兔子静脉介词模型模仿再血管化手术使用静脉移植评估动脉血压下的影响增生
Summary
本议定书旨在通过将静脉置于动脉血压下，在使用静脉移植进行静脉移植手术后衰减静脉内血性增生的策略，从而在实验中产生静脉内质增生。
Abstract
虽然静脉移植在缺血性疾病的再血管化手术中通常用作自体移植物，但由于动脉血压暴露，导致静脉增生加速，长期的体量仍然很差。本协议旨在通过将兔形静脉与ipsi侧胡萝卜动脉相交，建立实验性静脉增生。该协议不需要在身体躯干深处进行外科手术，切口的程度有限，对动物的侵入性较小，允许在植入后进行长期观察。这个简单的程序使研究人员能够研究减轻植入静脉移植的暗示增生进展的策略。使用这种协议，我们报告了微RNA-145（miR-145）的影响转导，已知微RNA-145能够控制血管平滑肌肉细胞（VSMC）从增殖到收缩状态的表型，进入收获的静脉移植物。我们通过VSMC的表型变化，在植入手术前通过转导miR-145，证实了静脉移植的体内增生衰减。在这里，我们报告一个侵入性较低的实验平台，以研究可用于减轻静脉移植在再血管手术中的风险增生的策略。
Introduction
全世界因动脉粥样硬化而患缺血性疾病的患者数量正在增加1。尽管目前心血管疾病的医学和外科治疗取得了进步，但缺血性心脏病，如心肌梗死，仍然是导致发病率和死亡率的主要原因2。此外，以四肢血流量减少为特征的外周动脉疾病诱发严重的肢体缺血，其中约40%的患者在诊断后6个月内失去双腿，死亡率高达20%3。
再血管化手术，如冠状动脉旁路移植（CABG）和周围动脉旁路手术，是缺血性疾病的主要治疗选择。这些手术的目的是提供一个新的血液途径，提供足够的血液流向动脉的硬骨或阻塞病变的远端部位。虽然原位动脉移植，如CABG的内部胸动脉，是首选作为旁路移植，因为预期的更长的光度，静脉移植，如自体性沙芬静脉，是常用的，因为更高的可及性和可用性4。静脉移植的薄弱环节是动脉移植率比动脉移植率低，在动脉压力下加速增生，导致静脉移植病6。
静脉移植疾病通过以下三个步骤发展：1） 血栓;2） 暗示性增生;和3）动脉粥样硬化7。为了解决静脉移植病，已经进行了大量的基础研究。到目前为止，除了抗血小板和降脂疗法外，没有建议,在近期指南9、10、11、1210中进行冠状动脉或外周9再血管化手术后进行二次预防。11,12因此，为了克服静脉移植疾病，特别是静脉增生，需要建立相关的实验平台，供进一步研究。
肌增多是一种适应现象，发生在对周围环境变化的反应中，血管平滑肌肉细胞 （VSMC） 在肌阵中增殖、积累和生成细胞外基质。因此，它为移植异质7提供了基础。在超塑性内皮中，VSMC承担增殖，生产而不是收缩，称为”象形变化“8。控制静脉移植的VSMC表型，预防静脉移植疾病，是一项关键的研究目标，并为此课题8进行了许多基础研究。然而，一项旨在实现VSMC表型药理控制的随机对照临床研究显示，结果有限。此外，没有标准化的疗法来防止内量增生。更多的基础研究，包括动物模型研究，是必要的。
为了促进这一领域的研究，建立一个动物模型，在动脉血压下重述静脉移植，允许长期的术后观察是至关重要的。Carrel等人建立了将外管静脉植入胡萝卜动脉14的一种狗模型。之后，各种静脉移植物被用于调查动脉血压变化的生理和病理影响，包括下部静脉卡巴移植到胸腔或腹部主动脉，或移植到股动脉15、16、1716,17的沙赫诺静脉。15这些模型建在较大的动物，如猪或狗，适合模仿静脉移植疾病在临床的情况下。然而，建立一个动物模型，可以准备没有特殊的手术技术，以较低的成本将是研究人员试图开发新的治疗策略，通过VSMC表型控制在体内衰减体内的增生。最初，在神经外科18、19,19领域，将血管插入兔子的胡萝卜动脉。此后，它应用于20、21,21年恶性增生研究。初始模型仅由静脉互置组成，从而节省时间。此外，随后的研究表明，静脉移植的制备也影响了静脉增生22。Davies等人评估了气球导管损伤对兔子静脉介词模型23、24,24中刺激性增生的影响。虽然气球导管损伤，除了静脉介词更相关的临床设置，一个更可重现的模型也要求。因此，江等人考察了差流量环境对突增增生的影响，建立了远端分支结扎程序，作为可重现的模型25。然而，他们在静脉移植介位时采用了袖口技术，在临床环境中似乎不同于手工缝合的麻醉。在本协议中，我们报告一个可重复的，临床相关，和广泛的程序，准备兔子静脉介词模型，以评估动脉血压下的内量增生。
