【作者】 张小琴；

【导师】 侯月梅；

【作者基本信息】 新疆医科大学 ， 内科学， 2008， 博士

【摘要】 目的:心脏内在神经脂肪垫与心脏外在神经迷走神经之间,以及心脏内在神经脂肪垫之间可能存在网络调控关系,在心房几个脂肪垫中间,可能存在“主控区”,而消融“主控区”可能在房颤的神经消融治疗中起着重要作用。左心房-肺静脉肌袖作为房颤发生维持的重要基质,在肺静脉消融治疗房颤中起着举足轻重的作用,而肌袖中分布的神经纤维对于起源肺静脉房颤治疗存在重要作用。本课题包括:1)消融单独SANFP、AVNFP以及SANFP联合AVNFP消融,研究SANFP、AVNFP之间以及与左右迷走神经干之间的网络调控关系,对左右心房及肺静脉电生理特性的影响,以及SAN-FP+AVN-FP联合消融后左右心房及肺静脉电生理特性的影响,旨在探索窦房结脂肪垫、房室结脂肪垫之间以及它们与双侧迷走神经干网络调控关系的机制,为临床射频消融心脏脂肪垫治疗心脏脂肪垫消融提供科学依据;2)通过基础高频刺激、给予阿托品后高频刺激、给予阿替洛尔高频刺激肺静脉-左心房肌袖,记录上述三种状态现APD90、EAD,从左心房.静脉肌袖肌袖组织交感神经与迷走神经,离体组织动作电位的角度揭示肺静脉—左心房中植物神经的分布特点;3)研究肺静脉—左心房交界部的肺静脉肌袖的分布模式,尤其是是否存在肌袖的不连续分布,而且这种不连续分布是否在四根肺静脉中或者在肺静脉内的不同部位存在定位特征,为临床射频消融治疗房颤提供理论依据。方法:1)本课题第一部分:18只犬分为二组:第一组9只犬为优先消融SANFP(sinus atrial node fat pad),第二组9只犬为优先消融AVNFP(atrialventricular node fat pad)。二组实验犬均经过以下方法:①分别于左右股静脉置入四极导管,记录右心室(RV)、右心房(RA),经左侧股动脉记录HIS电位,经右侧股动脉监测动脉血压;分离双侧迷走神经干;先左侧开胸,固定左侧肺静脉、左心耳导管;关左侧胸后暴露右侧胸腔,固定右肺静脉、右心房导管;通过解剖及电生理刺激确定SANFP、AVNFP。②第一组进行以下实验:分别以0.5v、1v、2v、4v(1000bpm,脉宽2ms)电压水平刺激左右迷走神经干、SANFP、AVNFP,测定窦性心率、AH间期、肺静脉ERP、房颤自主维持时间(以下简称指标);消融SANFP后以同样的条件刺激左右迷走神经干、AVNFP,测定上述指标;SANFP+AVNFP联合消融后同样条件刺激左右迷走神经干,测定指标。第二组实验电生理检查:分别以0.5v、1v、2V、4v(1000bpm,脉宽2ms)电压水平刺激左右迷走神经干、SANFP、AVNFP,测定指标;消融AVNFP后以同样的条件刺激左右迷走神经干、SANFP、AVNFP,测定指标;AVNFP+SANFP联合消融后同样条件刺激左右迷走神经干,测定指标。2)本课题第二部分:6只犬以3%的戊巴比妥钠(20mg/kg)静脉注射麻醉后,开胸后取心脏、分离肺静脉,完整剪下左心房-肺静脉肌袖,通过基础高频刺激、给予阿托品后高频刺激、给予阿替洛尔高频刺激肺静脉-左心房肌袖,记录上述三种状态下APD90、EAD。3)本课题第三部分:8只犬,以3%的戊巴比妥钠(20mg/kg)静脉注射麻醉后,取左侧卧位,,沿左侧第四肋间开胸,心肺整体取下。①心脏连同部分肺组织取下,钝性分离周围组织,暴露肺静脉;测定RSPV-LSPV、RIPV-LIPV、RSPV-RIPV、LSPV-LIPV之间的距离范围;②剪开左心耳,暴露四个肺静脉口,依次分离LSPV、LIPVR、RSPV、RIPV,并测定的肌袖的长度;③分离LSPV-LA、LIPVR-LA、RSPV-LA、LSPV-LA,每条肺静脉分别做组织学切片、HE染色电镜检查。结果:1)①优先消融SANFP的电生理结果:不同电压水平刺激左右侧迷走神经心率减慢效应大于左侧迷走神经,SANFP单独消融后刺激右侧迷走神经干减慢窦性心率的作用明显降低但仍有减慢效应。SANFP+AVNFP联合消融后,与消融前对比具有统计学意义,与消融SANFP单独消融后对比无统计学意义;消融SANFP刺激左侧迷走神经干房室传导能力较消融前增强与未消融前对比,具有统计学意义,但刺激右侧迷走神经房室传导能力无改变;SANFP+AVNFP联合消融后,刺激左侧迷走神经干房室传导能力较单独消融及未消融时增强(0.93±0.12 vs034±0.15,0.93±0.12 vs 0.70±0.23)具有统计学意义;刺激右侧迷走神经房室传导能力增强(0.75±0.16 vs 0.28±0.20,0.75±0.13 vs 0.22±0.13),具有统计学意义;右侧迷走神经刺激AF自主维持时间:消融SANFP后1v、2v、4v电压水平刺激右侧迷走神经干房颤自主维持时间较消融前明显缩短具有统计学意义;SANFP+AVN-FP联合消融后,刺激右侧迷走神经房颤自主维持时间与单独消融后相比延长;消融SANFP以及SANFP+AVN-FP联合消融后,刺激左侧迷走神经房颤自主维持时间与未消融前相比无统计学意义;心外膜脂肪垫消融前后刺激迷走神经干对心房肌和肺静脉ERP的影响:左右侧迷走神经干刺激心房肌和肺静脉的ERP均缩短,SANFP消融后刺激迷走神经干肺静脉及左右心房ERP缩短效应消失(P＜0.05),心房肌到肺静脉间的ERP离散度与基础相比没有差异,SANFP+AVNFP消融后刺激迷走神经干,仍有肺静脉和心房肌ERP的缩短及心房肌到肺静脉间的ERP离散度的增加,但未达统计学意义P＞0.05。②优先消AVNFP的电生理结果:消融AVNFP后刺激右侧迷走神经干,与未消融前相比,窦性心率改变(85.6±22.4 vs 51.6±20.4,79.8±20.6 vs 59.2±10.7),具有统计学差异;AVNFP单独消融与AVNFP+SANFP联合消融刺激右侧迷走神经干窦性心率改变(99±20.1 vs 51.6±20.4,97.6±20.3 vs 59.2±10.7),P＜0.05,有统计学差异;单独消融AVNFP与AVNFP联合SANFP消融,刺激左侧迷走神经干窦性心率改变,P＞0.05,无统计学差异。AVNFP单独消融后左侧迷走神经干,窦性心率减慢效应,与消融前对比无统计学意义,但AVNFP+SANFP联合消融后,与消融前对比窦性心率分别(105.2±15.8 vs 83.4±23.2,113.2±19.2 vs 85.4±14.8),具有统计学意义;AVNFP消融后刺激左右侧迷走神经干房室传导能力较消融前增强具有统计学意义,AVNFP+SANFPP联合消融后,刺激左侧迷走神经干房室传导能力较单独AVNFP消融后相比房室传导能力改变无统计学意义,但与基础状态消融前相比,房室传导能力增强具有统计学意义;AF自主维持时间在2、4v刺激时总体趋势缩短,基础状态与AVNFP联合SANFP消融,刺激右侧迷走神经干窦性心率改变,P＞0.05,无统计学意义。