【作者】 姜云发；

【导师】 傅向华；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 博士

【摘要】 急性心肌梗死(acute myocardial infarction, AMI )是常见的危重症之一,其主要病理学机制为冠状动脉的完全闭塞,开通梗死相关动脉(infarction related artery, IRA)、尽早恢复血流是治疗AMI防治心肌缺血性损伤的核心治疗手段,它能迅速恢复缺血心肌的血流灌注,挽救大量濒死心肌,改善心功能和降低病死率。多项临床试验观察发现,一些AMI病人虽经溶栓和血管成形术后心外膜冠状动脉再通,但生存率并没有显著提高。IRA通畅率与生存率之间不一致的原因可能在于心外膜冠状动脉血流的通畅并不意味着心肌组织水平再灌注得到完全恢复,微循环仍然阻塞,心肌得不到有效的血流灌注,表现为无复流(no-reflow)现象。急性心肌梗死患者行经皮冠脉介入治疗(percutaneous coronary intervention,PCI)术后,仍有10~30%的病人存在着no-reflow,血流灌注未完全达到心肌组织水平,且必将影响到其后左室重构进程和心脏恶性事件的发生。no-reflow作为一种并发症,使住院死亡和心肌梗死发生率增加5～10倍。而心肌在较长时间缺血造成一定损伤后,再恢复供血不但不减轻或逆转损伤,反而加重损伤,即缺血/再灌注损伤(ischemia/reperfusion injury,I/RI)。随着对I/RI和无复流现象研究的不断深入,逐渐认识到微循环功能障碍是I/RI的重要发病环节之一。冠脉造影显示的no-reflow现象其本质也属于心肌微循环灌注障碍,是微血管损伤和心肌组织灌注不足的严重表现,是梗死进展、心室重构、心功能恢复障碍的重要预测指标,因此心肌微循环水平灌注是目前评估再灌注治疗成功与否的主要指标之一。保护心肌微循环功能,减轻或防止I/RI以及no-reflow的发生将是PCI治疗面临的严峻困难和挑战,也是介入心脏病学领域研究的重点和难点。以往对冠状动脉病变程度的评价主要依靠冠状动脉造影,心肌组织水平灌注评价仅仅根据TIMI血流或TIMI计帧分级(TFCs)方法,其存在明显的局限性,不足以说明冠脉生理及微循环功能状况。随着冠状动脉压力阶差测定技术的发展,特别是冠脉血流储备分数(fractional flow reserve,FFR)概念的提出,经压力导丝测定的FFR已被用于评价狭窄血管的功能及介入治疗效果的评价。FFR不受心率、血压等血流动力学因素的影响,是反映心外膜冠脉病变的最佳指标,但FFR又有它的局限性,在存在微血管病变的情况下,微血管病变会限制冠脉获得最大充血状态,此时应结合冠状动脉血流储备(coronary flow reserve,CFR)的测定。CFR是反应冠脉血流动力学改变尤其是冠脉功能的敏感指标之一,除与心外膜冠脉狭窄程度有关,还与相应区域冠脉微循环的生理状态有关,由冠脉内多普勒导丝血流测定CFR准确可靠。两者结合他们所提供的有关心肌缺血多大程度上是由心外膜冠脉狭窄所致,多大程度上是由冠脉微血管病变所致的信息是互补的,从而能更深入地研究主要累及心外膜冠脉的病变和主要累及冠脉微血管的病变,更为客观直接评价冠脉微循环。如何保证尽早恢复缺血心肌组织血流灌注的同时,又减轻或防止IRI及no-reflow现象发生一直是困扰心血管介入医生的难题。由于其发病机制不清,并且是多种因素共同作用的结果使治疗效果常不满意。目前的治疗药物如硫氮卓酮、硝普钠、腺苷等虽有一定效果,但该类药物尚有明显降低冠脉平均灌注压和周围血压,以及过缓性心律失常及其它全身反应的副作用。因此寻求减轻I/RI及改善no-reflow理想的药物和方法,成为介入医生面临的重大课题。山莨菪碱(Anisodamine)为我国从茄科植物唐古特莨菪中分离出的一种生物碱,具有M-受体阻断作用,具有提高冠脉灌注压、改善微循环、降低血小板聚集、抑制血栓形成、保护心肌的作用,而且大剂量应用治疗休克有可靠的安全性和有效性,本研究有鉴于此,通过球囊扩张闭塞、再灌注、微导管超选前降支注入微血栓,阻塞冠脉血流,建立急性缺血-再灌注-无复流模型,采用冠脉内压力导丝和多普勒导丝两者结合的方法探讨缺血/再灌注以及no-reflow状态下微循环障碍的特征及可能机制;同时观察冠脉内注射山莨菪碱对缺血/再灌注心肌的保护作用;以及观察山莨菪碱对no-reflow现象微循环障碍的改善效果,探讨冠脉内压力导丝和多普勒导丝两者结合评价心肌微循环功能的可行性和可靠性,为临床防治冠脉再通后的IRI及no-reflow现象提供理论和实验依据。本研究的内容分为以下四个部分:第一部分冠脉内压力和多普勒血流测定评价猪缺血/再灌注心肌微循环功能障碍的研究目的:本研究旨在应用与人类心血管生物学特性更为接近的小型猪作为模型动物,通过选择性球囊扩张阻断冠状动脉前降支(LAD)血流,嗣后恢复血流灌注的方法,造成拟似急性心肌梗死缺血/再灌注损伤模型,应用冠脉内压力和多普勒血流同步测定方法,评价猪缺血/再灌注心肌微循环功能的变化特征。方法:8头3～5个月龄的约克型猪,采用自身对照的方法。在全麻和无菌条件下,采用非开胸方法,Seldinger法从左、右侧深股动脉途径置入6F动脉鞘,以4F冠脉造影管,(内径0.97mm)行冠状动脉造影。6F导引导管超选LAD,经指引导管分别送入压力导丝和多普勒导丝,应用压力及血流测量仪器进行冠脉口及远端压力和血流速度测定。根据LAD直径选择相应大小球囊,缺血预适应后持续闭塞左前降支45min,后撤出球囊,恢复冠脉血流灌注,缺血/再灌注制模成功。分别记录基础状态、冠脉再灌注即刻、30分钟和60分钟冠脉内压力阶差和血流频谱变化,获取静息和最大充血状态下主动脉压力(Pa)、冠脉远端压力(Pd)、平均峰值流速(average peak velocity, APV),冠脉微血管阻力(MR),同时获取FFR、CFR参数。同时于基础状态、再灌注30min、60min、120min、180min分别从冠脉内取血测定心肌坏死标记物CK-MB、Tn,应用5F四腔温敏球囊漂浮导管测量肺毛细血管楔嵌压(PCWP),心输出量(CO),经左侧股动脉途径4Fpigtail连续监测左室收缩压(LVSP)、左室舒张末期压力(LVEDP)、十二导联同步记录心电图(ECG)等参数。最大充血状态由冠脉指引导管向冠脉内注射罂粟碱后获得。以血压-心率指数(PRI)来反映制模前后心肌耗氧量的变化。实验结束后即刻切取左心室前壁缺血区、正常区及两者交界区心肌组织各6块行光镜病理学检查。结果:1、8头约克型猪中有7头制模后完成全部实验,模型成功率为87.5%。2、球囊扩张充盈时,ECG可见胸前导联广泛ST段抬高,可与高大的T波形成单向曲线。再灌注后,抬高的ST段有所下降。3、制模成功后CK-MB和cTn I均明显增高(CK-MB:16.85±4.52ng/ml vs 164.21±30.78ng/ml,p<0.01; cTnI: 13.96±4.42ng/ml vs 118.21±27.51ng/ml, p<0.01)。4、冠脉压力、多普勒导丝测定结果显示:缺血/再灌注后各个时间点静息及充血状态下Pa、Pd较基础状态降低,以再灌注即刻最明显,分别为b-Pa(109.3±11.4 vs 89.7±9.5,P<0.05),h-Pa(108.9±11.6 vs 89.4±9.3, P<0.05), b-Pd(109.1±10.9 vs 89.2±9.6, P<0.05),h-Pd (108.4±11.3 vs 89.3±7.9, P<0.05),Pa与Pd保持一致变化。FFR制模前后无变化,分别为0.99±0.03 vs. 0.99±0.04, P>0.05,APVb制模后较制模前升高(17.3±7.9 vs 21.0±2.9, P<0.05),MR降低(7.42±1.65 vs 4.77±0.85, P<0.01);最大充血状态下APV较基础状态降低(62.6±12.3 vs 40.5±10.1, P<0.01),MR较基础状态增加(2.01±0.31 vs 2.51±0.12, P<0.05)。CFR较基础状态明显降低(3.69±0.76 vs 1.93±0.35, P<0.01)。5、心肌耗氧量及血流动力学的变化:制模后心肌耗氧量显著下降。LVEDP较基础状态以及球囊扩张时均升高(P<0.05);PCWP升高,CO下降。6、病理学检查:缺血坏死区心肌组织显示肌纤维肿胀、网状结构变化及局灶性坏死。小结:选择性球囊高压充盈扩张阻断LAD血流然后释放球囊的方法,可以造成急性心肌梗死缺血/再灌注损伤模型,具有直观、简便、重复性好、低创伤性、闭胸、成功率高等优点;冠脉内压力和多普勒血流两者结合,同步测定是目前客观,可靠评价冠脉缺血/再灌注后心肌组织微循环功能的方法;缺血/再灌注心肌微循环功能表现为静息血流速度增快,冠脉阻力减低,最大充血时血流速度减低,阻力增加,冠脉血流储备减低,再灌注心肌耗氧量减少。第二部分猪急性心肌梗死后无复流心肌微循环冠脉内压力和多普勒血流特征目的:通过选择性球囊高压充盈扩张阻断LAD血流后,冠脉内注入无菌复合微血栓悬液的方法,造成拟似AMI再灌注后no-reflow模型,应用冠脉内压力和多普勒血流同步测定,评价猪AMI再灌注后的no-reflow心肌微循环功能的变化特征。