pressure gauge to give the stepping CRD from20to80mmHg. NTS neurons were identified if their spontaneous activities excited(ON cell) or inhibited(OFF cell) by CRD, electroacupuncture (EA,2mA,0.5ms,10Hz) of AA, PC6, ST36was conducted for30s for further discrimination.
3.3Results
3.3.1The echelonment responses of NTS neurons to CRD
The NTS “ON cell”neuronal discharges increased from205.52±30.12/30s to232.2±32.89/30s,279.13±41.28/30s,303.83±40.42/30s,365.81±43.05/30s by stepping CRD stimulation of20to80mmHg, and compared to the basis of discharges, the NTS neuronal discharges were significantly increased by21.10±5.23/30s,56.79±11.93/30s,86.17±13.38/30s,124.27±15.00/30s.
The NTS "OFF cell"neuronal discharges decreased from196.67±42.36/30s, respectively, to186.36±45.01/30s,123.92±34.10/30s,83.75±23.49/30s,49.83±18.27/30s by stepping CRD stimulation of20to80mmHg, and compared to the basic discharges reduced by25.82±13.43/30s,72.72±14.77/30s,112.91±22.37/30s,146.83±29.83/30s.
There were2neurons that had excited then inhibited responses to CRD, and3neurons that had firstly inhibited then excited responses to CRD. They were not statistically analyzed.
3.3.2The effects of EA on NTS CRD related neurons discharges activities before and after CRD
EA of AA, PC6, ST36can inhibit the "ON cell" NTS CRD related neuronal discharges before and during CRD stimulation.
EA of AA, PC6, ST36can excite the "OFF cell" NTS CRD related neuronal discharges before and during CRD stimulation.
4Conclusions
There are pressor reflex-related and depressor reflex-related neurons in NTS, these neurons'responses to these blood pressure reflexes can be excitation and inhibition, showing the diversity of the reaction. These neurons can be excited by AA. ST36more significantly compared with the acupuncture of PC6, in accordance with the antihypertensive effects of AA and ST36, indicating that the NTS plays an important central integrate role during acupuncture regulating blood pressure process, to regulate blood pressure in central integration of neural signals (cross talk).
Acupuncture of different acupoints had different effects on gastric motility, acupuncture of AA and gastrointestinal different segmental ST36and PC6can significantly enhance gastric motility, acupuncture of gastrointestinal similar neural segmental CV12can attenuate gastric motility. After i.v. different sympathetic subtypes" receptor agonists, the impact of acupuncture on gastric motility declined; MA affected gastric motility more significantly than EA; while acupuncture of different acupoints on gastric motility changes, AA, ST36can promote gastric motility significantly better than PC6. After i.v. parasympathetic cholinergic drugs, there is no significant change in the effects of different acupoints on gastric motility. Within the NTS, there exists neurons with synchronous changes of gastric motility, different autonomic receptor agonists can excite such neurons in the rat, mainly mediating acupuncture of AA, PC6, ST36enhancement in gastric motility and CV12gastric motility inhibition effect, acupuncture effects after the autonomic receptor agonist has no significant difference in the effects of such neurons during physiological state, mainly inhibited discharges of NTS neurons.