上述研究结果10s、30s、60s之间无统计学差异;在SANFP消融后刺激迷走神经状态下,其中右房、左房/RSPV的ERP与基础状态比较具有延长,P＜0.05,有统计学意义;SANFP联合AVNFP消融后与基础状态比较具有延长,P＜0.05,有统计学意义;但SANFP单独消融与SANFP联合AVNFP消融比较,P＞0.05,无统计学意义。2)犬肺静脉心肌袖的出现率为100%,肺静脉心肌袖的肌纤维走行都有斜行、环行和纵行3种,且都以环行和斜行为主;肺静脉肌袖纤维以内环外纵的基本模式分布,随向远处的延伸,肌袖层分布逐渐减少;在同一横断层或不同层面,肌袖细胞的分布存在呈不均一性,肺静脉肌袖细胞的电镜下超微结构与心房肌细胞相似。3)高频刺激肺静脉组织出现动作电位时程缩短、超极化及EAD,触发肺静脉灶性放电,与未刺激比较,肺静脉APD90明显缩短,具有统计学意义。阿托品注射液阻滞迷走神经后,再次高频刺激抑制动作电位时程缩短,无超极化出现,无触发活动。阿替洛尔注射液阻滞交感神经后,再次高频刺激动作电位时程缩短,无触发活动。结论:1)提示迷走神经干刺激对窦房结频率的控制须经心外膜脂肪垫进行,SAN-FP为主控区,AVN-FP为辅助区,右侧迷走神经干的迷走效应大于左侧迷走神经干;迷走神经干刺激对心房肌和肺静脉ERP和ERP离散度及AF诱发的影响是通过心外膜脂肪垫进行的,AVN-FP为主控介导区。左右迷走神经—SAN-FP、AVN-FP的低级神经调控中,SAN-FP对于心房电生理特性的调控起着关键性作用,提示消融SAN-FP是神经消融的重要靶点,该结论有助于实验室制作迷走神经介导房颤模型以及临床选择有效GP消融位点参考,以控制高电压刺激的不利影响。2)肌袖中分布着丰富的神经纤维,包括迷走神经与交感神经;肺静脉这种神经分布特点是环行肺静脉消融去神经的生理基础。3)肺静脉肌袖结构的特殊性可能是形成肺静脉局部折返的组织学基础,是肌袖性心律失常发生和维持的重要机制,肺静脉肌袖组织分布了丰富的神经丛,房颤的发生有赖于交感神经与迷走神经的兴奋性增高。更多还原

【Abstract】 Objectives:1) This study sought to systematically investigate the interactions between the extrinsic and intrinsic cardiac autonomic nervous system(ANS) in modulating electrophysiological properties and atrial fibrillation(AF) initiation,including sinus node ruction,A-V conduction and the maintanence of AF adjusted by stimulating cervical vagua trunck and ablating SANFP or AVNFP for understanding the mechanism of functional interactions between cervical vagua trunck and ganglionated plexi within the intrinsic cardiac ANS.Systematic ganglionated plexi(GP) ablation to evaluate the extrinsic and intrinsic cardiac ANS relationship has not been detailed.2) To determine if autonomic nerve stimulation within canine atrium and pulmonary vein sleeves initiates arrhythmia formation.3) To explore the possible relationship between the ultrastructural characteristics of pulmonary veins and the pathogenesis of atrial fibrillation originating from pulmonary veins.Methods:1)(1) Eighteen adult mongrel dogs was divided to two groups,both vagosympathetic stimulation was performed by applying high-frequency electrical stimulation(0.5v、1.0v、2.0v、4.0v) to each of the vagosympathetic trunk via a stimulator.The chest was opened via left lateral thoracotomy to record the potentail of LSP,VLIPV.Then closed the left chest,and open the right lateral thoracotomy to recorded he potential of RSPV and RIPV,and further expose the SANFP,AVNFP.With unilateral vagosympathetic trunk stimulation(0.5v、1.0v、2.0v、4.0v,100ms,0.1ms duration),sinus rate(SR),AH interval and the duration of AF were compared before and after sequential ablation of SANFP,SANFP+AVNFP during stimulating cervical vagosympathetic trunks repectively.In another group,with the same stimulation,sinus rate(SR),and AH interval were compared before and after sequential ablation of AVNFP,AVNFP+SANFP during stimulating cervical vagosympathetic trunks.2) Extracellular bipolar and intracellular microelectrode recordings were obtained from isolated superfused canine pulmonary veins(N=24) and right atrium(N=5) during local autonomic nerve stimulation.3)The myocardial sleeves of pulmonary veins from 6 dogs were serially