方法:12头约克型猪,采用自身对照的方法,应用4F JL3.5、JR4.0冠脉造影导管分别行左、右冠脉造影。6F指引导管超选LAD,分别送入压力导丝和多普勒导丝,球囊扩张堵塞冠脉45min,撤除球囊间歇注入无菌复合微血栓悬液,应用可量化的TIMI血流计帧数(TIMI frame count,TFC)、心肌组织灌注分级(TIMI myocardial perfusion grade,TMPG),评价心肌组织灌注水平,以TIMI血流≤2级或TFC﹥36.2帧、TMPG≤1级作为AMI-no reflow模型成功标准。应用压力及血流测量仪器进行冠脉口及远端压力和血流速度测定,分别评价记录无复流前后静息和最大充血状态下Pa、Pd、APV,MR,同时获取FFR、CFR参数。同时应用4F Pigtail导管连续监测LVEDP、LVSP以及左室内压上升和下降最大速率(the peak of left ventricular pressure risefall,±dp/dtmax);应用5F四腔温敏球囊漂浮导管至肺动脉测量PCWP,热稀释法测心输出量(cardiac output,CO);监测心电图的变化,并于基础状态、无复流30min、60min、120min、180min,冠脉内抽取血液标本,测定CK-MB、cTn I。实验结束后心肌组织取材行光镜病理学检查。结果:1、制模共有9头约克型猪成活,并达到AMI-no reflow动物模型标准,模型成功率为75%(9/12),平均无菌复合微血栓悬液注射次数为3.3±0.4次,TIMI血流均为≤2级,TMPG≤1级。2、No reflow模型ECG可见胸前导联ST段弓背向上抬高,可与高大的T波形成单向曲线,然后R波逐渐降低。3、所有制模成功后cTn I和CK-MB均较无菌复合微血栓悬液注入前明显增高(P<0.01)。4、AMI-no reflow后即刻、30分钟、60分钟所测PCWP、LVEDP均较前升高,且有显著性差异(P<0.05); LVSP由no reflow前的132.5±10.8 mmHg降低至no reflow后的113.0±14.2mmHg (P<0.05);5、随着AMI-no reflow出现,心率减慢、心肌耗氧量(PRI)明显下降(P<0.05)。6、No reflow后各个时间点静息及充血状态Pa、Pd较基础状态降低,以no reflow30min最明显,分别为b-P(a83.2±6.4 vs 109.6±12.7, P<0.05),h-Pa (82.0±9.5 vs 109.2±12.1, P<0.05), Pa与Pd保持一致变化。FFR制模前后无变化(P>0.75)。静息及最大充血状态下APV制模后较制模前明显降低(11.0±8.9 vs 17.3±7.9;20.4±10.6 vs 62.6±12.3, P<0.05),静息及最大充血状态下冠脉微血管阻力(b-MR和h-MR)显著升高(7.42±2.31 vs 9.47±3.57;2.01±0.61 vs 4.96±0.91, P<0.05);CFR较基础状态明显降低(3.61±0.76 vs 1.85±0.31, P<0.05)。7、病理检查:缺血坏死区表现为肌纤维肿胀、网状结构变化及局灶性坏死。小结:采用选择性球囊扩张阻断LAD血流及恢复血流灌注后冠脉内注射无菌复合微血栓悬液方法制作no reflow模型,具有直观、简便、重复性好、低创伤性、闭胸、成功率高等优点,为心肌微循环障碍的研究提供了较好的实验动物模型;冠脉内压力和多普勒血流两者结合同步测定,能够直接、客观地评价no reflow冠脉微循环功能状态,表现为冠脉血流储备分数正常,静息和最大充血状态下血流速度减慢,冠脉阻力增加,冠脉血流储备减低。第三部分冠脉内分次注射山莨菪碱对猪急性心肌梗死后无复流心肌微循环影响及冠脉内压力和多普勒血流评价目的:本研究在前期实验的基础上,应用冠脉内压力导丝和多普勒导丝同步测定,评价冠脉内分次注入山莨菪碱对AMI-no reflow心肌微循环的影响,为临床应用提供实验依据。方法:9头4±1个月龄的约克猪,采用前期方法成功制备AMI-noreflow模型,随机分为Saline对照组4头,Anisodamine组5头。两组即刻冠脉内分3次分别给予生理盐水5 ml/次、山莨菪碱2000μg/次冠脉内注射,每次间隔5分钟,即刻、5分钟和10分钟分三次行CAG,应用TFC和QCA测量系统分别对两组冠状动脉内给药后不同时间点的TFC和TMPG进行定量分析比较。经指引导管分别送入压力导丝和多普勒导丝,同步评价记录两组用药前后静息和最大充血状态下Pa、Pd、APV、MR,并获取FFR和CFR参数。应用5F四腔温敏球囊漂浮导管测量no reflow形成后即刻、用药后即刻、5分钟、10分钟后PCWP的变化,热稀释法测量CO。采用4F pigtail于用药前和用药后即刻、5分钟、10分钟监测LVSP、LVEDP。结果:1、Anisodamine组冠脉内给药后即刻、5、10分钟TFC分别较给药前减少52.44%、54.30%、54.20%,且冠脉血流达到TIMI2+-3级(P<0.05),与Saline组各时间点同期比较明显减少(P均<0.05)。2、冠脉内给生理盐水后各个时间点,no reflow状态下静息及最大充血状态Pa、Pd、FFR、APV、b-MR和h-MR以及CFR没有变化。冠脉内应用山莨菪碱6000ug后,各个时间点静息及最大充血状态Pa、Pd较no reflow时明显改善,以给药后5min最明显,分别为b-Pa:98.1±11.6 vs 86.1±10.5, P<0.05,h-Pa: 92.1±10.6 vs 85.3±10.1, P<0.05, Pd与Pa保持一致变化。FFR用药前后无变化(P>0.75)。静息及最大充血状态下APV用药后较no reflow时明显增加(P<0.05),静息及最大充血状态下b-MR和h-MR显著降低(7.62±2.23 vs 9.19±4.08;2.86±0.47 vs 4.89±0.86, P<0.05)。CFR较基础状态明显改善(2.98±0.75 vs 1.88±0.60, P<0.05)。除FFR外其余各指标与生理盐水组比较各时间点均有显著性差异,以用药后5min最显著(P<0.05)。3、Anisodamine组冠脉内给药后较用药前、Saline组血压、冠脉平均压均明显升高,心率增快(P均<0.05)。Anisodamine组在冠脉内用药后PCWP、LVEDP均较用药前及Sline组明显降低(P<0.01),CO明显增加(P<0.01)。4、Anisodamine组在冠脉内给药后LVSP、±dp/dtmax升高,均较Saline组差异有显著性(P<0.05)。小结:冠脉内应用山莨菪碱可明显增加静息及最大充血状态下无复流冠脉平均峰值血流速度,降低静息及最大充血状态无复流冠脉微循环阻力,增加冠脉血流储备,迅速改善微循环障碍,改善心肌组织灌注。第四部分冠脉内预先注射山莨菪碱对猪缺血/再灌注心肌微循环保护作用的研究目的:在建立球囊扩张选择性阻断LDA再恢复血流灌注约克猪在体急性心肌梗死再灌注模型基础上,通过预先冠脉内注射山莨菪碱,应用冠脉内压力和多普勒血流同步测定技术,评价冠脉内直接注射山莨菪碱对猪缺血/再灌注心肌的保护作用。方法:10头约克型猪随机分为2组:山莨菪碱组(Anisodamine组,5头),制备模型前冠脉内分次给予山莨菪碱;生理盐水组(Saline组,5头),先予冠脉内注射等量生理盐水。6F导引导管超选LAD,经指引导管分别送入压力导丝和多普勒导丝,制备缺血/再灌注损伤模型。应用压力及血流测量仪器进行冠脉口及远端压力和血流速度测定。球囊闭塞左前降支(LAD)45分钟造成心肌缺血,撤除球囊恢复冠脉血流再灌注;分别记录两组制模前后基础状态、冠脉再灌注即刻、30分钟和60分钟冠脉压力和血流频谱。获取两组静息和最大充血状态下Pa、Pd、APV、MR等参数,同时获取FFR、CFR参数值。同时于基础状态、再灌注30min、60min、120min、180min于冠脉内取血测定两组cTn I。应用5F四腔温敏球囊漂浮导管测量两组制模前后PCWP,CO,经左侧股动脉途径4Fpigtail连续监测LVSP、LVEDP。以PRI来反映两组制模前后心肌耗氧量的变化。同时监测制模前后心电图的变化,对两组心肌梗死面积进行比较。结果:1、两组制模成功率无差异。2、两组制模后心电图ΣST均显著升高,Anisodamine组较saline组ΣST抬高幅度明显降低(27.5±2.2 vs 16.2±1.7,P<0.05)。3、冠脉内给药后对血流动力学的影响显示:两组再灌注后LVEDP、PCWP均较前升高,而LVSP、CO较前明显下降(P<0.05)。但再灌注后各时间点Anisodamine组较Saline组灌注后各参数指标有显著性差异(P<0.05)。4、与基础状态相比,两组制模成功后cTnI和CK-MB均增高,两组比较有显著性差异。5、两组冠脉压力、多普勒导丝测定微循环变化显示:两组缺血/再灌注后各个时间点静息及充血状态下Pa、Pd较基础状态降低,但Saline组降低更为明显,与Anisodamine组相比均有显著差异(P<0.05),Pa与Pd保持一致变化。FFR制模前后两组均无变化,均大于0.75。静息APVb升高,b-MR降低,Anisodamine与Saline组有显著差异(P<0.05),b-MR(5.96±1.98 vs. 4.77±1.65,P<0.05);APVh较基础状态降低,h-MR较基础状态增加,两组之间差异有显著性。两组CFR较基础状态均明显降低,但Saline组更显著(P<0.05)。6、心肌耗氧量(PRI)的变化:Saline组较基础状态显著下降,Anisodamine组亦显著下降,但明显高于Saline组(13.8±2.81 vs. 0.2±1.7, P<0.05)。7、Anisodamine组心梗死面积较saline组明显减小(21.3±3.8% vs. 34.6±3.2%, P<0.05)。小结:预防性冠脉内注入山莨菪碱可降低前向血流阻力,增加微血管灌注,对冠脉微循环功能具有保护作用,从而保护缺血/再灌注心肌。更多还原

