使用跨女性访问将人类大小的冠状动脉支架植入大鼠腹主动脉
Summary
该协议描述了人类冠状动脉支架植入大鼠的腹部主动脉与apoE-/- 背景使用跨股骨访问。与其他动物模型相比，穆林模型具有吞吐量高、可重复性高、易处理性和外壳性好等优点，分子标记物广泛可用。
Abstract
皮下冠心病干预 （PCI） 结合冠状支架的部署，代表了冠状动脉疾病干预治疗的黄金标准。支架内恢复症 （ISR） 是由支架内新辛蒂莫组织过度增殖决定的，并限制支架的长期成功。各种动物模型被用来阐明支架内复发 （ISR） 背后的病理生理过程，其中猪冠状动脉和兔子胆道模型最常使用。Murine 模型具有吞吐量高、易于处理和外壳、可重复性和分子标记的广泛可用性等优点。非丙蛋白E缺乏（apoE-/-）小鼠模型已广泛用于研究心血管疾病。然而，支架必须小型化才能植入小鼠体内，这涉及到其机械和（潜在）生物特性的重大变化。使用猿类-/-大鼠可以克服这些缺点，因为apoE-/-大鼠允许评估人类大小的冠状动脉支架，同时提供一个异位表型。这使得它们成为在支架植入后研究 ISR 的极好可靠的模型。在这里，我们详细描述了将市售的人类冠状动脉支架植入具有apoE-/-背景的老鼠的腹部主动脉，使用跨股骨访问。
Introduction
皮下冠心病干预（PCI），结合冠状支架的部署，代表了冠状动脉疾病干预治疗的黄金标准1。然而，支架的长期成功可以限制在支架内复发（ISR）的发生，这是由支架2，3内新意组织过度增殖决定的。ISR 可能需要用冠状动脉旁路或重新 PCI 进行重新干预。为研究ISR提出了各种动物模型的建议，每个模型都有优点和缺点。最常用的猪冠状动脉和兔肠动脉模型的主要缺点，虽然在支架植入4、5后出现与人类明显相似的病变，但动物和住房成本高，这带来了后勤困难，特别是在长期研究中，以及处理和设备的限制。此外，猪和兔子细胞蛋白抗体的可用性有限。另一方面，Murine 模型具有吞吐量高、可重复性高、易于操作、外壳等主要优点，因此具有成本效益。此外，有较多的抗体可用。然而，虽然非丙蛋白电子缺乏症（apoE-/-）小鼠已被广泛用于动脉粥样硬化6，7，8的研究，他们不适合研究ISR，因为支架必须小型化植入小鼠，有可能改变支架的机械特性。此外，小鼠主动脉壁测量在幼鼠50μm和老老鼠9中85μm之间，支架必须使用低至2 atm的压力水平进行部署，这可能导致支架10的不良位。然而，大鼠允许植入市售的人类冠状动脉支架，并表现出类似于主动脉支架植入后较大的动物的血管愈合过程，首先由Langeveld等人11日报道。这项技术最初需要跨腹部通道，这就需要对主动脉进行物理收缩，以实现血液流动的暂时中断。为了避免潜在的相关船只伤害和炎症反应，该技术后来通过引入一个跨组织访问，这进一步导致动物12的存活率更高。
由于野生类型大鼠不发展动脉粥样硬化病变13，apoE-/-大鼠已产生使用核酶技术，如转录激活器一样的影响器核糖核酸（TALEN）14，聚类定期间歇短巴林德罗米重复（CRISPR/Cas9）15，和锌手指（ZF）16。自 2011 年以来，ApoE-/-大鼠已上市。提供外来背景，apoE-/-大鼠允许更现实地评估人类大小的冠状动脉支架，特别是关于ISR。
在此，我们通过跨性别访问路线描述该方法，并使用市售的薄支柱钴-铬药物脱脂支架 （DES），但是，它也可用于研究其他支架类型，如裸金属支架 （BMS） 或可生物降解支架。
Protocol
这些实验是根据德国动物福利法（TSchG）和2010/63/欧盟关于保护用于科学目的的动物的指令进行的。政府动物护理和使用委员会批准了这项研究的正式批准（第87-51.04.A065号议定书：兰德萨姆特·法尔·纳图尔，乌姆韦尔特·德·弗布劳彻舒茨·诺德海因-韦斯特法伦，德国雷克林豪森）。研究协议符合《实验室动物护理和使用指南》。术后疼痛治疗基于德国实验室动物科学协会 （GV-SOLAS） 的建议以及?…
Representative Results
该协议描述了使用跨股骨通道（图1）植入大鼠腹部主动脉的支架。这种动物模型的第一个中心点是，它允许部署人类大小的冠状动脉支架。一个市售的压接和气球安装冠状动脉支架可以放置到大鼠的腹部主动脉。因此，还可以应用与人类相同的支架部署原则。使用大鼠的另一个优点是提供转基因菌株，如apoE-/- 大鼠，这些菌株在商业上是可用的。 <p class="jove_conten…
Discussion
本协议描述了将人类大小的冠状动脉支架植入大鼠腹 主动脉。几个技术点值得强调。首先，应避免支架大小和主动脉大小之间的不匹配。放置过小的支架会导致支架失调，而植入对主动脉来说太大的支架则会导致血管过度拉伸、撕裂和损伤。因此，我们建议使用直径在 2.0 到 2.5 毫米之间的支架，并将植入压力保持在建议范围内，而不要过度拉伸支架。最合适的植入压力通常由支架制造商…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢安吉拉·弗伦德夫人在嵌入和幻灯片生产方面提供的宝贵技术援助。我们还要感谢实验室动物科学和实验外科研究所的塔杜斯·斯托平斯基先生在兽医工作中的有见地的帮助。
Materials
| Diet | |||
| SNIFF High Fat diet + Clopidogrel (15 mg/kg) | SNIFF Spezialdiäten GmbH, Soest | custom prepared | Western Diet |
| Drugs and Anesthetics | |||
| Buprenorphine | Essex Pharma | 997.00.00 | |
| ISOFLO (Isoflurane Vapor) vaporiser | Eickemeyer | 4802885 | |
| Isoflurane | Forene Abbott | B 506 | |