sectioned(2mm) transversely along the vessels.The odd number sections were fixed in 10%phosphate buffered formalin solution and the even number sections were fixed in 3%Glutaral for further electron microscopy observations.The myocardial sleeves of pulmonary veins were embeded in 10%by parrafen and sliced,then lices were tained with hemmatoxylin and eosin.The slices were examined by microscope.Results 1)(1)SANFP ablation significantly attenuated the SR response with right or left vagosympathetic stimulation. After SANFP ablation,AVNFP ablation produced further attenuation of SR slowing induced by right vagosympathetic stimulation.Left vagosympathetic stimulation produced similar but smaller effects on SR slowing compared with right vagosympathetic stimulation,left vagosympathetic stimulation significantly slowed atrial ventricular conductivity the during RAP,SANFP ablation attenuated the maximal effect but there was no the same effect in right vagosympathetic stimulation(0.34±0.13 vs 0.28±0.20). Compaed with SANFP ablation,SANFP+AVNFP ablation further attenuate slowing atrial ventricular conductivity effect(0.93±0.12 vs 034±0.15,0.93±0.12 vs 0.70±0.23). SANFP+AVNFP ablation induced significant changes on eliminating the atrial ventricular conductivity slowing effects.AF duration was shortened after SANFPablation during right vagosympathetic stimulation,after SANFP+AVNFP ablation,stimulation made AF duration became longer than SAN-FP ablation alone during RAP.Left vagosympathetic stimulation can make AF duration longer,but the differences before and after SANFP and SANFP+AVNFP ablation failed to achieve statistical significance at all voltage levels.Right and left vagosympathetic stimulation on ERP:at the level of 1v,left vagosympathetic stimulation shortened the ERP recorded from the right atrium and left atrial appendage(99ms±22 vs 141ms±16),LA(102ms±14 vs 135ms±12),RSPV(105ms±8 vs125ms±8).There is no significant difference between SANFP ablation and SANFP+AVNFP.(2) Compared with AVNFP ablation,AVNFP+SANFP ablation further produced attenuation of SR slowing induced by right vagosympathetic stimulation (99±20.1 vs 51.6±20.4,97.6±20.3 vs 59.2±10.7).There was significant difference in SR slowing between AVNFP ablation alone and AVNFP+SANFP ablation during left vagosympathetic stimulation at the level of 2v,4v(90.2.6±24.8 vs 83.4±23.2,95.6±18.8 vs 85.4±14.8);AVNFP ablation attenuated the maximal effect of slowing the atrial ventricular conductivityn during right or left vagosympathetic stimulation respectively (0.39±0.15 vs 0.74±0.33,0.23±0.13 vs 0.80±0.20).Compaed with SANFP ablation, AVNFP+SANFP ablation did not further attenuate slowing atrial ventricular conductivity effect(0.76±0.28 vs0.74±0.33,0.83±0.21 vs0.80±0.20).AF duration was shortened after SAN-FPablation at the level of 1v,2v,4v(0.75±1.5 vs 35±12,16.8±9.0 vs 60±0,2.5±1.5 vs 60±0),after AVNFP+SANFP ablation,stimulation made AF duration became longer than AVNNFPablation alone during RAP.Left