NTS CRD related neurons had two categories:first class exhibited excited reaction on the stepping CRD stimulation, called "ON cell"; second class exhibited inhibited reaction, called "OFF cell". These two types of neurons mediated acupuncture's anti-nociceptive effects. Acupuncture can make NTS CRD "ON cell" neurons inhibited,"OFF cell" neurons excited. NTS neurons participated the modulatory effect of EA on CRD induced visceral pain in anesthetized rat. Concerning the bilateral regulation of EA on the NTS neuronal activities excited or inhibited by CRD, there may exist cross-talk polysynaptic mechanism. These results suggest acupuncture can interfere in visceral sensation and pain through cross-talk, which is the brainstem nuclei mechanism of acupuncture analgesia.
针刺对内脏功能的调节是由脊髓及脊髓上中枢协同完成的。针刺引起的体表-内脏反射是其调节心血管、胃肠等内脏功能的重要神经反射形式,可分为脊髓固有反射及脊髓上反射两种形式。体表-自主神经反射分为体表-交感和体表-副交感反射两种,由器官的自主神经支配特性和刺激部位的脊髓节段性决定,同时受刺激强度和种类的影响。孤束核(nucleus of tractus solitary, NTS)位于延髓背侧,是迷走神经感觉传入的第一级中枢,参与心血管、呼吸、胃肠等多种内脏功能的调控,也是心血管压力感受性反射、胃肠感觉的第一级感觉中继站；同时NTS与迷走神经背核、延髓尾端腹外侧核(rostral Ventrolateral Medulla, rVLM)、延髓头端腹外侧核(caudal Ventrolateral Medulla, cVLM)、疑核等中枢核团有纤维联系,通过交感和副交感神经传出通路完成对内脏功能的调节。
针刺调节内脏功能,常见单一穴位或依据传统穴位配伍的多穴调节一脏的报道。例如内关对心血管功能的影响,足三里对胃肠功能的作用。未涉及穴位选取的规律性总结。本研究根据哺乳动物皮肤,肌肉,内脏神经分布的节段性特点,结合体表刺激引起躯体—植物神经反射的前期研究报道,探讨不同节段穴位,不同刺激方式引起的内脏植物神经反射的规律,力求从动物实验总结出针刺对特定内脏功能进行交感样和副交感样调节的规律。本研究通过电生理学方法在中枢鉴别出NTS与心血管、胃肠等内脏功能及结直肠扩张(colorectal distention, CRD)刺激相关的神经元,同时记录平均动脉压(mean arterial pressure, MAP)、胃运动,选取不同神经节段的穴位进行针刺刺激,观察不同穴位对MAP和胃运动的影响,并同步观察NTS与血压调节、胃运动、CRD刺激相关神经元放电活动,研究NTS在降压、调节胃运动和抑制CRD诱导内脏痛中的作用。
1针刺对MAP的影响及NTS与血压调节相关神经元在针刺降压中的作用
1.1实验目的
研究针刺对正常麻醉大鼠MAP、静脉注射去氧肾上腺素(phenylephrine,PE),硝普钠(sodium nitroprusside, NP)分别引起升压平台期、降压平台期时MAP的影响,并同步观察NTS与升压(hypertensive related neurons)、降压反射相关神经元(hypotensive related neurons)在针刺调节血压时放电活动的变化,探讨NTS中与升压、降压反射相关神经元在介导针剌降压中的作用。
1.2实验方法
本课题所有研究的动物实验过程均遵守美国国立卫生院倡导的实验动物保护和使用指导原则(Guide for Care and Use of Laboratory Animals, NIH),实验过程中对动物的处置遵照科技部发布的《关于善待实验动物的指导性意见》规定。
实验选用健康成年Sprague-Dawley雄性大鼠81只,体重300-380g之间,用10%的乌拉坦腹腔注射麻醉(urethane,1.0-1.2g/kg体重)。手术及实验中动物体温用动物恒温系统维持在37℃左右。大鼠麻醉后进行颈动、静脉插管、气管插管,并暴露延髓后,监测动脉血压和心率,同步记录MAP及NTS神经元放电,在微电极推进器下,找到自发放电稳定的NTS神经元,经静脉推注30s左右PE(4μg/kg)、NP (NP,20ug/kg),以判断该神经元对升压反射、降压反射的反应及鉴别出与升压反射和降压反射相关神经元。对升压反射和降压反射相关神经元,待自发放电稳定、MAP'恢复至正常后,记录30s放电脉冲数作为对照值,分别电针(波宽500ms,10Hz,1mA)“耳穴心”、“内关”、“足三里”各30秒。为排除针刺穴位顺序对神经元放电结果的影响,穴位针刺顺序随机选择,观察对MAP及血压调节相关神经元放电的影响。然后在大鼠颈静脉微量注射PE(6～121μg/kg,10-15min)或者NP(60～80μg/kg,10-15min)分别造成药物性血压升高、药物性血压降低模型各15min左右,在升压平台期、降压平台期再分别电针上述穴位,并观察对动脉血压和NTS神经元放电的影响。
1.3实验结果
1.3.1NTS与血压反射相关神经元的鉴别
麻醉大鼠颈静脉注射PE,完整记录120个NTS细胞放电(细胞记录不完整者不做统计,下同),其中70个细胞(58.3%)放电频率无变化；26个细胞(21.7%)放电频率减少,呈抑制反应；24个细胞(20.0%)放电频率增加,呈兴奋反应。
麻醉大鼠颈静脉注射NP,完整记录101个NTS细胞放电,其中52个细胞(51.5%)放电频率无变化；29个细胞(28.7%)放电频率减少,呈抑制反应；20个细胞(19.8%)放电频率增加,呈兴奋反应。
13.2电针刺激对麻醉大鼠生理状态下MAP及NTS神经元放电活动的影响
电针“耳穴心”和“足三里”能够明显降低实验动物的MAP,与此同时,NTS与升压、降压反射相关神经元的放电明显增加。电针“内关”虽然也能轻度激活NTS神经元放电,但对MAP没有明显影响。统计结果表明,正常血压状态下,电针“耳穴心”使动物的MAP由100.11±3.3mmHg下降为88.47±3.75mmHg,下降幅度为11.64±0.45mmHg(P<0.001)；电针“足三里”使MAP下降了5.74±0.64mmHg(P<0.001),电针“内关”对MAP无显著影响。不同穴位组间降压效应比较显示,电针“耳穴心”、“足三里”对正常MAP的影响大于“内关”(P<0.05)。
针刺不同穴位引起MAP变化,同时影响正常生理性血压状态下NTS与血压反射相关神经元的放电频率,这种影响主要以兴奋性为主。在MAP正常状态下,电针大鼠不同穴位,对血压反射有兴奋或抑制性反应的神经元放电频率发生明显变化。电针大鼠“耳穴心”(n=50),25个神经元(50%)放电频率增加,(净增加率为115.2±15.25%)。电针“内关”穴(n=50),24个神经元(48%)放电频率增加(净增加率为54.24±6.89%)。电针“足三里”穴(n=47),20个神经元(42.55%)放电频率增加(净增加率为103.35±14.78%)。组间比较显示,电针“耳穴心”和“足三里”两个穴位对神经元的激活百分比增加率显著高于“内关’穴(P<0.05)。
1.3.3电针刺激对PE引起血压升高状态MAP和NTS升压反射相关神经元放电活动的影响
大鼠颈静脉注射PE(12～16μg/kg,10～15min)造成药物性升压模型,MAP由99.28±3.82mmHg上升并维持在121.66±2.91mmHg(P<0.001)。电针“耳穴心”和“足三里”能明显降低实验动物升压平台期的MAP,与此同时,NTS与升压反射相关神经元的放电明显增加。电针“内关”虽然能轻度激活NTS神经元放电,但对MAP没有明显影响。统计结果表明,在PE引起的升压平台期,电针“耳穴心”使MAP由124.17±4.43mmHg下降到118.7±3.64mmHg,下降幅度8.30±0.79mmHg(P<0.001)；电针“足三里”使MAP下降6.07±0.79mmHg(P<0.05)；电针“内关”对MAP无明显影响。组间比较显示(one-way ANOVA),电针“耳穴心”、“足三里”的降压效应优于“内关”(P<0.05)。电针同一穴位对麻醉大鼠正常状态和PE引起血压升高状态下MAP的影响无明显差异(independent/test,P>0.05).