【Abstract】 Acute myocardial infarction(AMI) is a very common disease of the emergency disease. To reopen the infarction related artery(IRA) has become the key therapy(first choice) in the treatment of acute myocardial infarction. It can restore tissue perfusion rapidly, rescue more myocardium, improve heart function and decrease mortality. In recent years, although IRA of epicardial artery was reopened by thrombolysis or percutanous coronary intervention (PCI), many clinical trials showed that the survival rate did not improve in some patients with AMI. The reason may be that the repatency of epicardial artery does not always indicate the improvement of tissue perfusion. With gradually deepening research on AMI, recent studies showed that the related arteriole and capillary were damaged so seriously correspondingly once coronary artery occluded that disturbance of distal blood flow in microcirculation in ischemia area might still exist after revascularization, which was defined as slow reflow phenomenon(SRP) or no reflow penomenon(NRP). Now, reopening the infarction related artery in time with percutanous coronary intervention(PCI) is an effective and satisfactory therapy for most of AMI patients. But NRP exists in 10~30% patients with AMI after PCI whose myocardial tissue hasn’t gained complete perfusion, which will surely influence the remodeling process of left ventricle and the incidence of adverse cardiac events. The incidence rate of in-hospital mortality and reinfarction were 5-10 times increased in patients with NRP as a complication. With the restoration of blood to ischemic area , myocardium with long-term ischemia was damaged. It was defined as ischemia/reperfusion injury(I/RI). With gradually deepening research on I/RI, the microcirculation injury was the key step in I/RI. In nature, NRP is invalid reperfusion. The microcirculation dysfunction is its nature. The microcirculation dysfunction is also one of the predictor factors of continue ischemia of myocardium, ventricular remodeling and recovery obstacle of heart function. Therefore, to protect the microcirculation function and decrease the I/RI, to elucidate the mechanisim of NRP and choose the best method to prevent and treat NRP after PCI have become a great challenge in the field of coronary intervention.In the past, an assess the coronary stenoses was through coronary angiography .The evaluation of myocardial tissue perfusion only according to the judgement of TIMI flow grade , TIMI frame counts(TFCs) or TIMI myocardial perfusion grading(TMPG) has limitations. The shortcomings of these approaches to assess the physiological significance and microcirculation function of coronary artery have been recognized for decades. With the development of techniques, coronary pressure measurement has emerged over the past few years as a major step forward in the assessment of coronary artery disease. Especially the conception of fractional flow reserve(FFR) was advanced, it has been used to evaluate the function of artery stenoses and interventional therapy. It is the best index to assess the artery stenoses in epicardium because of no influence by heart rate or blood pressure. But it has limitations, microvascular disease may influence it. Coronary flow reserve(CFR) is one of a sensitive indexes to indicate the hemodynamics of coronary artery. It is not only related to the stenoses of artery in epicardium, but also related to the physiological status of the coronary microcirculation. Coronary Doppler flow wire is a correct method to obtain the CFR. But CFR can not distinguish the lesion between epicardial