动物模型,冠脉模型 z-bo 2022-12-17 691

       The procedure for preparing blood vessel samples of stent segments is as follows. Stent segment blood vessel hard tissue sectioning and staining device: timing constant temperature electromagnetic stirrer, digital display electric heating constant temperature drying oven, LEIKASP1600 hard tissue slicer.

       (1) Preparation of embedding solution: In a 250 ml container, add 100 ml methyl methacrylate, 2 g benzoyl peroxide, 2 ml dibutyl phthalate, mix well for at least 1 hour and mix well carry out. Store in a refrigerator at room temperature or 4°C.

　　 (2) Prepare the embedded container and base. Prepare a penicillin bottle and place the prepared label on the bottom of the bottle with the words facing outward. Put the prepared infusion into a vial and place about 1/4 of the vial. Tightly cap the bottle, inhale with a 5 ml syringe, put it in a desiccator, and heat it at 40-50°C for 24-48 hours for polymerization. Take out the hard object and check if it is fully polymerized. If it is not polymerized, continue heating. If it is fully aggregated, take it out and use it.

　　 (3) Dehydration: Take out the sample from the 10% formaldehyde fixative and put it in the sample holder. Put it in 80% alcohol solution and dehydrate for 1 hour. Dehydrate in 95% alcohol I solution. 1 hour: Put the sample in a 95% alcohol II solution for dehydration for 1 hour, put the sample in anhydrous alcohol I for 30 minutes, and put the sample in anhydrous alcohol II for 30 minutes.

　　 (4) Clear: Take out the sample from the absolute alcohol II, and then quickly put it into the xylene solution. The xylene soaking time is 2-5 minutes, depending on the size of the sample, and the sample is transparent.

　　 (5) Embedding: Take out the xylene solution, put the sample into the embedding bottle, close the stopper tightly, and then vacuum. Place the embedded vial in a dry box and heat at 42°C and 24-48°C to polymerize. time.

　　 (6) Preparations before sectioning: crush the penicillin vial (because the embedding fluid is fully polymerized, so it is usually not damaged), take out the embedded specimen and wash the glass ballast with water.

　　(7) Fix: Modify the plane so that the blade is perpendicular to the blood vessel of the stent. Tighten the screws. Otherwise, the blade may be damaged.

　　 (8) Shaving: First, cut out the plane and prepare for slicing. There is no need to adjust the thickness at this time. The speed is set to 6 to 7μm/sec, the water is washed off when slicing, and the temperature of the blade is reduced to achieve the purpose of cooling.

　　(9) Sectioning (Figure 5-14): After trimming, turn the knob to 350-360μm. At this time, the slice thickness is about 100 μm, and the speed is maintained at 6-7 μm/sec. Start slicing. Remember, when slicing, you need to take 1-2 of each of the near, middle and far stent segments in order to get the average value after the measurement.

　　 (10) Preparation before dyeing: After slicing, put the slices in the prepared plastic clamp, flatten them on two glass slides, put them in a drying oven, bake and iron.

　　(11) First, take out the baked and ironed parts, and then put them in the staining container (cell culture plate is recommended). After washing with water, add Hastelloy dye solution (37-40°C) and dye according to the sample. It usually takes 3 to 4 hours, depending on the coloring time. After alkaline coloring, take out the section, wash the excess dye with 70% ethanol and hydrochloric acid solution, and then put it back into the blue 1% ammonia solution or saturated lithium carbonate solution. The time depends on the color of the slice. After the bluing is completed, rinse off the ammonia and lithium carbonate, put in 50% alcohol and eosin, and dye for about 30 minutes. After coloring, wash the excess dye with 70% alcohol and hydrochloric acid solution, put it in the sample holder, and then bake the iron again. After baking and ironing, the 95% alcohol and 100% alcohol are dehydrated for 1 minute to make the xylene solution transparent for 1 minute, and then the resin is sealed.

　　2. Histomorphology and pathology score analysis Pathology analysis mainly includes inflammation score, injury score and endothelialization score. At the same time, the distance from the stent to the inner elastic layer and the ratio of proliferating smooth muscle cells are within the pathological analysis. When performing pathological integration, conventional HE staining usually does not meet the requirements and requires special staining methods, such as MOVAT staining, elastic plate staining, and Masson staining.

　　 (1) Histomorphometry: use LEIKA DFC300FX optical microscope and LEIKAQwinPLUS V3.2.1 image analysis software to analyze images. Determine the lumen area, the inner elastic layer or around the stent, the outer elastic layer and the new endometrial area = around the stent or inner elastic layer-vertical area.

　　 (2) Inflammation score: In a study of an animal over-expansion model, the placement of a stent caused severe damage to the blood vessel wall, and granulocytes and monocytes appeared immediately in the damaged area. In the second day and a few weeks, macrophages invade the neointima, gather around the scaffold filaments and form giant cells. However, anti-inflammatory treatments applied early can prevent monocyte recruitment and reduce neointimal hyperplasia. Based on this reasoning, the linear relationship between the number of monocytes and the area of the neointima indicates that monocytes play an important role in the process of restenosis. Activated macrophages affect the repair of damaged blood vessels by producing a series of mediators, including the interleukin family, tumor necrosis factor, MCP-1 and growth factors (such as platelet-derived growth factors). Carter et al. In a four-week histomorphology study, compared with a four-week animal study, Cypher and Yukon stents have significantly reduced neointimal area and stenosis rate compared with BMS. At the same time, the analysis of the inflammation score showed that the Cypher inflammation score was significantly higher than that of BMS and Yukon, but there was no significant difference between the latter two.