电针不同穴位引起升压平台期MAP变化,同时对NTS与升压反射相关神经元的放电频率产生影响,这种作用主要以兴奋性为主,使神经元放电增加。对升压反射有兴奋或抑制性反应的50个神经元中,完成升压平台期记录及针刺实验的共21个。电针大鼠耳甲区穴位,9个放电频率增加,净增加率为92.94±10.42%。电针“内关”穴,9个放电频率增加,净增加率为44.66±6.27%。电针“足三里”穴,8个放电频率增加,净增加率为86.73±5.57%。组间比较显示,电针“耳穴心”和“足三里”两个穴位对神经元的激活百分比增加率显著高于“内关”穴(P<0.05)。
1.3.4电针刺激对NP引起血压降低状态MAP和NTS降压反射相关神经元放电活动的影响
大鼠颈静脉注射NP(60～80μg/kg,10～15min)造成药物性降压模型,MAP由96.56±2.24mmHg下降并维持在78.29±1.84mmHg.电针不同穴位能明显降低实验动物降压平台期的MAP,与此同时,NTS与降压反射相关神经元的放电显著增加。统计结果表明,在NP引起的降压平台期,电针“耳穴心”使MAP由79.76±1.39mmHg下降到69.32±2.48mmHg,下降幅度10.44±1.60mmHg(P<0.001)；电针“内关”使MAP下降7.93±1.20mmHg(P<0.05)；电针“足三里”使MAP下降15.63±1.76mmHg(P<0.05)；组间比较显示(one-Way ANOVA),电针上述三个穴位的降压效应无差异(P>0.05)。电针“耳穴心”、“足三里”,对麻醉大鼠生理情况MAP及NP降压状态MAP的影响无明显差异,电针“内关”对降压平台期血压的降压效应好于生理状态(independent t test,P<0.05)。
电针不同穴位引起降压平台期MAP变化,同时,对NTS与降压反射相关神经元的放电频率产生影响,这种作用主要以兴奋性为主。电针“耳穴心”、“内关”和“足三里”使NTS内与降压反射有关神经元的放电增加。对降压反射有兴奋和抑制性反应的49个神经元中,完成升压平台期记录的共19个。电针大鼠耳甲区穴位,9个放电频率增加,净增加率为138.00±18.76%。电针“内关”穴,8个神经元放电频率增加,净增加率为96.75±20.51%。电针“足三里”穴,8个神经元放电频率增加,净增加率为100.75±31.87%。组间比较显示,电针三个穴位对神经元的激活百分比无显著差异(组间比较,one-way ANOVA, P>0.05)
2针刺对胃运动及NTS胃运动相关神经元放电活动的影响
2.1实验目的
观察针刺不同穴位对正常麻醉大鼠胃内压的影响,及静脉注射不同交感和副交感神经受体业型激动剂引起胃运动迟缓和亢进状态时针刺的效应,并同步观察NTS与胃运动相关神经元在针剌调节胃运动过程中放电活动的变化,探讨NTS中与胃运动相关神经元在针刺调节胃肠功能中的作用。
2.2实验方法
实验选用健康成年Sprague-Dawley雄性大鼠50只。实验前禁食12-18小时,手术及实验中动物体温用动物恒温系统维持在37℃左右。大鼠麻醉后进行颈静脉、气管插管,胃幽门部插入水囊连接压力换能器以记录胃内压,并监测血压、心率。
同步记录胃内压及孤束核与胃运动相关神经元,找到稳定孤束核神经元胞外放电后,记录背景放电30s,然后分别电针、手针“耳穴心”、“内关”、“中脘”、“足三里”四个穴位,同步观察对胃运动和孤束核神经元细胞外放电的影响。对针刺有反应的神经元,分为5组,分别静脉注射四种药物和生理盐水：p3体激动剂BRL37344Salt Hydrate4-8μ g/kg；β2受体激动剂盐酸克仑特罗0.05-0.1mg/kg;α：受体激动剂可乐定0.4-0.8mg/kg;拟胆碱药氨基甲酰胆碱0.2-0.4mg/kg和静脉注射0.2ml0.9%的生理盐水。上述药物引起胃蠕动迟缓或亢进的同时,NTS神经元放电活动发生兴奋或抑制的同步变化,则鉴定为孤束核与胃运动相关神经元。各组药物注射后,继续观察针刺不同穴位对胃运动和孤束核与胃运动相关神经元的影响。
2.3实验结果
2.3.1生理状态下针刺对胃运动和NTS胃运动相关神经元放电活动的影响
生理状态下,麻醉大鼠的胃运动频率为4-5次/min,波幅在50-100mmH2O。结果表明电针刺激“耳穴心”、“内关”、“足三里”都能促进麻醉大鼠胃蠕动(P<0.01),而电针“中脘”穴则明显减弱胃蠕动(P<0.001)。手针效果与电针效果一致(independent t test, P>0.05)。
针刺过程共记录133个NTS神经元胞外放电,对针刺上述4个穴位有反应的神经元104个；静脉注射p3受体激动剂、β2受体激动剂、α2受体激动剂、拟胆碱药有反应的共45个,以下分组讨论。在正常情况下,针刺对孤束核与胃运动相关神经元放电的影响以抑制为主。针刺不同穴位对孤束核与胃运动相关神经元的抑制作用无明显差异(one way ANOVA, LSD, P>0.05)
2.3.2使用β3受体激动剂后针刺对胃运动和NTS相关神经元活动的影响
静脉注射β3受体激动剂后,药物对胃运动抑制作用大约持续30-60min,胃运动蠕动波波幅下降了10-20mmH20,胃内压明显降低,幅度为65.4±7.20mmH2O(P<0.001)。药物后电针对胃运动没有影响(P>0.05)。手针刺激“耳穴心”、“足三里”使胃内压增高(P<0.05)；手针刺激“中脘”使胃内压降低(P<0.05)。与电针比较,手针“耳穴心”和“中脘”分别引起的胃运动的增强和抑制效应更为显著(P<0.05),其余穴位不同刺激无明显差异(independent t test, P>0.05)。
分别针刺4个穴位对p3受体激动剂静脉注射前后孤束核与胃运动相关神经元放电的影响无统计学差异(independent t test, P>0.05)。
2.3.2使用β2受体激动剂后针刺对胃运动和NTS相关神经元电活动的影响
静脉注射β2受体激动剂Clenbuterol(克伦布特罗)后,胃运动波幅明显减低了10-20mmH20,胃内压明显下降,幅度为51.18±9.93mmH2O(P<0.001)。电针刺激“耳穴心”使胃内压升高(P<0.05；电针刺激“内关”、“中脘”、“足三里”对胃运动无作用(P>0.05)。手针刺激“耳穴心”、“内关”、“足三里”能促进胃运动(P<0.01),手针“中脘”能抑制胃运动(P<0.05)。手针“内关”、“足三里”、“中脘”相比电针对胃运动的影响作用更明显。(independent t test, P<0.05)。
在Clenbuterol药物引起胃运动抑制状态,针刺不同穴位对药物前后孤束核与胃运动相关神经元的放电的影响无明显差异(independent t test, P>0.05)。
2.3.3使用α2受体激动剂后针刺对胃运动和NTS相关神经元放电活动的影响
静脉注射α2受体激动剂Clonidine后,胃运动的波幅降低了10-20mmH20,胃内压明显下降,幅度为79.07±24.59mmH20,持续30-60min(P<0.05)。药物后电针、手针“耳穴心”、“足三里”能促进胃运动(P<0.05),在Clonidine药物注射后引起胃运动迟缓状态下,两种刺激增强胃运动的作用无显著差异。手针“中脘”能抑制胃运动且比电针的抑制作用更显著(independent t test,P>0.05)。
针刺不同穴位对Clonidine药物静脉注射前后孤束核与胃运动相关神经元的放电的影响无明显差异(independent t test, P>0.05)
2.3.4使用拟胆碱药后针刺对胃运动和NTS相关神经元放电活动的影响
