动物模型,冠脉模型 中国实验动物信息网 2022-12-17 1045

　　制备支架节段的血管样品的程序如下。支架段血管硬组织切片和染色装置：定时恒温电磁搅拌器，数显电加热恒温干燥箱，LEIKASP1600硬组织切片机。

　　（1）包埋溶液的制备：在250 ml容器中，加入100 ml甲基丙烯酸甲酯，2 g过氧化苯甲酰，2 ml邻苯二甲酸二丁酯，充分混合至少1小时并充分混合完成。存放在室温或4°C的冰箱中。

　　（2）准备嵌入式容器和底座。准备一个青霉素瓶，将准备好的标签纸放在瓶子的底部，单词朝外。将准备好的浸液放入小瓶中，并放置约1/4的小瓶。盖紧瓶塞，用5 ml注射器吸气，放入干燥器中，在40-50°C加热24-48小时以聚合。取出硬物，检查其是否完全聚合。如果未聚合，请继续加热。如果已完全聚合，请取出并使用。

　　（3）脱水：从10％甲醛固定剂中取出样品，然后将其放入样品架中。放入80％酒精溶液中并脱水1小时。在95％酒精I溶液中脱水。 1小时：将样品置于95％酒精II溶液中脱水1小时，将样品置于无水酒精I中脱水30分钟，将样品置于无水酒精II中脱水30分钟。

　　（4）清除：从无水酒精II中取出样品，然后将其快速放入二甲苯溶液中。二甲苯浸泡时间为2-5分钟，具体取决于样品的大小，并且样品是透明的。

　　（5）包埋：取出二甲苯溶液，将样品放入包埋瓶中，紧紧塞住塞子，然后抽真空。将嵌入的小瓶放在干燥箱中，在42°C和24-48°C加热以聚合。时间。

　　（6）切片前的准备工作：压碎青霉素小瓶（因为包埋液已完全聚合，因此通常不会损坏），取出嵌入的标本并用清水洗涤玻璃镇流器。

　　（7）固定：修改平面，使刀片垂直于支架的血管。拧紧螺丝。否则可能会损坏刀片。

　　（8）剃须：首先，切出飞机并准备切片。此时无需调整厚度。速度设置为6到7μm/ sec，切片时将水洗掉，降低刀片的温度以达到冷却的目的。

　　（9）切片（图5-14）：修剪后，将旋钮旋转到350-360μm。此时，切片厚度约为100μm，速度保持在6-7μm/ sec。开始切片。请记住，切片时，您需要取近，中和远支架段中的每一个的1-2、以便在测量后获得平均值。

　　（10）染色前的准备工作：切片后，将切片放在准备好的塑料夹中，将它们压平在两个玻璃载玻片中，放入干燥炉中，烘烤并熨烫。

　　（11）首先，取出经烘烤和熨烫的部件，然后将其放入染色容器中（建议使用细胞培养板）。用水洗涤后，添加哈氏合金染料溶液（37-40°C）并根据样品染色。通常需要3到4个小时，具体取决于着色时间。碱性着色后，取出切片，将过量的染料用70％的乙醇和盐酸溶液洗涤，然后放回蓝色的1％氨溶液或饱和碳酸锂溶液中。时间取决于切片的颜色。上蓝完成后，冲洗掉氨水和碳酸锂，放入50％酒精和曙红中，染色约30分钟。着色后，用70％的酒精和盐酸溶液洗涤多余的染料，将其放入样品架中，然后再次烘烤铁。烘烤和熨烫后，依次使95％的醇和100％的醇脱水1分钟，使二甲苯溶液透明1分钟，然后密封树脂。

　　2、组织形态学和病理学评分分析病理学分析主要包括炎症评分，损伤评分和内皮化评分。同时，从支架到内部弹性层板的距离以及增殖的平滑肌细胞的比例均在病理分析之内。进行病理整合时，常规HE染色通常不符合要求，需要特殊的染色方法，例如MOVAT染色，弹性板染色，Masson染色。

　　（1）组织形态计量学：使用LEIKA DFC300FX光学显微镜和LEIKAQwinPLUS V3.2.1图像分析软件分析图像。确定管腔区域，内部弹性层或支架周围，外部弹性层以及新的内膜区域=支架周围或内部弹性层-垂直区域。

