| In recent years,it has been proved by basic and clinical researches that percutaneous coronary intervention (PCI) for acute myocardial infarction (AMI), is superior to medicines with higher repatency rate and less complications, such as cerebral hemorrhage, and is prior to CABG by it's little trauma and quick recovery. But, no-reflow phenomenon, in fact has no effective myocardium reperfusion , which increases the patients'MACE such as death, reinfarction and malignant arrhythmias by 5-10 times, is found after PCI in about 10-30% patients with AMI, although the infarction relative artery(IRA) has been reopened successfully. So researches have focused on how to make effective reperfusion to microcirculation of infarction related myocardium, increase the number of surviving myocardial cell, prevent acute ventricular remodeling, protect ventricular function and decrease the MACE. It is reported that many methods such as ligating the coronary artery, balloon occlude and microsphere perfusion have been applied to establish the animal model. To clearly understand no-reflow phenomenon, people also tried to establish the animal model by of dogs, sheep and rabbits, and so on. But up to now, the mechanism of no-reflow is still faint. Chinese wuzhishan minipig was selected in this study for its similarity to human being in cardiovascular biological features, which is one of optimal animals to copy human cardiovascular disease .In AMI-PCI no-reflow animal models ,there are many factors that associated with no-reflow closely. So it is important to measure and study these factors for treatment of no-reflow. Anisodamine was proved to have the function of antagonizing the spasm of cardiac artery, dilating vessels, improving the microcirculation of heart and lessening ischemia of myocardium, which showed two-way regulation effect to the tension of dysfunctional vessel, reconditioning dysfunctional vessel. Firstly, an animal model of AMI and reperfusion no-reflow (AMI-PCI no-reflow) was set up by dilating balloon to occlude the left anterior descending artery (LAD), reperfusing, minicatheter superchoosing LAD , and injecting the microthrombus. Then, anisodamine was injected to the coronary artery and its effect on no-reflow was studied. NO, MDA, vWF:AG, etc were measured to evaluate endothelial function and their influence to no-reflow. Anisodamine was injected to LAD to evaluate its influence to the change of NO, etc. Finally, the patients heart function and related factors of AMI no-reflow phenomenon were explored by the observation in clinical cases. The details are as follows: Part 1: The Establishment of Minipigs Model with AMI-PCI No-reflow . Objective: To establish Chinese minipigs AMI no-reflow models for its similarity to human being in cardiovascular biological features, and explore the feasibility of no-reflow phenomenon caused by superchoosing Minipig LAD, dilating balloon to occlude LAD, refusing myocardium and injecting the microthrombus with 4F minimal catheter technique in minipigs. Methods: A total of 20 wuzhishan minipigs (9±1 months old with body weight of 25±5Kg) were used. All cardiac catheterizations were performed under general anesthesia with sodium pentobarbital 20 mg/kg IV and sterile conditions. With Seldinger's technique and 4F Judkins angiographic catheter (inner diameter 0.97mm), left ventriculography and coronary angiography (CAG) were performed sequentially by femoral approach, at the same time, left ventricular hemodynamics parameter( LVEDP) was recorded. With Seldinger's technique and 6F Judkins angiographic catheter, LAD was superchosen and occluded by 2.5-3.0×15mm balloon which was chosen according to LAD diameter for 90 minutes, then the balloon was removed and microthrombus (3ml for each, injection time was 5s ) were injected into LAD. The injection was made 2 or 3 times based on CAG with 5 minutes interval. Microthrombus were made as follows: 10ml blood were taken from ear edge vein of minipigs, appended thrombin 10u, saved in 37°C warming box to formmicrothrombus. Then, microthrombus were chosen by boult 100μm. In sterile conditions, the emboli solution was prepared by suspending microthrombus (mean diameter 100 μm, ranged from 78-112μm) in 5ml of 0.9% N.S plus 5 ml of ultravist 370 contrast medium. The routine ECG was recorded with polygraphy. Heart rate, pressure-heart rate index (PRI), systolic and diastolic blood pressure were measured. The markers of myocardial damage, TnI and CK-MB, were quantified in all minipigs at different time points, namely: just before operation, post-reperfusion and after no-reflow phenomenon. Results: sixteen animals survived in whole experiment, fourteen of them were acute