artery and microvascular. So, it can reflect objectively the coronary microcirculation and the level of myocardial tissue perfusion by the intracoronary pressure and Doppler flow assess simultaneously.How to reduce or avoid I/RI and NRP when the myocardial perfusion was quickly and completely recovered is a difficult problem puzzling the interventional cardiologists. The effect of treatment on NRP was not satisfying because I/RI and NRP may be the result of interaction of many factors, the mechanism of which is complex and unclear. Some drugs such as dilthazam, adenosine, nitroprusside have used to treat NRP, but they have side effects of pressure and heart rate. Therefore, seeking for a ideal medicine or method to reduce the I/RI and improve NRP has become an important subject for the interventional cardiologists. Anisodamine is a alkaloid isolated from nightshade henbane in China. Being a M-choline receptor blocker, Anisodamine can not only increase coronary perfusion pressure, improve microcirculation, inhibit thrombus formation, but also show obvious effect and safety on shock with large dose of it. The purpose of this study is on the base of establishing acute ischemia-reperfusion injury model and NRP model in York swines, to observe the status of microcirculation in coronary artery, hemodynamics, as well as the effect on myocardial infarction size by the the intracoronary pressure wire and Doppler guidewire assessment simultaneously. At the same time, the effect on the microcirculation and the protection on I/RI were observed after intracoronary administration of Anisodamine in this study. The study is comprised of four parts described as follows:Part I: The study of the myocardial microcirculation function in ischemia reperfusion york swines by intracoronary pressure and Doppler flow assessment simultaneouslyObjective: Minipigs were selected in this study for the similarity to human being in cardiovascular biological features. The swine models of I/RI were established by superselecting left anterior descending artery (LAD) with 6F catheter, dilating balloon to occlude the flow of the coronary and withdrawing the balloon. The characteristics of the myocardial microcirculation in I/RI models were explored by intracoronary pressure wire and Doppler guidewire assess simultaneously.Methods: Total of 8 york swines (4±1 months) were included in this study. 6F catheter were performed in two sides deep femoral approach by Seldinger’s puncture. Left and right coronary angiography were performed by 4F micro-catheter technique. LAD was superselected with 6F guiding catheter , then the pressure wire and Doppler guidewire were transferred into coronary respectively through guiding catheter. The pressure and blood flow of the coronary artery were measured with ComboMap System Model 6800. LAD was occluded for 45 minutes by balloon which was chosen according to LAD diameter,and then perfused by withdrawing the balloon.The coronary pressure and flow velocity were recorded simultaneously at baseline, instant,30, 60 minutes after reperfusion. The aortic pressure (Pa),coronary distal pressure (Pd), average peek velocity (APV), microvascular resistance (MR) were obtained at rest and maximal hyperemia induced by intracoronary bolus of 10mg cardoverine. At the same time, fractional flow reserve (FFR) and coronary flow reserve (CFR) were obtained. CK-MB and TnI were measured in all swines at baseline, 30, 60, 120, 180min after reperfusion. Pulmonary capillary wedge pressure (PCWP) was measured by 5F Swan-Ganz floating catheter, and cardiac output(CO) was measured by thermodilution method. Left ventricular systolic pressure (LVSP) and left ventricular end diastolic pressure were monitored continuely with 4F Pigtail. At the same time ,ECG was recorded. The myocardium oxygen consumption was reflected by pressure rate index (PRI).The animals were sacrificed after the trial finished. Ischemic region, normal region and