动物造模,ST段抬高心肌梗死 z-bio 2021-04-26 879

　　Background: Thrombolytic therapy is essential to alleviate ST-segment elevation (STEMI), which accounts for 25%-40% of myocardial infarction cases. For STEMI patients who cannot receive thrombolytic therapy in time for percutaneous coronary intervention (PCI), it is recommended to follow the latest ACCF/AHA and ESC guidelines. Pre-hospital thrombolytic therapy is an important intervention to rescue ischemic myocardium, improve prognosis and provide more time for clinical treatment. Most patients can benefit from thrombolytic therapy, but the specificity, efficacy and safety of thrombolytic drugs need to be improved. There is an urgent need to develop new drugs with rapid action and less side effects, and it is very necessary to establish a reproducible and consistent animal model of coronary thromboembolism based on clinical conditions, especially the components of thrombus. However, in previous animal models, most coronary thrombosis is red or mixed embolism, with a different clinical environment. Prior to the use of coronary thrombosis aspiration techniques, it was not clear what constitutes coronary thrombosis within the time frame of thrombosis. Recently, it has been found that coronary thrombosis in STEMI patients is mainly composed of platelets, a small amount of fibrin, a small amount of red blood cells, white blood cells and cholesterol crystals.

　　This fibrin-rich thrombus is similar to cerebrovascular thrombosis. Kirchhof et al. A rabbit cerebral embolism model was created using white embolism and evaluated that thrombolytic drugs should not be used on the heart. The purpose of this work is to establish an ideal arterial thrombosis model to reflect the clinical symptoms of STEMI patients. Therefore, we compared the red and white embolism models, and injected thrombolytic drugs into the coronary arteries through the animal model catheters to study its effectiveness. Method: Animals: 21 male dogs, weighing 12-17kg

　　Embolization preparation: Based on previous research, some changes have been made. Four hours before the operation, 3 milliliters of autologous venous blood was drawn from each experimental animal to prepare individual matching emboli. White blood clot: venous blood, no anticoagulant. Centrifuge at 1500pm for 5 minutes at 4°C. After extracting the supernatant and injecting it into a silicone tube (diameter 2.5 mm), the blood clot was placed in a 37°C water bath for 0.5 hours. Push the clot into the sterile plate, automatically retract it through the needle tube for 3 hours (approximately 1.2 mm in diameter), and cut into 5 mm long cylinders. For red emboli: inject the blood in the syringe directly into the silicone tube and leave it at 37°C for 0.5 hours. The subsequent process is the same as creating the white plug. We also measured the concentration of fibrin, platelets and red blood cells in whole blood and supernatant. Fibrin parameters are measured by an automatic coagulation analyzer, and platelets and red blood cells are measured by an automatic blood cell analyzer. Coronary artery thromboembolism model: Anesthetize the animal with ketamine (35 mg/kg) and diazepa (15 mg/kg), and halve its dose every hour to maintain anesthesia. Fentanyl (0.03 mg/kg) is used for intraoperative and postoperative analgesia. After anesthesia, the animal was fixed on the operating table in a supine position, and the tracheal intubation was performed in the synchronized intermittent forced ventilation (SIMV) mode of assisted breathing. Parameters include tidal volume (10 ml/kg), respiratory rate (20 breaths/min), exhalation/inspiration (E/I) ratio (1:1.5-2) and oxygen saturation (55%). Monitor the occurrence of arrhythmia. Heparin and rt-PA were injected into the left forearm brachiocephalic vein. Insert the axillary artery branch of the right forelimb into the arterial sheath. Under X-ray guidance, a 5F catheter was inserted into the left coronary artery for coronary angiography. Place the 5F catheter near the first diagonal branch of the left anterior descending branch and inject a stopper to prevent blood from flowing through the inner and distal ends of the LAD. Since the catheter cannot be inserted deeply, the embolus can reach the diagonal branch. Therefore, the operator must handle carefully and prevent emboli from flowing into the blood vessel under lower pressure. Thrombolytic therapy: 60 minutes after LAD obstruction, 1000 U heparin sodium was injected every 2 hours thereafter. Use RT-PA infusion (0.4 mg/kg) as a loading dose, and continuous infusion of thrombolytics for at least 30 minutes (1.2 mg/kg). The remaining rt-PA was continuously infused for more than 60 minutes (0.8 mg/kg). The procedure complies with the drug instructions, and the dose used is clinically safe. Coronary artery perfusion measurement: Animals record an electrocardiogram every 15 hours before, during and after the embolization to determine the status of the embolus, and change the ST segment, T wave, etc. recording. On coronary angiography, the animal had previously received embolization injections to the right anterior oblique position and left anterior oblique coronary angiography. During injection, observe the degree of obstruction and/or autolysis every 30 minutes for 3 hours. .. 10, 20, 30, 60, 90, and 120 minutes after using rt-PA or arrhythmia or ECG changes, coronary angiography was also performed to evaluate the effect of thrombolysis. The reperfusion time is defined as the recanalization time confirmed by coronary angiography.