myocardial infarction with no-reflow(TIMI≤2), model success rate 70% (14/20), average balloon pressure 12±2 atmospheric pressure, average microthrombus injection 2.2±0.9 times, amount of microthrombus injection 7±1.8ml, mean diameter of microthrombus 100μm. Among failed models, 1 died from anesthesia accident,and 3 died from ventricular fibrillation. Summary: Chinese minipig is an ideal model of AMI no-reflow with superiority to dogs, sheep, and rabbits by its similarity to human being in cardiovascular biological features. The pathophysiological changes of coronary no-reflow phenomenon in minipig models is similar to that happened in clinical AMI patients, it is established by acutely occluding the coronary artery, reperfusing, microcatheter superchoosing LAD, and injecting the microthrombus. Meanwhile the AMI-PCI no-reflow model has such advantages as closed chest, simplicity, stability, mild trauma, lower mortality rate,and higher success rate . Part 2: Effect of anisodamine with inracoronary injection on AMI-PCI no-reflow in minipigs Objective: Anisodamine is a kind of antagonist of M receptor of cholinergic nerve with stronger antispastic action, which can release the spasm of microvessel, increase the tolerance of myocardial cells to ischemia and anoxia for its special pharmacological characteristics. In this study, anisodamine was injected into coronary LAD of minipig AMI no-refelowmodels, to evaluate the changes of no-reflow and hemodynamic, explore the feasibility of intracoronary anisodamine injection and its effect on minipig AMI-PCI no-reflow, comparing with diltiazem. Methods: Fourteen Minipigs models with no-reflow were divided into three groups : normal saline (N.S), diltiazem (DIL) and anisodamine (ANI) groups. Group N.S (4 animals) was set as control group, while Group DIL (5 animals) and group ANI (5 animals) as experiment group. In group N.S , 4 ml saline was injected into the LAD, two times, total amount 8ml. The change of blood flow was observed through CAG 1,3,5,10 minutes after injection. The velocity of injecting was 1ml per second. Time recording began when medicine fluid in the guiding catheter was injected into the coronary artery, CAG was performed 1,3,5,10 minutes after injection, at the same time, changes of intracoronary artery pressure and ECG were recorded. Group DIL: 1000ug diltiazem (concentration 250 ug/ml)was injected into the coronary artery, two times, total amount 2000ug. The change of blood flow was observed through CAG 1,3,5,10 minutes after injection. In group ANI, 2000ug anisodamine (concentration 500 ug/ml) was injected into the coronary artery, two times ,total amount 4000ug. The change of blood flow was observed through CAG 1,3,5,10 minutes after injection. All CAGs were performed at the same degree of cast angle, distance of tuber, and velocity of injecting contrast medium. Blood flow of TIMI level was measured by two cardiologists who did not know the drug administration in advance. Quantitative measurement of diameter was performed with the quantitative coronary angiography system at the same degree of cast angle, height of tuber and magnifying rate. The inner diameter of the guiding catheter was made as the reference. The vessel diameter was measured at the end-diastolic phase. The diameter of medial segment of LAD was measured at instantly after CAG 1,3,5,10 minutes after injection of saline, diltiazem and anisodamine respectively. The pressure of femoral artery of all the enrolled experimental minipigs was continuously measured from pre-model to the process of model making.5F balloon floating catheter of 4 chambers temperature sensitivity was inserted into pulmonary artery via femoral vein to measure mean right arterial pressure (mRAP), mean right ventricular pressure (mRVP), mean pulmonary artery pressure (mPAP) and pulmonary capillary wedge pressure (PCWP). Results: After application normal saline there is no change of the blood velocity and LAD inner diameter, but diltiazem had the stronger dilating function to the medial segment of LAD 3 minutes after its useed in the coronary artery. The medial diameter of LAD dilated from 2.12±0.38 mm to 2.51±0.42 mm(P<0.05). Application of anisodamine had dilating action to coronary artery, which lasted to about 10 minutes. The diameter of medial coronary artery with strongest dilated from 2.12±0.38 mm to 2.60±0.43 mm(P<0.05). TIMI frame was rectified by CAG. Comparing with group N.S,TIMI frame were decreased by 10.6%,20.4%,14.3%,21.5% at 1,3,5,10 minutes after administration of diltiazem respectively in group DIL (P<0.05).While in group ANI 1,3,5,10 minutes after administration of anisodamine, no-reflow phenomena decreased by 