borderline were sectioned respectively and 6 pieces of myocardial tissues were sent to check for pathology.Results: (1) According to the standards of I/RI, 7 animals achieved the I/RI model of AMI successfully, success rate of model establishing was 87.5% (7/8). (2) While the balloon was dilating , instant ECG showed that ST segment elevated and formed single-direction curve with high T wave, after reperfusion, ST segment was gradually depressed. (3) cTnI and CK-MB were increased significantly after I/RI models were successfully established [(cTnI 118.21±27.51 ng/ml vs 13.96±4.42 ng/ml),(CK-MB 164.21±30.78ng/ml vs 16.85±4.52ng/ml)]. (4) The Pa and Pd at rest and hyperemia were decreased significantly at different time after reperfusion, especially at reperfusion instant t[b-Pa(109.3±11.4 vs 89.7±9.5,P<0.05),h-Pa (108.9±11.6 vs 89.4±9.3, P<0.05), b-Pd ( 109.1±10.9 vs 89.2±9.6, P<0.05 ), h-Pd (108.4±11.3 vs 89.3±7.9, P<0.05)]. The change of Pd was the same to Pa. FFR did not change before and after reperfusion(0.99±0.03 vs.0.99±0.04, P>0.05). APVb was increased significantly, it was higher than that of before reperfusion(21.0±2.9 vs.17.3±7.9, P<0.05). b-MR was decreased after reperfusion, it was lower than that of before reperfusion ( 4.77±0.85 vs.7.42±1.65, P<0.01). APVh was lower than that of baseline(40.5±10.1 vs.62.6±12.3, P<0.01), while h-MR was increased significantly(2.51±0.12 vs.2.01±0.31, P<0.05). CFR was lower than that of baseline(1.93±0.35 vs.3.69±0.76, P<0.01). (5) MVO2 was decreased significantly. PCWP and LVEDP were increased significantly at instant, 30 and 60min after I/RI with AMI (P all<0.05). LVSP and CO were decreased after successful establishment of I/RI model. (6) Pathological examination showed the occurrence of myocardium fiber swelling, sarcolysis, reticular formation and local liquefaction necrosis.Conclusion: It is feasible to establish the model of I/RI by superselecting LAD under CAG ,dilating balloon and withdrawing balloon .The model of I/RI could be established in swines whose heart anatomy and characteristics of coronary artery were more similar to human beings. This model had advantages of direct-viewing, simplicity, reproducibility, mild trauma, closed chest and high achievement ratio. At present, it is an advanced, objective, and reliable method to evaluate the microcirculation in ischemia-reperfusion coronary by intracoronary pressure wire and Doppler wire assessment simultaneously. The manifestation of myocardial microcirculation in I/RI model is that : The APVb is increased,b-MR is decreased , APVh is decreased, h-MR is increased, CFR is obviously decreased, myocardial oxygen consumption is decreased.Part II: The study of the myocardial microcirculation function in no- reflow york swines by intracoronary pressure and Doppler flow assessment simultaneouslyObjective: This study was aimed to probe the feasibility of establishing no-reflow model in AMI Yorkpigs by balloon-occlusion-reperfusion- intracoronary injection of sterile microembolus via superselecting LAD with 6F guiding catheter. To explore the characteristics of the myocardial microcirculation in NRP models by intracoronary pressure wire and Doppler guidewire assessment simultaneously.Methods: Total of 12 york swines (4±1 months) were included in this study. 6F catheter were performed in two sides deep femoral approach by Seldinger’s puncture. Left and right coronary angiography were performed by 4F micro-catheter technique. LAD was superselected with 6F guiding catheter , than the pressure wire and Doppler guidewire were transfer into coronary artery respectively through guiding catheter. LAD was occluded for 45 minutes by balloon which was chosen according to LAD diameter, and then perfused by withdrawing the balloon. After deflating the balloon, the sterile microembolis were injected into LAD intermittently. The coronay blood flow and the level of myocardial tissues perfusion were quantitatively