肺动脉高压与右心室衰竭的大型动物模型：绵羊左肺动脉结扎与进行性主肺动脉束带
1Department of Thoracic Surgery,Vanderbilt University Medical Center, 2Department of Biomedical Engineering,Carnegie Mellon University, 3Department of Pediatrics,Columbia University Medical Center, Columbia University Irving Medical Center, 4Department of Medicine,Columbia University Medical Center, Columbia University Irving Medical Center, 5Department of Anesthesiology,Vanderbilt University Medical Center, 6Department of Biomedical Engineering,Vanderbilt University
Summary
本手稿描述了发展严重右心室压力过载的手术技术和实验方法，以模拟其适应性和适应不良表型。
Abstract
肺动脉高压 （PH） 中的失代偿性右心室衰竭 （RVF） 是致命的，药物治疗选择有限。开发和测试PH的新疗法需要一个临床相关的大型动物模型，该模型增加肺血管阻力和裂谷热。本手稿讨论了先前发表的绵羊PH-RVF模型的最新发展，该模型利用左肺动脉（PA）结扎和主PA闭塞。这种PH-RVF模型是一个多功能平台，不仅可以控制疾病的严重程度，还可以控制RV的表型反应。
成年绵羊（60-80 kg）接受了左PA（LPA）结扎，放置主PA袖带并插入RV压力监测器。PA袖带和RV压力监测器连接到皮下端口。受试者每周接受两次进行性 PA 束带，持续 9 周，依次测量 RV 压力、PA 袖带压力和混合静脉血气 （SvO2）。在该模型的起始和终点时，使用超声心动图评估心室功能和尺寸。在12名动物受试者的代表性组中，到第9周，RV平均压和收缩压分别从第1周的28±5和57±7 mmHg增加到44±7和93±18 mmHg（平均±标准差）。超声心动图显示 PH-RVF 的特征性表现，尤其是右心室扩张、壁厚增加和隔膜弓缩。SvO2 和 PA 袖带压力的纵向趋势表明，可以调整 PA 条带的速率以引发不同的 RV 表型。较快的PA条带策略导致SvO2急剧下降<65%，表明RV失代偿，而较慢，更节奏的策略导致生理SvO2维持在70%-80%。一只经历加速策略的动物在第9周出现了几升胸腔积液和腹水。这种慢性PH-RVF模型为研究分子机制，开发诊断生物标志物以及实现治疗创新以管理PH的RV适应和适应不良提供了有价值的工具。
Introduction
失代偿性右心室 （RV） 衰竭是肺动脉高压 （PH） 患者发病和死亡的主要原因。在 PH 值患者中，超过 50% 的住院治疗是 RV 衰竭的原因，也是该患者群体中常见的死亡原因1，2。虽然目前对PH的药物治疗可以提供时间性措施，但它们并不能逆转疾病的进展。因此，唯一的长期治疗方法是肺移植。为了探索和测试PH和RVF的新型医学治疗和干预措施，需要一个临床相关的动物模型来概括该疾病复杂的病理生理学。特别是，临床上非常需要为PH患者开发RV靶向疗法，以改善RV功能。迄今为止，大多数已发表的PH和RV功能障碍动物研究都依赖于小鼠和大鼠等小型哺乳动物3。另一方面，只有少数大型动物模型从异常后负荷中研究疾病和RV病理生理学4，5，6，7。此外，先前发表的大型动物模型均未包括对疾病严重程度的受控滴定的实验程序的描述，这些实验程序可鉴别地导致代偿与失代偿的RV失败表型。需要一种可以滴定以不同程度补偿诱导急性和慢性房车衰竭的PH动物模型来研究疾病机制，并开发，测试PH和RVF的新型诊断和治疗方法并将其转化为临床实践。这种模型在大型动物中对于机械循环支持装置的发展特别有价值8。
这里，提出了一种慢性大型动物PH-RVF模型，该模型在成年绵羊中使用左肺动脉（PA）结扎和进行性主PA束带9，10。左PA（LPA）的结扎会增加肺血管阻力并降低PA电容11，12。渐进式 PA 条带法可精确滴定疾病严重程度和房车适应性。该平台也可以很容易地用于纵向研究疾病进展为RV失代偿。执行该模型所需的程序和过程被作为对大型动物平台感兴趣的研究人员的资源，以开发PH和RVF的新疗法。
Protocol
范德比尔特大学医学中心的机构动物护理和使用委员会批准了该协议。所描述的程序是根据美国国家研究委员会的实验 动物护理和使用指南第8版进行的。实验过程的概述和时间表如图 1所示。补充表1描述了绵羊的性别、体重、品种、绵羊的来源以及可能有助于重现目的的其他相关信息。 1.手术前一天，准备动物 在外科手术前…
Representative Results
使用12只绵羊的代表性组来显示该模型对发展不同程度的PH-RVF的功效。在这些绵羊中，PA袖带的平均压力从第1周的32±20 mmHg增加到第9周的1002±429 mmHg。这导致房车平均压和收缩压分别从第1周的28±5和57±7 mmHg增加到第9周的44±7和93±18 mmHg。此外，将PA袖带压力曲线叠加到混合静脉血氧饱和度（SvO2）上，以证明该模型在微调疾病表型方面的疗效（图4）。具体而言，更快的…
Discussion
所提出的PH-RVF模型可以可靠地诱导不同程度的疾病严重程度，以匹配调查目标。结合使用两种不同的方法来诱导这种疾病模型。首先，LPA结扎用于增加肺血管阻力并降低PA电容11，12，从而在已经增加的RV后负荷状态下建立慢性模型的起点。然后，PA袖带的植入及其进行性膨胀有助于开发PH-RVF的靶向表型。控制PA气囊压力及其变化率可以差分地产生补偿或?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作由美国国立卫生研究院R01HL140231资助。我们感谢动物护理司的畜牧业和兽医护理。我们感谢SR Light实验室及其工作人员Jamie Adcock，Susan Fultz，Codi VanRooyen和José Diaz，感谢他们对大型动物手术的专门技术支持。