vagosympathetic stimulation can make AF duration longer,but the differences before and after AVNFP and AVNFP+SANFP ablation failed to achieve statistical significance at all voltage levels. Left vagosympathetic stimulation shortened the ERP recorded from the right atrium and left atrial appendage(99ms±22 vs141ms±16),LA(102ms±14 vs135ms±12),RSPV (105ms±8 vs125ms±8).There is no significant difference between AVNFP ablation and AVNFP+SANFP.2) Autonomic nerve stimulation decreased pulmonary vein sleeve action potential duration(APD90=160+/-17 to 92+/-24ms;P＜0.01) and initiated rapid (782+/-158 bpm) firing from early afterdepolarizations in 22 of 28 pulmonary vein preparations.The initial spontaneous beat had a coupling interval of 97+/-26 ms.Failure to induce arrhythmia was associated with a failure to shorten APD90(151+/-18 to 142+/-8ms;P=0.39).Muscarinic receptor blockade(atropine:3.2×10(-8) M) prevented APD90 shortening in 8 of 8 preparations and suppressed firing in 6 of 8 preparations, whereas beta1-adrenergic receptor blockade(atenolol:3.2×10(-8) M) suppressed firing in 8 of 8 preparations.The same stimulus trains produce atropine-suppressed APD90 shortening in superfused right atrial free wall but fail to produce triggered arrhythmia.3) The myocardial sleeves in pulmonary veins can be devided into or three strata,and lasted along pulmnary veins,the direction of myocardial sleeves appeared a change from circular to oblique and longitudinal.At the termials,they were encompassed and separated by connective tissue.3) The muscle fibers in myocardial sleeves were assembled with fasciculation and extened irregularly.The width of myocardial sleeves in RSPV,RIPV,LSPV and LIPV were 0.46±0.14,0.35±0.16,0.37±0.13,0.35±0.11 respectively,and the length were 10.75±3.15,9.46±2.20,9.20±2.88,11.41±2.8 in RSPV, RIPV,LSPV and LIPV.Conclusions:1)The study provides functional evidence that SANFP played an very important role in adjusting sinus node fuction,A-V conduction and the maintainence of atrial fibrillation.While SANFP fad pad was an cooperator, supporting clinical evidence that intercormections within the intrinsic cardiac ANS are critical elements in identifying the targets for atrial fibrillation ablation.Our study also subjected that cervical vagal nervous connected with anatomically and affected fuctionally on ganglionated plexi modulated sinus node,AV nodal function and atrial electrophysiological characteristics together.The GP function as the "integration centers" that modulate the autonomic interactions between the extrinsic and intrinsic cardiac ANS. 2) The data demonstrate triggered firing within canine pulmonary veins with combined parasympathetic and sympathetic nerve stimulation.3) The characteristic distribution of canis myocardial sleeves in pulmonary veins possibly provides modal foundation for the occurance of focal atrial fibriilation.The myocardial fibers are irregularly arranged, which may cause the electric conduction unevenly and nonisotropically.更多还原