　　Pathology research: Use scanning electron microscope (SEM) to analyze autologous embolism. The sample was automatically collected after 3 hours, the sample was washed 3 times with phosphate buffer, fixed with 2% glutaraldehyde for 120 minutes, and then washed 3 times with phosphate buffer. Then the sample was fixed for 120 minutes, stained with osmotic acid, washed, dehydrated in the order of ethanol concentration (50%, 70%, 90% 100%) for 40 minutes, and then added isoamyl acetate (50), ethanol (%, 70%, 90%). %, 100%). The thrombus was dried for 10 minutes and stretched to obtain a fractured surface for analysis. The blood clot was coated with gold and palladium and examined with a scanning electron microscope. The area of myocardial infarction was observed by 2,3,5-triphenyltetrazole chloride (TTC) staining. , Ketamine (35 mg/kg) and Diaz 1.5 (1.5 mg/kg) were combined intravenously to anesthetize the animals, and then 10% potassium chloride (15-20 ml) was injected to euthanize the animals. The heart was excised, the cut part was 10mm thick, and it was stained with 2,3,5-triphenyltetrazolium chloride (TTC). The infarct area is a non-TTC stained area, and the infarct area (%) is the ratio of the infarct area to the left ventricular area. Thrombolysis of LAD was also observed. Examine the skin, mucous membranes, heart, brain, lungs, liver, spleen and kidneys under a microscope to estimate the risk of bleeding.

　　Result: 21 dogs were used in the experiment. One animal died of ventricular fibrillation due to the duration of the catheter in the LAD. Two animals had oblique branch embolism. These three animals were excluded from the study. Finally, 18 animals were randomly divided into 3 groups. This includes the red plug group (n=6), the white plug group (n=6) and the white plug rt-PA group (n=6). ECG: All animals had normal ECG before surgery. Four patients in the Hongshuanzi group and the rt-PA group had transient ventricular premature beats, and sinus rhythm was reversed after defibrillation. In addition, as described below, these 6 animals are currently undergoing coronary artery recanalization (reperfusion) according to coronary angiography. Since the embolization injection, the ST segment and V1-V4T wave of Phragmites communis have increased. In the red thrombus group, the ST segment of the electrocardiogram decreased, and the T wave was low at 120 minutes, indicating that the thrombus was automatically dissolved. Neither situation was observed in the white embolism group. After dissolving the white emboli with rt-PA, the ST segment decreased and the T wave decreased. Coronary angiography: Preoperative coronary angiography showed that the coronary arteries of the experimental animals were normal. Three hours after LAD occlusion, recanalization occurred in the red embolization group, and the embolization position of the white embolization group was still located at the medial and distal ends. These results indicate that the white emboli may be suitable for subsequent experiments. In addition, after 2 hours of rt-PA treatment, 5 of the 6 dogs experienced reperfusion, but none of the 6 dogs in the control group experienced reperfusion. The average reperfusion time of the t-PA group was 43.2±7.4 minutes.

　　Pathology research: Use scanning electron microscope to observe the characteristics of various autologous embolism. White embolism is more difficult than complete thromboembolism because it contains more fibrin and fewer red blood cells in the same volume. After the autopsy, there were no obvious signs of bleeding on the skin, mucous membranes, heart, brain, lungs, liver, spleen, kidneys or other vital organs. In the measurement of the infarct area, the image J is used to obtain the color of each slice to quantify the degree of myocardial necrosis in the unstained part of the heart. The infarct area ratios of Baishuan group and rt-PA group were 11.61±0.64% and 4.48±0.52%, respectively. Coronary artery dissection thrombolysis is consistent with the results of coronary angiography.

　　Conclusion: A coronary thromboembolism model of white thrombosis with better stability, uniformity and higher success rate has been established for the first time. Importantly, this model has similar characteristics and is suitable for evaluating thrombosis and thrombolytic therapy in STEMI patients within the time frame of thrombolytic therapy. This model can be used to evaluate new thrombolytic drugs for the treatment of STEMI.