Materials
| 0.9% Sodium Chloride Irrigation Pour Bottle by Baxter Healthcare, 1000 mL | Medline | BHL2F7124 | Surgical Disposable |
| 0.25% Bupivacaine | Hospira Inc | 0409-1160-18 | Medication, Intra-Operative |
| 0.9% Normal Saline, 1000 mL | Baxter Healthcare Corp | 0338-0049-04 | Medication, Intra-Operative |
| 0.9% Normal Saline, 500 mL | Baxter Healthcare Corp., | 0338-0049-03 | Medication, Chronic PH |
| 16 mm Heavy Duty Occluder with actuating tubing | Access Technologies | OC-16HD | Surgical Disposable |
| 3-mL Skin Prep Applicator | Medline | MDF260400 | Surgical Disposable |
| 70% isopropyl alcohol prep pads | Medline | MDS090670 | Disposable, Chronic PH |
| Adhesive bandage tape | Patterson Veterinary | Disposable, Chronic PH | |
| Adson forceps | V. Mueller | NL1400 | Surgical Instrument |
| Allis tissue forceps | V. Mueller | CH1560 | Surgical Instrument |
| Aortic clamp, straight (bainbridge forceps) | V. Mueller | SU6001 | Surgical Instrument |
| Backhaus towel forceps | V. Mueller | SU2900 | Surgical Instrument |
| Bags, Infusion: Nonsterile Novaplus Infusion Bag, 500 mL | Medline | TCV4005H | Disposable, Chronic PH |
| Berry sternal needle holder | V. Mueller | CH2540 | Surgical Instrument |
| Blades, Electrode: Electrode Blade, 6.5", with 0.24 cm Shaft | Medline | VALE15516 | Surgical Disposable |
| Blades: Stainless-Steel Sterile Surgical Blade, Size #10 | Medline | B-D371210 | Surgical Disposable |
| Blades: Stainless-Steel Sterile Surgical Blade, Size #11 | Medline | B-D371211 | Surgical Disposable |
| Blades: Stainless-Steel Sterile Surgical Blade, Size #15 | Medline | B-D371215 | Surgical Disposable |
| BNC Male to BNC Male Cable | Digi-Key | 415-0198-036 | Equipment |
| Castroviejo needle holder | V. Mueller | CH8589 | Surgical Instrument |
| Cefazolin | Apotex Corp | 60505-6142-0 | Medication, Intra-Operative |
| Ceftiofur Crystalline Free Acid | Zoetis Inc | 54771-5223-1 | Medication, Post-Operative |
| Chest Drain, with Dry Suction, Adult-Pediatric | Medline | DEKA6000LFH | Surgical Disposable |
| Chest tube passer | V. Mueller | CH04189 | Surgical Instrument |
| COnfidence Flowprobes for Research (PAU-Series) | Transonic | 24PAU | Equipment, Perivascular Flow Probe |
| Cooley tangential occlusion clamp | V. Mueller | CH6572 | Surgical Instrument |
| Data Acquisition Hardware | ADInstruments | PowerLab 16/30 | Equipment |
| DeBakey Aorta clamp | V. Mueller | CH7247 | Surgical Instrument |
| DeBakey multi-purpose clamp | V. Mueller | CH7276 | Surgical Instrument |
| Debakey tissue forceps, 12’’ | V. Mueller | CH5906 | Surgical Instrument |