13.2%,25.3%,35.6%,33.8% respectively compared with group N.S (P<0.05). The heart rate decreased, but there were no obvious changes of blood pressure at 1,3,5,10 minutes after the administration of diltiazem infused in the coronary artery. The heart rate and blood pressure increased at 1,3,5,10 minutes after the administration of anisodamine in the coronary artery, which had statistic significance compared with Group N.S(P<0.05).The heart rate increasing of anisodamine was more apparent than that of diltiazem, but arterial and ventricular arrhythmias was not found. Summary: In our study, anisodamine was administrated intracoronary in minipigs with AMI-PCI no-reflow, showed that the forward flow achieved rapid and effective amelioration. Anisodamine could relieve the myocardium ischemia and increase heart rate to enhance cardiac output, perfusion of myocardium and coronary pressure. So anisodamine prevented no-reflow induced by AMI reperfusion through releasing coronary microvascular spasm,increasing the cardiac output and perfusion of coronary artery, improving heart function, decreasing mean pulmonary artery pressure and LVEDP. Meanwhile the occurrence of AMI-PCI no-reflow were obviously decreased. Anisodamine is superiority to diltiazem in improving AMI-PCI no-reflow phenomenon. Part 3: The changes of endothelial related factors induced by AMI–PCI and intracoronary anisodamine injection in minipigs Objective: NO, vWF:Ag factor and ET-1, etc are important markers of endothelial function. Decrease in serum NO represents endothelial dysfunction, while increase of vWF:Ag factor and ET-1 may be the manifestation of endothelial dysfunction. Malondialdehyde (MDA), as an index of systemic over-oxidation,and indirectly reflects the degree of cell damage, platelet aggregation denoting coagulation system activation due to AMI-PCI. In this study, the changes of these factors in coronary artery LAD during AMI-PCI and the impact of anisodamine were observed, meanwhile endothelial function was analyzed to explore the protecting effect of anisodamine for coronary artery endothelium. Methods: Twelve minipigs were randomized into two groups: group normal saline(N.S) and anisofamine(ANI). In group N.S, the balloon was dilated to occlude medial segment of LAD for 90 minutes, then balloon was removed and 2 ml saline was infused to coronary artery immediately, 2ml each time 10mins for interval . Restore coronary perfusion for 180 minutes. Blood samples were taken before coronary occluded ,at 5,30,60,120,180 min after reperfusion and when no-reflow phenomenon appear .In group ANI, The balloon was removed after coronary occlusion 90 minutes, 1000ug anisodamine (dilution to 2ml with saline) was infused into coronary artery within 5 seconds instead, 2ml each time 10 mins for interval. Restore coronary perfusion for 180 minutes. Blood samples were taken as group N.S. Samples were treated specially according to manual and saved in -70℃refrigerator for later use. NO, ET-1, vWF:Ag,and so on were measured. Finally, took out the heart to measure myocardial ischemic and infarction area . Results:For AMI-PCI minipig models there are great change of NO,ET-1, etc at the beginning of coronary reperfusion and 180mins later. NO decreased from 152.4±11.6 umol/L to 87.8±10.3umol/L and ET-I increased from 51.2±10.3 ng/L to 109.6±11.9 ng/L, MDA increased from 3.57±0.78 nmol/ml to 6.61±1.12 nmol/ml,vWF:Ag increased from 138.2±19.6 IU/Dl to 168.3±11.0 IU/dL,and PagT increased from 47±4% to 73±8%. But these changes were relatively small in group ANI. Platelet aggregation rate rised abruptly, while CRP was stable. Blood pressure was found drop down and heart rate rised after coronary reperfusion in group N.S, while no obvious blood pressure change in group ANI, heart rate rised more than group N.S.There was significant difference in ischemic and infarction area between group N.S and group ANI. Summary: AMI-PCI can affect vascular endothelial function. NO decreased when reperfusion started, vWF:Ag and ET-1 increased at the same time, these changes reached peak at 1.5-2 Hr, and then the effect disappeared slowly as reperfusion went on. AMI-PCI activated systemic coagulation, platelet aggregation rate raised as reperfusion, but the inflammation marker-CRP was less raised .Vascular endothelial dysfunction and systemic coagulation activation were the main reasons for no-reflow phenomenon during occlusion-reperfusion in AMI-PCI no-reflow minipig models. Anisodamine intracoronary infusion can protect vascular endothelial function and decrease the ischemic and infarction myocardium area . Part 4 : The related factors of no-reflow in AMI PCI patients and their influence on ventricular function