静脉注射拟胆碱药氨基甲酰胆碱后,胃运动频率明显升高至6-7次/min,胃运动波幅升高了200-300mmH2O,胃内压明显升高,幅度为121.27±29.53mmH20(P<0.01)。药物注射前后,电针和手针“耳穴心”、“内关”、“足三里”均能促进胃运动(P<0.01,P<0.05,P<0.001),电针和手针“中脘”均能抑制胃运动(P<0.01；P<0.01)。与电针相比较,在拟胆碱药引起胃运动亢进状态下,手针“耳穴心”和“中脘”的作用更显著(independent t test, P<0.05)。
针刺不同穴位对拟胆碱药静脉注射前后孤束核与胃运动相关神经元的放电的影响无明显差异(independent t test, P>0.05)。静脉注射上述四种交感和副交感受体业型激动剂对针刺调节孤束核神经元放电活动无影响。
3针刺对NTS与CRD相关神经元活动的影响与针刺治疗内脏痛的机制
3.1实验目的
给予麻醉大鼠梯度的CRD条件刺激,鉴别NTS中CRD“点燃”(ON cell)型和“熄火”(OFF cell)型神经元,观察针刺作为检验刺激对此类神经元放电活动的影响,探讨针刺在孤束核部位干预内脏痛的中枢神经系统机制。
3.2实验方法
正常雄性SD大鼠,监测心率、颈总动脉血压,记录孤束核神经元细胞外放电。大鼠经肛门在直结肠放置长度4-5cm左右的气囊,通过压力计随机给予梯度为20、40、60、80mmHg的结直肠扩张刺激(CRD),同时用电生理单细胞记录的方法记录和鉴别孤束核对CRD刺激有反应的相关神经元,参照Fields等的研究大鼠RVM内存在对伤害性热刺激引起的甩尾反射相关的“熄火”型(OFF cell)和“点燃”型(ON cell)神经元,对梯度CRD刺激产生递增的兴奋性反应的神经元,称为“点燃”型(ON cell),对梯度CRD刺激产生递减的抑制性反应的神经元,称为“熄火”型(OFF cell).鉴别出NTS中CRD相关神经元后,待放电恢复至基础水平,给予CRD刺激,NTS神经元放电发生明显"ON cell"或“OFFcell"型反应时,给予2mA、10Hz持续30s的电针不同穴位的刺激,观察针刺“耳穴心”、“内关”、“足三里”在正常及CRD条件刺激引起NTS放电变化时对孤束核与CRD "ON cell"型和"OFF cell"型神经元的影响。
3.3实验结果
3.3.1NTS神经元对梯度CRD刺激的反应
本实验在57只大鼠共记录到对CRD "ON cell"型神经元21个。在CRD20、40、60、80mmHg条件刺激时,NTS神经元放电由自发的205.52±30.12个/30s分别增加了21.10±5.23个/30s、56.79±11.93个/30s、86.17±13.38个/30s、124.27±15.00个/30s(P<0.001,P<0.01,P<0.001,P<0.001),为梯度增加反应。
另外记录到12个CRD "OFF cell"型神经元。在CRD20、40、60、80mmHg条件刺激时,NTS神经元放电由自发的196.67±42.36个/30S分别减少了25.82±13.43个/30s、72.72±14.77个/30s、112.91±22.37个/30s、146.83±29.83个/30s(P<0.05,P<0.01,P<0.01,P<0.05),为梯度抑制反应。
此外记录到2个对梯度CRD刺激产生先兴奋后抑制,3个先抑制后兴奋的神经元,在本实验中未纳入统计。
3.3.2电针对NTS与CRD相关神经元放电活动的影响
CRD刺激对NTS产生"ON cell"型反应的神经元,CRD刺激前和CRD过程中电针“耳穴心”、“内关”、“足三里”都以抑制反应为主。如CRD前电针“耳穴心”放电减少了34.73±3.37个/30s(P<0.01)；CRD刺激过程电针“耳穴心”使放电减少了45.55±4.17个/30s(P<0.001)。
对CRD刺激产生"OFF cell"型反应的神经元,在CRD刺激前和CRD刺激过程中电针上述3个穴位都以兴奋反应为主。如CRD前电针“耳穴心”使放电增加了184.26±27.35个/30s(P<0.001)；CRD刺激时电针“耳穴心”使放电增加了130.14±28.23个/30s(P<0.01)。
4结论
孤束核内存在升压、降压反射相关神经元,这些神经元可以分别被升压、降压反射兴奋和抑制,表现为反应的多样性。在生理状态下及PE引起的升压平台期,这些神经元被“耳穴心”、“足三里”激活的百分比与“内关”相比更为显著；在降压平台期,这些神经元被上述三个穴位激活的百分比无明显差异,与其针刺降压相应相一致。这说明NTS在针刺调节血压过程中发挥重要的中枢整合作用,通过神经信号交互调节(cross talk)的方式完成对血压的调节。
针刺不同穴位对胃运动的影响不同,针刺“耳穴心”及与胃肠异节段的“足三里”、“内关”能显著增强胃运动,针刺与胃肠同神经节段的“中脘”使胃运动减弱。使用交感神经不同业型神经受体激动剂后,针刺对胃运动的影响减弱；电针对胃运动的作用不如手针显著；而针刺不同穴位对胃运动的影响也发生变化,“耳穴心”、“足三里”促进胃运动的效果比“内关”明显。使用副交感神经的拟胆碱药物后,不同穴位对胃运动的作用与药物前相比无明显变化。NTS内存在与胃运动同步变化的神经元,不同植物神经受体激动剂对此类神经元的影响以兴奋为主,介导了针刺“耳穴心”、“内关”、“足三里”的胃运动增强效应和“中脘”的胃运动抑制的效应,使用植物神经受体激动剂后针刺对此类神经元的效应与生理状态下无显著差异,以抑制NTS神经元放电为主。
NTS与CRD刺激相关神经元有两类：第一类对梯度的CRD刺激产生兴奋反应,表现为神经元胞外放电梯度增加,为“点燃”型神经元(ON cell);第二类对梯度的CRD刺激产生抑制反应,表现为神经元胞外放电梯度减少,为“熄灭”型神经元(OFF cell).这两类神经元介导了针刺拮抗CRD痛刺激的效应。针刺能够使孤束核CRD"ON cell"型神经元的放电抑制,而使孤束核CRD"OFF cell"型神经元的放电增加。提示针刺可以通过交互调节(cross talk)的方式在孤束核干预内脏感觉和痛觉,是针刺镇痛的脑干核团机制所在。
The Miraculous Pivot (it is one of the earliest medical classics in China) stated."Twelve regular channels belong to the zang-fu organs internally, and connect to the extremities and joints externally." Acupuncture and Moxibustion can treat visceral diseases to modulate visceral function by stimulating the acupoins. Acupuncture and Moxibustion's treatment of kinds of visceral diseases such as cardiovascular, gastrointestinal diseases are very effective, and has certain acupoints specificity.
