在一只兔中产生两个具有不同血流动力学的囊状弹性蛋白酶消化动脉瘤
Summary
该协议描述了创建具有两个具有不同血流动力学（残端和分叉星座）的弹性蛋白酶消化动脉瘤的兔子模型的步骤。这允许在单个动物内具有不同血管结构和血流动力学状况的动脉瘤中测试新型血管内装置。
Abstract
具有接近人颅内动脉瘤的血流动力学、形态学和组织学特征的临床前动物模型在理解病理生理过程以及开发和测试新的治疗策略方面起着关键作用。这项研究旨在描述一种新的兔动脉瘤模型，该模型允许在同一动物内创建两个具有不同血流动力学条件的弹性蛋白酶消化的囊状动脉瘤。
五只平均体重为4.0（±0.3）公斤，平均年龄为25（±5）周的雌性新西兰白兔接受了显微外科残端和分叉动脉瘤的形成。一个动脉瘤（残端）是由右颈总动脉（CCA）暴露在其起源头躯干造成的。在CCA原点应用了一个临时夹子，另一个在2厘米以上。用局部注射100U弹性蛋白酶处理该段20分钟。通过将弹性蛋白酶处理的动脉袋缝合到右侧CCA与左侧CCA的端对侧吻合口中，产生了第二个动脉瘤（分叉）。创建后立即通过荧光血管造影控制通畅性。
平均手术时间为221分钟。在同一只动物中产生两个动脉瘤在所有兔子中都是成功的，没有并发症。除一个分叉动脉瘤外，所有动脉瘤在手术后立即未愈合，由于弹性蛋白酶孵育和立即腔内血栓形成，该动脉瘤显示出极端的组织反应。在手术和长达一个月的随访期间未观察到死亡。发病率仅限于一过性前庭综合征（一只兔子），该综合征在一天内自发恢复。
这里首次证明了创建具有残端和分叉血流动力学特征以及高度退化壁条件的双动脉瘤兔模型的可行性。该模型允许在不同流动条件下基于动脉瘤生物学研究自然病程和潜在的治疗策略。
Introduction
颅内动脉瘤是一种严重的疾病，破裂后死亡率达到50%，10-20%的患者长期残疾1。在过去的十年中，血管内治疗方案发展迅速，但与此同时，复发率也在增加，卷曲后动脉瘤再通率高达33% 2，3。为了更好地了解动脉瘤闭塞和再通的潜在病理生理学，以及开发和测试新的血管内装置，目前需要可靠的临床前模型，其血流动力学、形态学和组织学特征与人颅内动脉瘤相似4，5，6.截至今天，还没有明确的模型作为临床前试验的标准，研究人员可以使用大量的物种和技术7，8。
然而，兔子是一个特别感兴趣的物种，因为它的颈部动脉和人类脑血管之间的大小和血流动力学相似性，以及其相似的凝血和溶栓特征。颈总动脉 （CCA） 上具有弹性蛋白酶消化囊状动脉瘤的几种模型在流动条件、几何特征和壁特征方面显示出与人颅内动脉瘤的定性和定量相似性9，10，11，12。本研究旨在描述一种在同一动物中同时使用残肢和分叉弹性蛋白酶消化动脉瘤创建新的兔动脉瘤模型的技术。手术技术的灵感来自Hoh等人13和Wanderer等人14的技术，略有修改，以提供良好的标准化和可重复性，并确保低死亡率和发病率。
Protocol
注意：该实验已获得瑞士伯尔尼州地方动物护理委员会的批准（申请号BE108/16），所有动物护理和程序均按照机构指南和3R原则15，16进行。数据根据 REACH 指南报告。围手术期管理由董事会认证的兽医麻醉师进行。在这项研究中，雌性新西兰白兔，平均体重为4.0（±0.3）公斤，平均年龄为25（±5）周，被饲养在22-24°C的室温下，进行12小时的光照/黑暗循环?…
Representative Results
残端和分叉动脉瘤的产生在所有5只新西兰大白兔中均成功，没有术中并发症。在手术期间或24±2天的随访期间未观察到死亡。一只兔子经历了术后并发症，伴有前庭综合征和右侧失明。动物在24小时后完全自发恢复。这种并发症不会干扰其正常活动（自由运动，水和食物摄入，与其他动物的相互作用），并且不需要任何特殊治疗。没有自发性动脉瘤破裂。 平均手术时间为221分…
Discussion
最常见的动脉瘤形成技术包括通过开放或血管内方法在右侧CCA的起源处创建残端动脉瘤。该模型已被验证为一种稳定的非生长动脉瘤，随时间20，21 保持开放状态。第二种可能的技术涉及通过手术在左侧吻合右侧CCA并在分叉处缝合动脉瘤袋14，22，23来建立动脉分叉动脉瘤。尽管这两?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢Hans Rudolf Widmer教授，Luca Remonda博士和Javier Fandino教授对这项工作的科学支持和技术贡献。特别感谢Olgica Beslac在手术过程中的建议和Kay Nettelbeck的帮助。此外，我们感谢Daniela Casoni DVM，博士和医学兽医。Luisana Garcia，PD Alessandra Bergadano博士和Carlotta Detotto博士，感谢他们敬业的兽医支持。
Materials
| 4-0 resorbable suture | Ethicon Inc., USA | VCP292ZH | |
| 4-0 resorbable suture | Ethicon Inc., USA | VCP304H | |
| 6-0 non absorbable suture | B. Braun, Germany | C0766070 | |
| 9-0 non absorbable suture | B. Braun, Germany | G1111140 | |
| Adrenaline | Amino AG | 1445419 | any generic |
| Amiodarone | Helvepharm AG | 5078567 | any generic |
| Anesthesia machine | Dräger | any other | |
| Aspirin | Sanofi-Aventis (Suisse) SA | 622693 | any generic |
| Atropine | Labatec Pharma SA | 6577083 | any generic |
| Bandpass filter blue | Thorlabs | FD1B | any other |
| Bandpass filter green | Thorlabs | FGV9 | any other |
| Biemer vessel clip (2x) | B. Braun Medical AG, Aesculap, Switzerland | FD560R | any other |
| Bipolar forceps | any other | ||