Objective: To explore the clinical features, related risk factors and their influence on the heart function of no-reflow phenomenon during PCI treatment in AMI patients Methods: 252 patients with primary acute myocardial infarction (159 males and 93 females, 56.9±12.1 years old) were consecutively admitted into our hospital, all of them were consist with the diagnostic criteria of AMI: chest pain≥30 minutes; ST segment elevation in more than two adjacent leads of ECG; and increased myocardial enzyme and tropnin.Patients of each group were performed left and right coronary artery angiography in the cathelab .The conditions of the coronary artery lesion were analyzed through image to make sure of the infarction related artery, the infarction related position and the degree of stenosis that measured through quantitative computer angiography, at the same time, the collateral circulation was judged. The degree of forward flow post-PCI of infarction related artery was measured with TIMI degree method. Left ventricular angiography was performed with pigtail at RAO 30°and LAO 60°. The ventricular aneurysm was diagnosed according to the criteria of CASS and Meizlish JL, etc with following features: adynamic or retrograde dynamic of some left ventricular. 12 leads ECG was recorded before PCI and after the admission of the patient. Myocardial enzyme were measured once every 4 hours during the 1st 24 hours after AMI and every 8 hour in the next 48 hours. The peak value of CK-MB was considered as the enzyme marker of infarction area. Radionuclide angiocardiography was performed with SPECT imager of SOPHY DST. Images were collected at LAO 30-45°, which was the best position to differentiate the left and right ventricle. 32 frames were collected during each movement cycle of the heart. All the data were stored after being collected. The parameters of ventricular function were measured with the special analysis software made for angiocardiography heart function analysis, which can semi-automaticly draw out the region of interest, time-radiation activity curve got by deducting the background of end-systole. According to the principle of count-volume method, the parameters of left ventricle function can be obtained by the end-systolic and end-diastolic counts. Results: Among all the factors related to CAG and PCI, the rate of no-reflow phenomenon after PCI was decreased in the patients with well collateral circulation, pre-infarction angina (6.9% vs 17.7%, P<0.05).while increased in the patients with total occlusion lesion,or heart failure (19.2% vs 8.0%,P<0.05).Direct stenting could decrease the incidence of no-reflow(5.6% vs 16.6%,P<0.05). The features of no-reflow in coronary artery lesion mainlyoccurred in the proximal and middle segment. The incidence of ventricular aneurysm had an increasing trend in patients with no-reflow phenomenon. The independent risk factors were as follows: no preinfarction angina, the number of pathologic Q wave, total occluded proximal segment of LAD without obvious collateral circulation and the peak value of CK-MB. The nuclide blood pond of heart image showed that the systolic and diastolic functions were significantly damaged. PSD, PS and FWHM of patients in the no-reflow group were obvious higher than that in the reflow group. The incidence of MACE of in-hospital patients was obviously increased in the patients with no-reflow than that with reflow(28.3% vs 14.0%). Summary: The occurrence of AMI-PCI no-reflow was influenced by many factors, which were mainly related with proximal occlusion of LAD, number of leads with pathologic Q wave, peak value of enzyme CK and without pre-infarction angina. Direct stenting could decrease the incidence of no-reflow. The cardiac systolic and diastolic function and systolic synchronization of left ventricular could be damaged by no-reflow phenomenon. Patients with no-reflow had more MACE and poor prognosis. Conclusion 1 Chinese wuzhishan minipig is an ideal model of acute myocardial infarction no-reflow with superiority to dogs, sheep, and rabbits by its similarity to human being in cardiovascular biological features. The pathophysiological changes of coronary no-reflow phenomenon in wuzhishan minipig AMI-PCI models is similar to those happened in clinical AMI patients, for it is established by acutely occluding the coronary, reperfusing, microcatheter superchoosing left anterior descending, and injecting the microthrombus. Meanwhile the model has such advantages as simplicity, closed chest, stability, mild trauma, lower mortality rate and higher success rate . 2 Anisodamine were injected into LAD of Chinese wuzhishan minipigs...
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