　　（2）炎症评分：在一项动物过度膨胀模型的研究中，放置支架对血管壁造成了严重损害，在受损区域会立即出现粒细胞和单核细胞。在第二天和几周内，巨噬细胞侵入新内膜，聚集在支架丝周围并形成巨细胞。但是，早期应用的抗炎治疗可阻止单核细胞募集，可减少新内膜增生。基于此推理，单核细胞数量与新内膜面积之间的线性关系表明，单核细胞在再狭窄过程中起重要作用。活化的巨噬细胞通过产生一系列介体，包括白介素家族，肿瘤坏死因子，MCP-1和生长因子（如血小板衍生的生长因子），来影响受损血管的修复。卡特等。在一项为期四周的组织形态学研究中，与一项为期四周的动物研究相比，Cypher和Yukon支架与BMS相比，其新内膜面积和狭窄率明显降低。同时，对炎症评分的分析表明，Cypher炎症评分显着高于BMS和育空地区，但后两者之间没有显着差异。

　　积分方法：炎症评分（0分=无炎性细胞：1分=散在的炎性细胞； 2分= 25％-50％的血管周围支架分被50％炎性细胞包围3分= 25％大约50％的血管周围支架点被炎症细胞完全包围，评估了支架引起的炎症反应。

　　（3）损伤评分：Schwartz等人提出血管损伤与炎症和新内膜增生之间存在密切关系。uss0等人研究了小型猪中过度冠状动脉扩张的模型，发现随着扩张比率的增加（1.0：1至1.4：1），新内膜增生也显着增加。然而，值得注意的是，除了在支架插入期间的损伤之外，支架对血管壁的长期刺激还会加剧血管损伤。卡特等。动物研究发现，为期4周的组织形态学研究将Cypher和Yukon支架与BMS进行了比较。在伤害评分分析中，Cypher炎症评分显着高于BMS和Yukon评分，但后两者之间没有显着差异。  积分方法：损伤积分：0分=内部弹性板损坏； 1分=内部弹性板损坏； 2分=内部弹性板和中等损坏：3分=外部弹性板损坏。

　　（4）内皮化评分：目前，大多数学者认为，药物洗脱支架中的内皮细胞延迟愈合与晚期血栓形成密切相关。除了抑制细胞增殖的药物的作用外，长期刺激高分子量聚合物还会显着减慢内皮化过程。 Virmani等人在动物研究中发现，裸支架的内皮化在植入后28天就完成了，但是药物洗脱支架的表面显示出不完全的内皮化和连续的纤维素沉积。它是在使用Cipher和Taxus进行的上市前早期动物实验中，它在移植后28天几乎完全被内皮化。但是，在上述活检病理学和血管镜检查中，人体状况远非如此。对于上述情况的更合理的解释是，所选的28天评估期似乎不是*佳的。在一项通过扫描电子显微镜研究内皮化程度的动物研究中，在兔血管模型中的过程耗时21天，而裸露支架的表面被置于带有裸露支架的小型猪模型中。 14天后，发现它已完全治愈。

　　整合方法：内皮化评分：根据内皮细胞包围的管腔周长，将内皮化评分定义为三个级别（级别1 = 25％，级别2 = 25％？75％， 3级≥75％）。

　　（5）从支架到内部弹性薄片的*大距离：不完全支架术（ISA）是指支架与血管壁的分离，除了支架与血管壁之间的血流（不包括血管分叉）。可以检测到的迹象其机制包括积极的血管重塑，血栓斑块消退或溶解，支架扩张不完全，炎症和组织坏死以及慢性支架收缩。尽管临床研究存在争议，但由于支架与血管壁支架之间的间隙，血小板和纤维素理论上会附着在血栓形成组织上，从而增加了支架内血栓形成的可能性。除了应用IVUS测试来评估支架的附着力差外，病理检查还可以测量支架与内部弹性板之间的距离（间隙，GW），以评估支架的附着力差。是一个指标。 Jabara等人在动物研究中使用小型猪冠状动脉模型评估了可降解的聚合物涂层紫杉醇洗脱支架。一项为期一个月的病理研究发现，药物支架组的GW显着高于裸支架组的GW。 3个月未观察到上述结果。病理检查发现支架和内部弹性层之间有血栓，浸润的炎性细胞和坏死组织。

　　（6）扫描电子显微镜：电子显微镜样品的制备：①沿血管的纵轴切割支架，并在4°C下用3％戊二醛固定2小时。 （2）用0.2 mol/L的冲洗溶液冲洗3次，总共3个小时。 ③用蒸馏水冲洗一次。 ④用50％，70％和90％的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.
丙酮溶液逐渐脱水。每步15分钟。 ⑤用100％丙酮溶液脱水3次，每次15分钟。在乙酸异戊酯中浸泡30分钟。 a在干燥器中干燥2-3小时。用真空喷涂在24个样品的表面上喷涂3-5分钟24K纯金。使用扫描电子显微镜检查标本，观察内皮化程度，尤其是支架丝附近的内皮愈合情况，拍摄数码照片，并通过软件计算内皮化程度。