evaluated by TIMI frame counts (TFC) TMPG and coronary Doppler flow wire. The model of NRP of AMI was considered as success while TIMI blood flow being less than grade 2 or TFC more than 36.2 counts or TMPG less than grade 1. The pressure and blood flow of the coronary were measured with ComboMap System Model 6800. The coronary pressure and flow velocity were recorded simultaneously at baseline, instant, 30 and 60 minutes after NRP . The Pa, Pd, APV, MR were obtained at rest and maximal hyperemia. At the same time FFR and CFR were obtained. The maximal hyperemia was induced by intracoronary bolus of 10 mg cardoverine . CK-MB and TnI were measured in all swines at baseline, 30, 60,120 and 180min after NRP. PCWP was measured by 5F Swan-Ganz floating catheter, and CO was measured by thermodilution method. LVSP and left ventricular end diastolic pressure were monitored continuously with 4F Pigtail. At the same time ECG was recorded. The myocardium oxygen consumption was reflected by PRI.The animals were sacrificed after the trial finished. Ischemic region, normal region and borderline were sectioned respectively. 6 pieces of myocardial tissues were sent to check for pathology. Results: (1) (1) According to the standards of NRP, 9 animals achieved the NRP model of AMI successfully, success rate of model establishing was 75% (9/12), average times of injection of microembolus was 3.0-4.0. (2) While the model of NRP was established, instant ECG showed that ST segment elevated and formed single-direction curve with high T wave, and R wave was gradually depressed. (3) cTnI and CK-MB were increased significantly after NRP models were successfully established[(cTnI 120.18±27.25 ng/ml vs 13.69±4.37 ng/ml),(CK-MB 171.34±32.75ng/ml vs 16.87±4.54ng/ml)]. (4) PCWP and LVEDP were increased with statistical significance at instantly, 30, 60min after NRP with AMI (P all<0.05). LVSP and CO were decreased after successful establishment of NRP model.(5) MVO2 was decreased significantly. (6)The Pa and Pd at rest and hyperemia were decreased significantly at different time after NRP, especially at 30 min after NRP [b-Pa(83.2±6.4 vs 109.6±12.7, P<0.05),h-Pa (82.0±9.5 vs 109.2±12.1, P<0.05)]. The change of Pd was same to Pa. FFR did not change before and after NRP(0.98±0.06 vs.0.99±0.04, P>0.05). APV at rest and hyperemia were all decreased significantly, they were lower than that before NRP(11.0±8.9 vs. 17.3±7.9;20.4±10.6 vs. 62.6±12.3, P<0.05). b-MR was decreased after reperfusion, it was lower than that before NRP(4.77±1.65 vs.7.42±2.31, P<0.05 ) .MR at rest and hyperemia were all increased significantly(9.47±3.57 vs. 7.42±2.31;4.96±0.91 vs.2.01±0.61, P<0.05). CFR was lower than that of baseline(1.85±0.31 vs.3.61±0.76, P<0.05). (7) Pathological examination showed the occurrence of myocardium fiber swelling, sarcolysis, reticular formation and local liquefaction necrosis.Conclusion: It is feasible to establish the model of NRP by superselecting LAD under CAG , dilating balloon, withdrawing balloon and injecting microembolus. The model of NRP could be established in swines whose heart anatomy and characteristics of coronary artery were more similar to human being. This model had advantages of direct-viewing, simplicity, reproducibility, mild trauma, closed chest, high achievement ratio. It might provide better experimental animal model for the research on microcirculation dysfunction after AMI. It is a advanced, objective, and reliable method to evaluate the microcirculation in NRP models by intracoronary pressure wire and Doppler wire assessment simultaneously. The manifestation of myocardial microcirculation in NRP is that : FFR is normal before or after NRP, The APV at rest and hyperemia is decreased,b-MR and h-MR are increased , CFR is obviously decreased.Part III: The effects of anisodamine on the coronary microvascular disfunction in no-reflow swines by intracoronary pressure and Doppler flow assessment