兔子缺血和再灌注损伤模型
Summary
本研究展示了兔子急性局部心肌缺血和再灌注损伤的高度可重复动物模型，使用左侧微型开胸术治疗生存病例或中线胸骨切开术治疗非生存病例。
Abstract
这里的方案提供了一种简单、高度可重复的方法，用于非生存和生存实验在兔子中诱导原 位 急性区域心肌缺血。新西兰白成年兔用阿托品、乙酰丙嗪、布托啡醇和异氟烷镇静。对动物进行插管并置于机械通气状态。将静脉导管插入边缘耳静脉以输注药物。用肝素、利多卡因和乳酸林格氏液预先给动物用药。进行颈动脉切断术以获得动脉管路以监测血压。通过连续实时分析监测和记录选定的生理和机械参数。
在动物镇静并完全麻醉的情况下，进行第四个肋间小左开胸术（存活）或中线胸骨切开术（非存活）。打开心包，找到左前降支 （LAD）。
聚丙烯缝合线绕过LAD动脉的第二或第三对角线分支，聚丙烯长丝穿过一个小乙烯基管，形成圈套。动物经受30分钟的局部缺血，通过收紧圈套来闭塞LAD。心肌缺血可通过心外膜局部发绀在视觉上确诊。局部缺血后，结扎松动，心脏重新灌注。
对于生存和非生存实验，都 可以通过 超声心动图 （ECHO） 测量缩短分数来评估心肌功能。对于非生存研究，可以使用植入缺血区域的三个数字压电超声探头和使用根尖插入的左心室 （LV） 导管的左心室发育压力 （LVDP） 收集的超声显微测量数据可以连续获取，分别用于评估区域和整体心肌功能。
对于生存研究，切口闭合，进行左针胸腔穿刺术以清除胸腔空气，并实现术后疼痛控制。
Introduction
心血管疾病是世界上的主要死亡原因，每年导致超过1800万人死亡1,2,3。急性心肌梗死 （MI） 是一种常见的医疗急症，当血凝块或一块动脉粥样硬化斑块阻塞冠状动脉的血流时就会发生。这会导致动脉灌注区域的局部心肌缺血。
本研究描述了一种方案，该方案利用一种简单可靠的方法在兔模型中创建原位急性区域心肌缺血，用于非生存和生存实验。该方法的最初目标是评估线粒体移植对调节心肌坏死和增加缺血事件后缺血后心脏功能的影响。先前的研究表明，在缺血发作和氧气供应减少后，线粒体改变和高能磷酸盐水平迅速下降，导致心脏能量储存急剧下降4.研究人员试图使用药物干预和/或程序技术来改善缺血后功能并减轻心肌组织坏死，但这些技术提供的心脏保护有限，对线粒体损伤和功能障碍的影响最小 5,6,7。我们的团队和其他人之前已经表明，线粒体损伤主要发生在缺血期间，并且随着再灌注期间线粒体呼吸功能的保留，收缩恢复可以增强，心肌梗死面积减小 8,9,10。因此，我们假设线粒体从未受缺血影响的组织移植到再灌注前的缺血区域将提供一种减少心肌坏死和增强心肌功能的替代方法。在此，我们详细介绍了用于测试该理论的协议以及从我们的初步研究分析中获得的代表性结果。
此外，一些研究人员还关注了其他主题，这些主题对于确定心肌缺血再灌注损伤的影响和建立适当的治疗干预措施至关重要。其中一个研究领域是预处理。心肌缺血预处理是一种由短暂缺血应激激活的心脏保护机制，可在随后的长期缺血发作期间降低心肌细胞坏死率。这些机制可以通过缺氧或冠状动脉闭塞激活。Mandel 等人证明，缺氧-高氧预处理有助于维持一氧化氮代谢物的平衡，减少内皮素-1 的过度产生，并支持器官保护11。此外，还探讨了远程缺血预处理的概念，这是一种单器官预处理提供全身保护的现象。Ali 等人发现，在接受择期开腹主动脉瘤修复术的患者中，通过间歇叉夹住髂总动脉作为刺激进行远程预处理可降低术后心肌损伤、心肌梗死和肾功能损害的发生率12。