Acupuncture modulating visceral diseases through coordination of spinal and supraspinal centers. Acupuncture-induced somato-viscera reflexes have two patterns of propriospinal reflex and supraspinal reflex. Somato-autonomic nervous reflexes are divided into two kinds:the somato-sympathetic and the somato-parasympathetic reflex, dominated by the autonomic nervous characteristics of the organs and the spinal cord segments of the stimulated parts, as well as the impact of stimulus intensity and type. The medullary dorsal nucleus of solitary tract (NTS) is the first level of the central vagus nerve afferent involved in the regulation of cardiovascular, respiratory, gastrointestinal and other visceral functions of cardiovascular baroreceptor reflex, the relay station of gastrointestinal senses. NTS has fibric connections with the dorsal nucleus of the vagus nerve, the rostral Ventralateral Medulla (rVLM,), the caudal Ventrolateral Medulla (cVLM), nucleus ambiguous (NA) and other central nuclei of the fiber contact, to complete the regulation of visceral functions through the sympathetic and parasympathetic efferent pathways.
Acupuncture regulating visceral function was commonly reported using single point or multi-points according to the traditional acupuncture compatibility. For example, the impact of Neiguan (PC6) on cardiovascular function, Zusanli (ST36) on gastrointestinal function, yet the summary of the regularity of acupuncture points selected is not involved in. In this study, according to the segmental characteristics of mammalian skin, muscle, and splanchnic nerve distribution, combined with a preliminary study of surface stimulation of the body, we tried to explore the law of the visceral autonomic reflex caused by the different segments of acupuncture points, the different stimulation, and strive to summarize the sympathetic and parasympathetic-like regulation of the law of acupuncture on the specific function of internal organs from animal experiments. This study identified the central NTS neurons that related to the cardiovascular, gastrointestinal and colorectal distention (CRD) by electrophysiological methods, simultaneously recording the blood pressure, gastric motility, to stimulate by selecting the different acupoints of the body segments, and to observe the NTS-related neuronal activity, to study the role of NTS related neurons in the effects of acupuncture reducing MAP, modulating gastric motility and inhibiting CRD-induced visceral pain.
1The effect of acupuncture on MAP and the role of NTS blood pressure regulation related neurons in the acupuncture reducing blood pressure
1.1Objective
To observe the effects of acupuncture on MAP in three conditions of anaesthized rats:physiological, pressor reflex, depressore reflex caused by i.v. PE, NP and the firing changes of the NTS pressor reflex related neurons, depressor reflex related neurons, to demonstrate the role of NTS blood pressure regulation related neurons in acupuncture reducing MAP.
1.2Methods
All the experimental process of animals in this study was in accordance with '"Guide for Care and Use of Laboratory Animals, NIH", the disposal of animals was in accordance with "Guidance to treat experimental animals", released by the Ministry of Science and Technology.