| Isotonic (0.9%) NaCl solution | DeltaSelect GmbH | PZN 00765145 | |
| Ringer's lactate solution | Baxter Deutschland GmbH | 3775380 | |
| (S)-ketamine | CEVA Germany | ||
| Xylazine | Medistar Germany | ||
| Consumable supplies | |||
| 10 mL syringes | BD Plastipak | 4606108V | |
| 2 mL syringes | BD Plastipak | 4606027V | |
| 6-0 prolene suture | ETHICON | N-2719K | |
| 4-0 silk suture | Seraflex | IC 158000 | |
| Bepanthen Eye and Nose Ointment | Bayer Vital GmbH | 6029009.00.00 | |
| Cotton Gauze swabs | Fuhrmann GmbH | 32014 | |
| Durapore silk tape | 3M | 1538-1 | |
| Poly-Alcohol Skin Desinfection Solution | Antiseptica GmbH | 72PAH200 | |
| Sterican needle 18 G | B. Braun | 304622 | |
| Sterican needle 27 3/4 G | B.Braun | 4657705 | |
| Tissue Paper | commercially available | ||
| Surgical instruments | |||
| Graefe forceps curved x1 | Fine Science Tools Inc. | 11151-10 | |
| Graefe forceps straight | Fine Science Tools Inc. | 11050-10 | |
| Needle holder Mathieu | Fine Science Tools Inc. | 12010-14 | |
| Scissors | Fine Science Tools Inc. | 14074-11 | |
| Semken forceps | Fine Science Tools Inc. | 11008-13 | |
| Small surgical scissors curved | Fine Science Tools Inc. | 14029-10 | |
| Small surgical scissors straight | Fine Science Tools Inc. | 14028-10 | |
| Standard pattern forceps | Fine Science Tools Inc. | 11000-12 | |
| Vannas spring scissors | Fine Science Tools Inc. | 15000-08 | |
| Equipment | |||
| Dissecting microscope | Leica MZ9 | ||
| Temperature controlled heating pad | Sygonix | 26857617 | |
| Equipment for stent implantation | |||
| Drug-eluting stent Xience 2,25mm x 8mm | Abbott Vascular USA | 1009544-18 | |
| Guide wire Fielder XT PTCA guide wire: 0.014" x 300cm | ASAHI INTECC CO., LTD Japan | AGP140302 | |
| Inflation syringe system | Abbott 20/30 Priority Pack | 1000186 | |
| Tissue processing and analysis | |||
| 30% H2O2 | Roth | 9681 | Histology |
| Ethanol | Roth | K928.1 | Histology |
| Giemsas Azur-Eosin-Methylenblau | Merck | 109204 | Histology |
| Graphic Drawing Tablet | WACOM Europe GmbH | CTL-6100WLK-S | |
| Roti Histofix, Formaldehyd 4% buffered | Roth | P087 | Histology |
| Technovit 9100 | Morphisto | 12225.K1000 | Histology |
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Cite This Article
Cornelissen, A., Florescu, R., Schaaps, N., Afify, M., Simsekyilmaz, S., Liehn, E., Vogt, F. Implantation of Human-Sized Coronary Stents into Rat Abdominal Aorta Using a Trans-Femoral Access. J. Vis. Exp. (165), e61442, doi:10.3791/61442 (2020).