与其他物种的模型相比，兔子模型具有潜在的优势，并且几十年来一直用于多种不同的场景，包括心律失常的诱导、全球和区域缺血模型以及心脏收缩研究等13,14,15。虽然兔子的心脏比狗或猪的心脏小，但它足够大，可以以更低的成本轻松进行外科手术13.兔子心脏经常被使用，因为它与人类的心脏非常相似;事实上，它具有相似的代谢率，表达β-肌球蛋白重链，并且缺乏显着的心肌黄嘌呤氧化酶16。本文所描述的诱导局部心肌缺血的技术是简单、可重复且具有成本效益的。这种方法允许非生存和生存病例，因为仅诱导局部缺血而不是全局缺血，并且所需的材料是非特异性的。可以使用两种不同的手术方法（即胸骨切开术和微型开胸术），从而在研究设计方面为操作员和实验方案提供了更大的自由度。此外，该手术不需要使用体外循环。在这种情况下，冠状动脉旁路移植术的微创方法已成为需要多血管血运重建的患者的宝贵替代方案17,18。该模型可用于研究这些方法之间的差异，并为外科实习生提供基于动物的学习工具。此外，利用该模型进行心导管插入术可能有助于生理学研究和/或外科训练。
我们的模型为诱导区域心肌缺血并随后测量梗死大小、心肌功能和细胞变化的应用提供了一种方法。通过该协议，我们已经能够通过检查细胞器的内化、耗氧量、高能磷酸盐合成以及细胞因子介质和蛋白质组学途径的诱导来评估细胞功能和对缺血的适应以及拟议的治疗干预（即线粒体移植）的几个标志物。这些结果对于保持心肌能量、细胞活力和心脏功能非常重要，并允许客观评估缺血再灌注损伤后的心脏保护技术。该模型可用于研究缺血后心肌病理学和恢复领域的类似生物学途径和替代方案。
该方案的目标是提供一种高度可重复的方法，以诱导兔子原位急性局部心肌缺血，用于非生存和生存实验。该模型提供了一种高生存率、低术中死亡率和最低发病率的方法19。已经使用放射性标记材料、造影剂、磁共振成像或计算机模拟描述了急性区域心肌缺血的其他模型20,21,22。我们的方案提供了一种可靠且简单的方法，该方法具有成本效益，始终如一的可重复性，并且技术要求低，因此可以由没有手术专业知识的研究人员执行。该协议适用于使用左侧迷你开胸术的生存项目或使用中线胸骨切开术的非生存模型。
Protocol
Representative Results
Discussion
我们的方案展示了一种在兔子中进行急性局部心肌缺血的可靠方法。左侧微型开胸手术方法非常适合生存病例，必须尽量减少切口和相关疼痛。重要的是，拔管前不需要利尿剂治疗，非生存组术中或术后 4 周时没有死亡率。当方案的设计需要非生存病例时，或者当需要更详细地监测整体和区域心肌功能时，可以使用中线胸骨切开术（图 1）。
该方案最关…
Declarações
The authors have nothing to disclose.