Eighty one healthy adult Sprague-Dawley male rats were used, weighing300-380g, anesthetized with10%urethane intraperitoneal injection of anesthetic (urethane,1.0～1.2g/kg body weight). During Surgery and experiments, animals thermostat system was used to maintain animal body temperature around37℃. Simultaneously NTS neurons and arterial blood pressure were recorded, looking for stabile spontaneous discharges of the NTS neurons under the control of microelectrode thruster. To identify the neurons'responses to the pressor reflex, depressor reflex reaction, intravenous injection of phenylephrine (PE)4μg/kg, sodium nitroprusside (NP)20ug/kg were administered. The discharges of NTS neurons responded to blood pressure reflex were recorded,30s discharges pulse number were recorded as control, then respectively EA of "Auricular acupoint"(AA),"Neiguan"(PC6)","Zusanli"(ST36) each for30seconds. To exclude the impact of acupuncture order on neuronal firing results, the acupuncture order randomly selected to observe the impact of acupuncture on the MAP and the discharge of NTS neurons. Microinjection of PE in the rat jugular vein caused drug-induced high blood pressure, or microinjection of NP in the rat jugular vein caused drug-induced low blood pressure. During the step-up platform, step-down platform, we observed the impact of EA on arterial blood pressure and the discharge of NTS neurons.
1.3Results
1.3.1The identification of the NTS blood pressure reflex related neurons
After jugular vein injection of PE in normal anesthetized rats, a complete of120NTS neuron discharges were recorded (the remaining cells were not completely recorded, and did not do statistic analysis), in which the discharge frequency of70cells (58.3%) did not change; the discharge frequency of26cells (21.7%) reduced, exhibitng inhibited response; the discharge frequency of24cells (20.0%) increased, exhibitng excited reaction.
After jugular vein injection of NP in normal anesthetized rats, a complete of101NTS neuron discharges were recorded, in which the discharge frequency of52cells (51.5%) did not change; the discharge frequency of29cells reduced (28.7%), showing inhibited responses;20cells (19.8%) increased, showing excited reaction.
Among them,50cells responded to the pressor reflex, in which31cells responded to depressor reflex. In24Pressor reflex excitated cells,9had inhibited reaction to NP,7no reaction,8excited; In26pressor reflex inhibited cells,13excited,1no response,12inhibited.
1.3.2The effects of electro acupuncture (EA) on MAP and NTS neurons' discharges in anesthetized rats under physiological conditions.
EA of A A and ST36can significantly reduce the MAP of the experimental animals, at the same time, discharges in the NTS pressor reflex neurons increased significantly. EA of PC6can also mildly activate NTS neural discharges, but almost no effect on the MAP. The statistical results showed that during the state of normal blood pressure, EA of AA reduced MAP of the animals from100.11±3.3mmHg to88.47±3.75mmHg, decreased by11.64±0.45mmHg (P<0.001); EA of ST36decreased normal MAP for5.74±0.64mmHg (P<0.001),EA of PC6had no significant effect on the normal MAP. The comparison of the antihypertensive effects of different acupoint groups, EA of AA, ST36is greater than PC6under normal MAP condition (P<0.05).
MAP changed during acupuncture at different acupoints, at the same time, the NTS pressor reflex related neurons'discharge activities were excited. EA of AA, PC6and ST36increased NTS pressor reflex related neural discharges. When MAP restored to normal state, EA of AA increased discharge frequencies of25neurons of pressor reflex excitatory or inhibitory response of50neurons (net increase in the rate was115.2±15.25%). EA of PC6,24neurons discharge frequency increased (net increase in the rate of54.24±6.89%). EA of ST36,20neurons'firing rate increased (net increase was103.35±14.78%). The results of the comparison between groups showed that the EA of AA and ST36two points compared to PC6, the neuron activation percentage increased even more significantly (P<0.05).
1.3.3The effects of EA on MAP and NTS neurons discharges in PE caused MAP increased state rats
In the rats'carotid, intravenous injection of PE (12～16μg/kg,10～15min) caused drug-induced step-up model, the MAP increased and maintained from normal99.28±3.82mmHg to121.66±2.91mmHg. EA of AA and ST36can significantly reduce the step-up platform of MAP in experimental animals. At the same time, the discharges of NTS pressor reflex related neurons increased significantly. EA of PC6also can mildly activate NTS neural discharges, but almost no significant effect on the MAP. The statistical results showed that during the PE pressor plateau, EA of AA reduced step-up platform of MAP from124.17±4.43mmHg to118.7±3.64mmHg, decreased by8.30±0.79mmHg (P<0.001); EA of ST36decreased the step-up platform of the MAP by6.07±0.79mmHg (P<0.05); EA of PC6had no significant effect on step-up platform of MAP. Between the depressor effect of EA of AA and ST36to PC6, the former two acupoints had better pressor effects (one way ANOVA, P>0.05).
EA of A A, PC6, ST36on the MAP states of normal anesthetized rats and PE caused high blood pressure state, had no significant difference (independent t test, P>0.05).
EA caused MAP changes during the step-up platform, at the same time, EA excited the discharged of NTS pressor related neurons. EA of AA, PC6and ST36increased the NTS pressor reflex related neurons'discharges. Of the50NTS pressor reflex excitatory and inhibitory neurons, a total of21NTS discharges were recorded during the step-up platform. EA of AA, nine discharge frequencies increased, the net increase rate was92.94±10.42%,7had no change in discharge frequency, five discharge frequency decreased. EA of PC6increased nine neurons firing rate, a net increasing rate was44.66±6.27%, seven discharge frequencies did not change,5discharge frequencies reduced. EA of ST36, eight neuronal firing frequencies increased, the net increasing rate was86.73±5.57%, eight discharge frequencies did not change, five discharge frequencies were reduced. The results of the comparison between groups showed that the EA of AA and ST36excited the neuronal discharges more significantly compared to PC6(P<0.05).