Protocol
注：所有对动物进行的外科手术均应按照《实验室动物护理和使用指南》（www.nap.edu/catalog/5140.html）或其他适当的道德准则进行。议定书应在继续之前得到有关机构动物福利委员会的批准。 1. 动物的准备 购买体重2.7~3.0公斤的雄性日本白兔（或体型相当的兔子）。 在12小时的光暗周期中使兔子适应1周，并在手术前定期喂兔子周食。 2. 麻?…
Representative Results
图 1A显示了静脉间位手术（上面板）后 2 周内成功进行插值增生的代表性图像。下面板显示微RNA-145负载聚（乳酸-共甘油酸）纳米粒子的治疗效果，这些纳米粒子衰减了内量增生（下面板）。图1B显示了使用磷酸盐缓冲盐水控制（PBS）、控制微RNA（Cont-miR）和微RNA-145（miR-145）组对照组之间的内量增生的比较。微RNA含有聚（乳酸-共甘油酸）纳米粒子。微R…
Discussion
本协议旨在提供一个实验平台，用于测试 VSMC 的各种分子或基因干预，以控制表型从增殖状态到收缩状态，进而衰减体内静脉内刺激增生的进展。利用这个模型，我们在手术后2周成功地制备了内皮增生（图1A），并指明了微RNA-145控制VSMC表型26、27,27的治疗潜力，验证了本发明作为模型，以进一步研究静脉增生的衰减。
<p class="jo…Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了日本教育、文化、体育、科学和技术部的研究资助（25462136） 的支持。
Materials
| 10% Povidone-iodine solution | Nakakita | 872612 | Surgical expendables |
| 2-0 VICRYL Plus | Johnson and Johnson | VCP316H | Surgical expendables |
| 4-0 Silk suture | Alfresa pharma | GA04SB | Surgical expendables |
| 8-0 polypropylene suture | Ethicon | 8741H | Surgical expendables |
| Cefazorin sodium | Nichi-Iko Pharmaceutical | 6132401D3196 | Antibiotics |
| Fogarty Catheter (2Fr) | Edwards Lifesciences LLC | E-060-2F | Surgical expendables |
| Heparin | Nipro | 873334 | Anticoagulant |
| Intravenous catheter (20G) | Terumo | SR-OT2051C | Surgical expendables |
| Isoflurane | Fujifilm | 095-06573 | Anesthesia |
| Lidocaine hydrochloride | MP Biomedicals | 193917 | Anesthesia |
| Pentobarbital sodium | Tokyo Chemical Industry | P0776 | Anesthesia |
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Citar este artigo
Nishio, H., Minatoya, K., Masumoto, H. A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure. J. Vis. Exp. (159), e60931, doi:10.3791/60931 (2020).