simultaneouslyObjective:To investigate the effect of intracoronary administration of Anisodamine on NRP with saline as contrast by intracoronary pressure wire and Doppler guidewire assessment simultaneously. It will maybe provide evidence for clinical use. Methods Total of 9 york swines (4±1months old ) were included in this study, 9 York swines with stable NRP were randmized into saline group(n=4), Anisodamine group(n=5). Left and right coronary angiography was performed by 4F angiography catheter to observe the distribution and shape of coronary artery. After the NRP models were established, saline 5ml, Anisodamine 2000μg were injected into LAD three times with 5 minutes interval in the two groups respectively, and coronary flow velocity was quantitatively measured by TFCs and TMPG at instant, 5, 10 min after administration. LAD was superselected with 6F guiding catheter , then the pressure wire and Doppler guidewire were transferred into coronary artery respectively through guiding catheter. The pressure and blood flow of the coronary were measured with ComboMap System Model 6800. The coronary pressure and flow velocity in two groups were recorded simultaneously at baseline, instant,5 and 10 minutes after administration. The Pa, Pd, APV, MR were obtained at rest and maximal hyperemia. At the same time FFR and CFR were obtained. The maximal hyperemia was induced by intracoronary bolus of 10mg cardoverine. PCWP was monitored with 5F Swan-Ganz floating catheter and CO was measured with thermodilution method. 4F pigtail was inserted into left ventricular via femoral artery. The LVSP and LVEDP were measured instant, 5, 10 minutes after injection of saline, Anisodamine respectively.Results: (1)TFCs were significantly decreased by 52.44%、54.30%、54.20% instant, 5,10 min after intracoronary Anisodamine compared with that of baseline and saline group (P<0.05). At same time coronary blood flow reached TIMI2+-3 grade (P<0.05). (2) There were no difference in Pa and Pd at rest and hyperemia , FFR, APV at rest and hyperemia ,b-MR and h-MR and CFR after saline administration. While in Anisodamine group, after administration of 6000 ug Anisodamine, the Pa and Pd at rest and hyperemia were all increased significantly at different time, especially at 5min after administration[ b-Pa (98.1±11.6 vs 86.1±10.5, P<0.05),h-Pa (92.1±10.6 vs . 85.3±10.1, P<0.05)]. There were significance between two groups [b-Pa(98.1±11.6 vs. 86.7±11.7,P<0.05),h-Pa (92.1±10.6 vs. 86.5±10.3,P<0.05)]. The change of Pd was same to Pa. FFR did not change between before and after administration in two groups. APVb and APVh were all increased significantly in Anisodamine group. There were significance between two groups [APVb(15.7±13.7 vs. 11.3±8.7, APVh (46.3±12.2 vs.20.6±10.6, P<0.05)]. b-MR and h-MR were decreased obviously in Anisodamine group ( 7.62±2.23 vs.9.19±4.08 ; 2.86±0.47 vs.4.89±0.86, P<0.05), they were better than those of saline group [b-MR (7.62±2.23 vs. 8.98±2.87,P<0.05), h-MR (2.86±0.47 vs. 4.89±0.88, P<0.05]. CFR was increased , it was higher than that of baseline and saline group[(2.98±0.75 vs.1.88±0.60, P<0.05), (2.98±0.75 vs. 1.83±0.41,P<0.05)].(3) Blood pressure, mean coronary pressure and heart rate were significantly increased after administration of Anisodamine compared with those of baseline and saline group (P<0.05). PCWP and LVEDP were significantly decreased while CO was significantly increased after administration of anisodamine compared with that of baseline and saline group (P<0.01). (4) LVSP and±dp/dtmax were increase significantly in Anisodamine group after intracoronary administration medicine compared with those of baseline and saline group(P<0.05).Conclusion: Administration of intracoroanary Anisodamine could increase APVb and APVh in NRP microcirculation, decrease b-MR and h-MR, improve CFR in NRP and improve microcirculation dysfunction .Part IV: Protective effects of anisodamine on the coronary microcirculation