1.3.4The effects of EA on MAP and NTS neurons discharges of rats in the NP caused MAP reduced state
In rat jugular vein injection of the NP (60～80μg/kg, and10～15min) caused drug-induced step-down MAP model, the MAP reduced from the normal96.56±2.24mmHg to78.29±1.84mmHg. EA of AA, PC6and ST36can significantly reduce the step-down platform of MAP in experimental animals. At the same time, neurons in the NTS step-down reflex related neurons discharges increased significantly. Statistical results showed that the EA of AA in NP caused step-down platform of MAP dropped from79.76±1.39mmHg to69.32±2.48mmHg, decreased by10.44±1.60mmHg (P<0.001); In NP caused step-down platform of MAP, MAP decreased by7.93±1.20mmHg by EA of PC6(P<0.05);15.63±1.76mmHg by ST36decreased by (P<0.05); Comparison between groups showed that (one way ANOVA), the depressor effect of EA at these three points had no significant difference (P>0.05).
EA of AA and ST36can reduce the MAP during physiological and NP induced depressor conditions and there was no significant difference between these two states, EA of PC6can reduce the MAP in the NP induced depressor conditions, which was better than in the physiological state.(Independent t test, P<0.05).
EA caused the changes of the MAP during the step-down platform, at the same time, EA excited the discharge frequencies of neurons in the NTS. EA of AA, PC6and ST36can increase the discharges of NTS depressor reflex related neurons. In the49excitatory and inhibitory response neurons,19NTS discharges were recorded during the step-down platform. During EA of AA, nine discharge frequencies increased, the net increase in the rate was138.00±18.76%, seven discharge frequencies did not change,3discharge frequencies reduced. EA of PC6, eight neuronal firing frequencies increased, the net increase rate was96.75±20.51%, and seven had no change in the discharge frequencies, and four reduced discharge frequencies. During EA of ST36, eight neurons increased the discharge frequencies, the net increase rate was100.75±31.87%, eight firing rate did not change,3discharge frequencies reduced. The results of the comparison between groups showed that EA of these three acupoints on the activation percentage had no significant difference (between the two groups, One-way ANOVA, P>0.05).
2The role of NTS gastric motility related neurons in acupuncture adjusting gastrointestinal function
2.1Objective
To investigate the effect of acupuncture of different acupoints on physiological gastric pressure and the inhibited or excited conditions caused by i.v different sympathetic and parasympathetic subtype receptor agonists, and synchronizely on the firing rates of NTS gatric motility related neurons, to discuss the role of NTS gastric motility related neurons in acupuncture regulating gastrointestinal functions.
2.2Methods
Fifty Sprague-Dawley male rats were used, weighing300～380g, anesthized with10%urethane intraperitoneal injection (urethane,1.0～1.2g/kg body weight). Fasted for18-20hours before the experiment, animal body temperature during surgical and experimental periods maintained at around37℃by an animal thermostatic system.
Simultaneous recording intragastric pressure and NTS gastric motility related neurons, the stabile discharges of NTS were recorded for background30s, and then EA, manual acupuncture (MA) of AA, PC6, CV12and ST36. The acupuncture responses of neurons divided into5groups were injected intravenously with four drugs:β3agonist, BRL37344the Salt Hydrate4-8μg/kg; β2receptor agonist, Clenbuterol Hydrochloride0.05-0.1mg/kg; α2receptor agonist, Clonidine Hydrochloride0.4-0.8mg/kg; Cholinomimetics, carbamyl-B-methylcholine chloridecrystal0.2-0.4mg/kg and the fifth group of intravenous0.2ml0.9%saline. Of these drugs to slow or facilitate gastric motility, at the same time, the NTS neuron discharge activities had excitatory or inhibitory changes, and then they were identified as NTS gastric motility related neurons. Continue to observe the different effects of acupuncture points on the gastric motility and the NTS gastric motility related neurons after drug injection.
2.3Results
2.3.1The effects of acupuncture on gastric motility and the NTS gastrointestinal motility related neurons under physiological conditions
The results showed that EA of AA, PC6, ST36stimulation can promote gastric motility (P<0.01), EA of CV12significantly reduced the gastric motor function (P0.001). MA had the same results with EA (Independent t test, P>0.05).
Recorded a total of133NTS neuron extracellular discharges,29had no response to MA,104Neurons responded;45neurons responded to intravenous injection of one of the four drugs.
Acupuncture inhibited neurons in the NTS gastric motility related neurons in normal conditions. Acupuncture of the four acupoints inhibiting effects on the NTS gastric motility related neurons had no significant difference (P>0.05, ONE WAYANNOVA of LSD).
2.3.2The effects of β3adrenergic agonist on acupuncture regulating gastric motility and NTS related neurons activities
After intravenous injection of β3adrenergic agonist, EA of AA, PC6and ST36no longer appeared to promote gastric motility, the inhibitory effect of EA of CV12on gastric motility disappeared (P>0.05). MA of AA, ST36increased gastric pressure (P<0.05). MA of CV12decreased the intragastric pressure (P<0.05). MA of PC6in promoting gastric motility disappeared. MA of AA enhanced gastric motility more significantly than EA of AA. MA of CV12had better inhibitory effect on gastric motility than EA. The exciting effect of EA and MA on rest of the acupoints had no significant difference (independent t test, P>0.05).
After the β3agonist BRL37344drugs intravenously, the acupuncture effects on NTS related neurons were still inhibiting.
2.3.3The effects of β2adrenergic agonist on acupuncture regulation of gastric motility and NTS neurons discharge activities
After intravenous drug administration, EA of AA increased intragastric pressure (P<0.05), EA of PC6, CV12and ST36had no effect on gastric motility (P>0.05). MA of AA, PC6and ST36can promote gastric motility (P<0.01), MA of CV12can inhibit gastric motility (P<0.05). MA and EA of AA had no significant difference in the enhancement of gastric motility, MA in the abdominal had more significant inhibitory effect on gastric motility than EA of CV12. MA of PC6and ST36had better gastric motility enhancement than EA of PC6and ST36.(Independent t test, P <0.05).
Acupuncture of the four points on the NTS gastric motility related neurons discharges before and after Clenbuterol drugs had no significant difference (independent t-test).