Acknowledgements
使用该方案的原始研究得到了美国国家心脏、肺和血液研究所资助 HL-103642 和 HL-088206 的支持
Materials
| #10 blade | Bard Parker | 371210 | |
| #11 blade | Fisher Scientific | B3L | |
| 22 G PIV needle | BD Insyte | 381423 | |
| Acepromazine | VETONE | NDC 13985-587-50 | 0.5 mg/kg IM and IV |
| Aline pressure bag | Infu-Stat | 2139 | |
| Angiocath | Becton Dickinson | 382512 | |
| Arterial Catheter | Teleflex | MC-004912 | |
| Atropine | Hikma Pharmaceuticals | NDC 0641-6006-01 | 0.01 mg/kg IM |
| Betadine and 70% isopropyl alcohol | McKesson | NDC 68599-2302-6 | |
| Blood gas machine | Siemens | MRK0025 | |
| Bovie | Valleylab | E6008 | |
| Bulldog clamps | World Precision Instruments | 14119 | |
| Bupivacaine | Auromedics | NDC 55150-249-50 | 3 mg/kg IM |
| Butorphanol | Roxane | NDC 2054-3090-36 | 0.5 mg/kg IM |
| Clear acetate sheet | Oxford Instruments | ID 51-1625-0213 | |
| Clipers | Andis | AGC2 | |
| DeBakey forceps | Integra | P6280 | |
| Echocardiography machine | Philips | IE33 F1 | |
| Electrocardiography machine | Meditech | MD908B | |
| Endotracheal tube | Medline | #922774 | |
| Fentanyl | West-Ward | NDC 0641-6030-01 | 1–4 µg/kg transdermal patch |
| Formaldehyde solution 10% | Epredia | 94001 | |
| Glass plates | United Scientific | B01MUHX6MR | |
| Heparin Sodium | Sagent | NDC 69-0058-02 | 1000U in 1 mL 3 mg/kg |
| Hot water blanket | 3M | 55577 | |
| Isoflurane | Penn Veterinary Supply, INC | NDC 50989-606-15 | 1%–3% |
| Ketamine | Dechra | NDC 42023-138-10 | 10 mg/kg IV |
| Lab Chart 7 Acquisition Software | Adinstruments | ||
| Lactated Ringer's solution | ICUmedical | NDC 0990-7953-09 | 10 mL/kg/h |
| Laryngoscope | Welch Allyn | 68044 | |
| Left ventricule lumen catheter 3Fr | McKesson | 385764-EA | |
| Lidocaine (1%) | Pfizer | 4276-01 | 1–1.5 mL/kg IV |
| LVDP transducer | Edward | PDP-ED | |
| Marking pen | Viscot | 1451SR-100 Unsterile | |
| Mayo scissors | Mayo | S7-1098 | |
| Medetomidine | Entireoly Pets Pharmacy | NDC 015914-005-01 | 0.25 mg/kg IM |
| Metzenbaum scissors | Cole-Parmer | UX-10821-05 | |
| Monastra. Blue pigment 98% | Chemsavers | MBTR1100G | |
| Monocryl 5-0 | Ethicon | Y463G | |
| Mosquito clamp | Shioda | 802N | |
| PDS 3-0 | Ethicon | 42312201 | |
| Piezoelectric sonomicrometry crystals | Sonometrics | Small 2mm round | |
| Plegets | DeRoyal | 32-363 | |
| Povuine Iodine Prep Solutions | Medline | MDS093940 | |
| Precision vaporized system face mask | Yuwell | B07PNH69BF | |
| Prolene 3-0 | Ethicon | 8665G | |
| Proline 5-0 | Ethicon | 8661G | |
| Pulse oximetry probe | Masimo | 9216-U | |
| Rib spreader | Medline | MDS5621025 | |
| S12 Pediatric Sector Probe | Phillips | 21380A | |
| Sonomicrometer | Sonometrics | BZ10123724 | |
| Sterile gauze | Medline | 3.00802E+13 | |
| Sterile towels | McKesson | MON 277860EA | |
| Sternal retractor | Medline | MDS5610321 | |
| Sutures for closure | J&J Dental | 8698G | |
| Telemetriy monitor | Meditech | MD908B | |
| Temperature probe | Omega | KHSS-116G-RSC-12 | |
| Triphenyl tetrazolium chloride (1%) | Millipore | MFCD00011963 | |
| Ventilator | MedGroup | MSLGA 11 | |
| Vicryl 2-0 | Ethicon | V635H | |
| Vinyl tubing | ABE | DISW 3001 |
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