function in ischemia- reperfusion swinesObjective: To investigate the effect of intracoronary anisodamine on coronary microcirculation function in ischemia-reperfusion swine model by intracoronary pressure wire and Doppler wire assessment simultaneously.Methods: Total of 10 swines were divided into saline group (n=5) and Anisodamine group (n=5). Two milliliter saline and 2 mg Anisodamine were injected into LAD respectively in the two groups. Left and right coronary angiography were performed by 4F micro-catheter technique. LAD was superselected with 6F guiding catheter , then the pressure wire and Doppler guidewire were transferred into coronary respectively through guiding catheter. The pressure and blood flow of the coronary were measured with ComboMap System Model 6800. LAD was occluded for 45 minutes by balloon which was chosen according to LAD diameter, and then perfused by withdrawing the balloon. The coronary pressure and flow velocity were recorded simultaneously at baseline, 30 and 60 minutes after reperfusion . The Pa, Pd, APV, MR were obtained at rest and maximal hyperemia in two groups. At the same time FFR and CFR were obtained. The maximal hyperemia was induced by intracoronary bolus of 10mg cardoverine. CK-MB and TnI were measured in all swines at baseline, 30, 60, 120 and 180min after reperfusion . PCWP was measured by 5F Swan-Ganz floating catheter, and CO was measured by thermodilution method. LVSP and left ventricular end diastolic pressure were monitored continuously with 4F Pigtail. At the same time ECG was recorded. The myocardium oxygen consumption was reflected by PRI. The animals were sacrified after the trial finished. Ischemic region, normal region and borderline were sectioned respectively, the size of which was 0.3cm×0.3cm, and 6 pieces of myocardial tissues were sent to check for pathology. All parameters were compared between two groups.Results: (1) Success rate of model establishing was no difference. (2) ΣST of ECG was obviously increased in two groups, but it was obviously decreased after preventive intracoronary administration of Anisodamine compared with that of contrast group (P<0.05). (3) PCWP and LVEDP were significantly increased while CO was significantly decreased in two groups, but they were better in anisodamine group than those in saline group (P<0.05). (4) cTnI and CK-MB were all increased significantly in two groups, but were increase significantly in saline group compared with that of anisodamine group (P<0.05). (5) The Pa and Pd at rest and hyperemia were all decreased significantly at different time after reperfusion, especially at reperfusion instant in two groups with obvious in Saline group.The change of Pd was same to Pa. FFR did not change before and after reperfusion in two groups. APVb was increased significantly in two groups, it was higher than that of before reperfusion, but it was different between two groups(18.8±8.2 vs. 21.0±9.9,P<0.05). B-MR was decreased after reperfusion, it was lower than that of before reperfusion, there were difference between two groups (5.96±1.98 vs. 4.77±1.65,P<0.05). APVh was lower than that of baseline, while h-MR was increased significantly. CFR was lower than that of baseline in two groups, but it was higher in Anisodamine group than that of in Saline group(2.59±0.41 vs. 1.93±0.35,P<0.05). Myocardial oxygen consumption was decreased more in saline group than in anisodamine group. (6) The necrosis area to the LV areas were (34.6±3.2) % and (21.3±3.8)% in saline and Anisodamine group respectively,which had statistical significance in Anisodamine group compared with that of saline group(P<0.05).Conclusion: Preventive intracoronary administration of Anisodamine can maintain coronary mean perfusion pressure, decrease the microcirculation resistance of coronary artery, increase the reperfusion of myocardial microcirculation, protect the coronary microcirculation function and ischemic-reperfusion myocardium.更多还原