2.3.4The effects of α2receptor agonist on acupuncture in regulating gastric motility and NTS neurons discharge activities
After intravenous drug administration, EA of A A and ST36can promote gastric motility (P<0.05), EA of PC6, CV12had no significant effect on gastric motility (P>0.05). MA of AA, ST36can promote gastric motility, PC6had no effect on gastric motility (P>0.05). MA and EA had no significant difference on the gastric motility on AA, PC6and ST36after Clonidine injection, MA in the abdomen caused inhibitory effect on gastric motility was more significant than EA in the abdomen (independent t test, P>0.05).
Before and after the intravenous injection of Clonidine,acupuncture at different acupoints reduced NTS gastric motility related neurons (P<0.01, P<0.05).
The impact of acupuncture four acupoints on NTS gastric motility related neurons discharges before and after Clonidine drug injection had no significant difference (independent t test, P>0.05).
2.3.5The effects of Cholinomimetics on acupuncture regulating gastric motility and NTS neurons discharge activities
Before and after the intravenous injection of Cholinomimetics, EA of AA, PC6, ST36can promote gastric motility (P<0.01, P<0.05), EA of CV12can inhibit gastric motility (P<0.01). MA of AA, PC6, ST36obviously can promote gastric motility (P <0.001), MA of CV12in the abdomen significantly inhibited gastric motility (P <0.01). MA and EA of ST36, PC6had no difference in gastric motility enhancement.
Acupuncture at different acupoints inhibited NTS gastric motility related neurons (P<0.01, P<0.05). There was no difference in the effects of acupuncture on NTS gastric motility related neuron discharges (independent t test).
Of the four intravenously injected autonomic agonist drugs, none of them had effects on acupuncture regulating NTS neurons (P<0.001).
3The effects of NTS colorectal distention (CRD) related "ON cell","OFF cell" neurons and their roles in the acupuncture treatment of visceral pain
3.1Objective
To identify NTS CRD related "ON cell" or “OFF cell” neurons by graded CRD in anaesthized rats, to observe the effects of acupuncture on these NTS CRD related neurons, to discover the central nervous mechanism of acupuncture interfering vescral pain in the NTS.
3.2Methods
In normal male SD rats, heart rate, blood pressure of the carotid artery, NTS neuronal discharges were monitored. A4cm ballon was placed in rectum connected to a pressure gauge to give the stepping CRD from20to80mmHg. NTS neurons were identified if their spontaneous activities excited(ON cell) or inhibited(OFF cell) by CRD, electroacupuncture (EA,2mA,0.5ms,10Hz) of AA, PC6, ST36was conducted for30s for further discrimination.
3.3Results
3.3.1The echelonment responses of NTS neurons to CRD
The NTS “ON cell”neuronal discharges increased from205.52±30.12/30s to232.2±32.89/30s,279.13±41.28/30s,303.83±40.42/30s,365.81±43.05/30s by stepping CRD stimulation of20to80mmHg, and compared to the basis of discharges, the NTS neuronal discharges were significantly increased by21.10±5.23/30s,56.79±11.93/30s,86.17±13.38/30s,124.27±15.00/30s.
The NTS "OFF cell"neuronal discharges decreased from196.67±42.36/30s, respectively, to186.36±45.01/30s,123.92±34.10/30s,83.75±23.49/30s,49.83±18.27/30s by stepping CRD stimulation of20to80mmHg, and compared to the basic discharges reduced by25.82±13.43/30s,72.72±14.77/30s,112.91±22.37/30s,146.83±29.83/30s.
There were2neurons that had excited then inhibited responses to CRD, and3neurons that had firstly inhibited then excited responses to CRD. They were not statistically analyzed.
3.3.2The effects of EA on NTS CRD related neurons discharges activities before and after CRD
EA of AA, PC6, ST36can inhibit the "ON cell" NTS CRD related neuronal discharges before and during CRD stimulation.
EA of AA, PC6, ST36can excite the "OFF cell" NTS CRD related neuronal discharges before and during CRD stimulation.
4Conclusions
There are pressor reflex-related and depressor reflex-related neurons in NTS, these neurons'responses to these blood pressure reflexes can be excitation and inhibition, showing the diversity of the reaction. These neurons can be excited by AA. ST36more significantly compared with the acupuncture of PC6, in accordance with the antihypertensive effects of AA and ST36, indicating that the NTS plays an important central integrate role during acupuncture regulating blood pressure process, to regulate blood pressure in central integration of neural signals (cross talk).
Acupuncture of different acupoints had different effects on gastric motility, acupuncture of AA and gastrointestinal different segmental ST36and PC6can significantly enhance gastric motility, acupuncture of gastrointestinal similar neural segmental CV12can attenuate gastric motility. After i.v. different sympathetic subtypes" receptor agonists, the impact of acupuncture on gastric motility declined; MA affected gastric motility more significantly than EA; while acupuncture of different acupoints on gastric motility changes, AA, ST36can promote gastric motility significantly better than PC6. After i.v. parasympathetic cholinergic drugs, there is no significant change in the effects of different acupoints on gastric motility. Within the NTS, there exists neurons with synchronous changes of gastric motility, different autonomic receptor agonists can excite such neurons in the rat, mainly mediating acupuncture of AA, PC6, ST36enhancement in gastric motility and CV12gastric motility inhibition effect, acupuncture effects after the autonomic receptor agonist has no significant difference in the effects of such neurons during physiological state, mainly inhibited discharges of NTS neurons.
NTS CRD related neurons had two categories:first class exhibited excited reaction on the stepping CRD stimulation, called "ON cell"; second class exhibited inhibited reaction, called "OFF cell". These two types of neurons mediated acupuncture's anti-nociceptive effects. Acupuncture can make NTS CRD "ON cell" neurons inhibited,"OFF cell" neurons excited. NTS neurons participated the modulatory effect of EA on CRD induced visceral pain in anesthetized rat. Concerning the bilateral regulation of EA on the NTS neuronal activities excited or inhibited by CRD, there may exist cross-talk polysynaptic mechanism. These results suggest acupuncture can interfere in visceral sensation and pain through cross-talk, which is the brainstem nuclei mechanism of acupuncture analgesia.
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