Integration method: inflammation score (0 points = no inflammatory cells: 1 point = scattered inflammatory cells; 2 points = 25%-50% of the perivascular stent points are surrounded by 50% inflammatory cells 3 points = 25% approximately 50 % Of the stent spots around blood vessels were completely surrounded by inflammatory cells, and the inflammatory response caused by the stent was evaluated.

　　 (3) Damage score: Schwartz et al. proposed that there is a close relationship between vascular injury and inflammation and neointimal hyperplasia. Uss0 et al. studied the model of excessive coronary artery dilation in minipigs and found that as the expansion ratio increased (1.0:1 to 1.4:1), neointimal hyperplasia also increased significantly. However, it is worth noting that in addition to the damage during the stent insertion, the long-term stimulation of the stent to the blood vessel wall can also aggravate the blood vessel damage. Carter et al. Animal studies found that a 4-week histomorphology study compared Cypher and Yukon scaffolds with BMS. In the injury score analysis, the Cypher inflammation score was significantly higher than the BMS and Yukon scores, but there was no significant difference between the latter two. Integration method: Damage points: 0 points = internal elastic plate damage; 1 point = internal elastic plate damage; 2 points = internal elastic plate and medium damage: 3 points = external elastic plate damage.

　　 (4) Endothelialization score: At present, most scholars believe that the delayed healing of endothelial cells in drug-eluting stents is closely related to late thrombosis. In addition to the effects of drugs that inhibit cell proliferation, long-term stimulation of high molecular weight polymers can significantly slow down the endothelialization process. Virmani et al. found in animal studies that the endothelialization of the bare stent was completed 28 days after implantation, but the surface of the drug-eluting stent showed incomplete endothelialization and continuous cellulose deposition. It was used in early pre-market animal experiments conducted with Cipher and Taxus, and it was almost completely endothelialized 28 days after transplantation. However, in the above-mentioned biopsy pathology and angioscopy, the human condition is far from this. A more reasonable explanation for the above situation is that the selected 28-day evaluation period does not seem to be optimal. In an animal study using scanning electron microscopy to study the degree of endothelialization, the process in a rabbit blood vessel model took 21 days, and the surface of the bare stent was placed in a miniature pig model with a bare stent. After 14 days, it was found that it was completely cured.

　　Integration method: Endothelialization score: According to the circumference of the lumen surrounded by endothelial cells, the endothelialization score is defined as three levels (Level 1 = 25%, Level 2 = 25%? 75%, Level 3 ≥ 75%).

　　 (5) The maximum distance from the stent to the inner elastic sheet: Incomplete stenting (ISA) refers to the separation of the stent and the vessel wall, except for the blood flow between the stent and the vessel wall (not including vascular bifurcation). The mechanisms of detectable signs include aggressive vascular remodeling, regression or dissolution of thrombotic plaque, incomplete stent expansion, inflammation and tissue necrosis, and chronic stent contraction. Although there are controversies in clinical studies, platelets and cellulose theoretically adhere to the thrombosis tissue due to the gap between the stent and the vascular wall stent, thereby increasing the possibility of thrombosis in the stent. In addition to applying the IVUS test to evaluate the poor adhesion of the stent, pathological examination can also measure the distance (gap, GW) between the stent and the inner elastic plate to evaluate the poor adhesion of the stent. It is an indicator. Jabara et al. evaluated a degradable polymer-coated paclitaxel-eluting stent in an animal study using a miniature pig coronary artery model. A one-month pathology study found that the GW of the drug stent group was significantly higher than that of the bare stent group. The above results were not observed for 3 months. Pathological examination revealed thrombus, infiltrating inflammatory cells and necrotic tissue between the stent and the inner elastic layer.

　　 (6) Scanning Electron Microscope: Preparation of Electron Microscope Samples: ① Cut the stent along the longitudinal axis of the blood vessel and fix it with 3% glutaraldehyde for 2 hours at 4°C. (2) Rinse 3 times with 0.2 mol/L rinse solution for a total of 3 hours. ③ Rinse once with distilled water. ④ Use 50%, 70% and 90% acetone solution to gradually dehydrate. Each step is 15 minutes. ⑤ Dehydrate with 100% acetone solution 3 times, 15 minutes each time. Soak in isoamyl acetate for 30 minutes. a Dry in a desiccator for 2-3 hours. Use vacuum spray to spray 24K pure gold on the surface of 24 samples for 3-5 minutes. Use a scanning electron microscope to examine the specimens, observe the degree of endothelialization, especially the healing of the endothelium near the stent wire, take digital photos, and calculate the degree of endothelialization through software.