| Bispectral index (neonatal) | any other | ||
| Blood pressure cuff (neonatal) | any other | ||
| Bycilces spotlight | any other | ||
| Clamoxyl | GlaxoSmithKline AG | 758808 | any generic |
| Dexmedetomidine | Ever Pharma | 136740-1 | any generic |
| Elastase | Sigma Aldrich | E7885 | |
| Electrocardiogram electrodes | |||
| Ephedrine | Amino AG | 1435734 | |
| Esmolol | OrPha Swiss GmbH | 3284044 | |
| Fentanyl (intravenous use) | Janssen-Cilag AG | 98683 | |
| Fentanyl (transdermal) | Mepha Pharma AG | 4008286 | |
| Fluoresceine | Curatis AG | 5030376 | |
| Fragmin | Pfizer PFE Switzerland GmbH | 1906725 | |
| Heating pad or heating forced-air warming system | |||
| Isotonic sodium chloride solution (0.9%) | Fresenius KABI | 336769 | |
| Ketamine | Pfizer PFE Switzerland GmbH | 342261 | |
| lid retractor | Approach | ||
| Lidocaine | Streuli Pharma AG | 747466 | |
| Longuettes | |||
| Metacam | Boehringer Ingelheim | P7626406 | Medication |
| Methadone | Streuli Pharma AG | 1084546 | Sedaton |
| Micro-forceps curved | Ulrich Swiss, Switzerland | U52-015-15 | |
| Micro-forceps straight 2x | Ulrich Swiss, Switzerland | U52-010-15 | |
| Microscissors | Ulrich Swiss , Switzerland | U52-327-15 | |
| Midazolam | Accord Healthcare AG | 7752484 | |
| Needle 23 G | arteriotomy | ||
| Needle holder | |||
| O2-Face mask | |||
| Operation microscope | Wild Heerbrugg | ||
| Papaverin | Bichsel | topical application | |
| Povidone iodine | Mundipharma Medical Company | any generic | |
| Prilocaine-lidocaine creme | Emla | ||
| Propofol | B. Braun Medical AG, Switzerland | General anesthesia | |
| Pulse oxymeter | |||
| Rectal temperature probe (neonatal) | |||
| Ringer Lactate | Bioren Sintetica SA | Infusion | |
| Ropivacain | Aspen Pharma Schweiz GmbH | 1882249 | Local anesthesia |
| Scalpell | Swann-Morton | 210 | |
| Small animal shaver | |||
| Soft tissue forceps | |||
| Soft tissue spreader | |||
| Stainless steel sponge bowls | |||
| Sterile micro swabs | |||
| Stethoscope | |||
| Surgery drape | |||
| Surgical scissors | |||
| Syringes 1 mL, 2 mL, and 5 mL | |||
| Tris-Buffer | Sigma Aldrich | 93302 | Elastase solution |
| Vascular clip applicator | B. Braun, Germany | FT495T | |
| Vein and arterial catheter 22 G | |||
| vessel loop | Approach | ||
| video camera or smartphone | |||
| Vitarubin | Streuli Pharma AG | 6847559 | |
| Yasargil titan standard clip (2x) | B. Braun Medical AG, Aesculap, Switzerland | FT242T |
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Cite This Article
Boillat, G., Franssen, T., Grüter, B., Wanderer, S., Catalano, K., Casoni, D., Andereggen, L., Marbacher, S. Creation of Two Saccular Elastase-Digested Aneurysms with Different Hemodynamics in One Rabbit. J. Vis. Exp. (170), e62518, doi:10.3791/62518 (2021).