| Debakey vascular tissue forceps 7 3/4’’ | V. Mueller | CH5902 | Surgical Instrument |
| Debakey vascular tissue forceps, 9’’ | V. Mueller | CH5904 | Surgical Instrument |
| Electrosurgical Generator | Covidien | Force FX-C | Equipment |
| Endotracheal Tube, 10mm | Patterson Veterinary | 07-882-9008 | Surgical Disposable |
| Enrofloxacin | Norbrook Laboratories Limited | 55529-152-05 | Medication, Intra-Operative |
| Fentanyl Transdermal Patch | Apotex Corp | 60505-7007-2 | Medication, Pre-Operative |
| Ferris smith tissue forceps | V. Mueller | SU2510 | Surgical Instrument |
| Finochietto rib spreaders, large | V. Mueller | CH1220-1 | Surgical Instrument |
| Finochietto rib spreaders, medium | V. Mueller | CH1215-1 | Surgical Instrument |
| Flexsteel ribbon retractor, 1” x 13” | V. Mueller | SU3340 | Surgical Instrument |
| Flexsteel ribbon retractor, 2” x 13” | V. Mueller | SU3346 | Surgical Instrument |
| Foerster sponge forceps, curved | V. Mueller | GL660 | Surgical Instrument |
| Gauze Sponges: Sterile X-ray Compatible Gauze Sponges, 16-Ply, 4" x 4" | Medline | PRM21430LFH | Surgical Disposable |
| Gerald-DeBakey forceps | V. Mueller | CH04242 | Surgical Instrument |
| Glassman Allis | V. Mueller | SU6152 | Surgical Instrument |
| Halsted mosquito forceps | V. Mueller | SU2702 | Surgical Instrument |
| Harken clamp | V. Mueller | CH6462 | Surgical Instrument |
| Heat Therapy Pump | Gaymar/Stryker | TP-400 | Equipment |
| Heparin | Fresenius Kabi, | 63323-540-31 | Medication, Chronic PH |
| Hospira Primary IV Sets, 80" | Patterson Veterinary | 07-835-0123 | Surgical Disposable |
| Hypertonic saline 3% | Baxter Healthcare Corp., | 0338-0054-03 | Medication, Chronic PH |
| Hypodermic Needle with Bevel and Regular Wall, 20 G x 1" | Medline | B-D305175Z | Disposable, Chronic PH |
| Interface Cable, Edwards LifeScience Transducer to ADInstruments Bridge Amplifier | Fogg System | 0395-2434 | Equipment |
| Intravenous Infusion Pump | Heska | Vet/IV 2.2 Infusion Pump | Equipment |
| Isoflurane | Patterson Veterinary | 14043-704-06 | Medication, Pre-Operative |
| Kantrowitz thoracic clamp, 9-1/2” | V. Mueller | CH1722 | Surgical Instrument |
| Kelly hemostats | V. Mueller | 88-0314 | Surgical Instrument |
| Lidocaine HCl, 2.46% | PRN Pharmacal, | 49427-434-04 | Medication, Chronic PH |
| Ligaclip Multiple-Clip Appliers by Ethicon | Medline | ETHMCS20 | Surgical Disposable |
| Loop, Vessel, Mini, Red, 2/pk, Sterile | Medline | DYNJVL12 | Surgical Disposable |
| Lorna non-perforating towel forceps | V. Mueller | SU2937 | Surgical Instrument |
| Mayo dissecting scissors, curved | V. Mueller | SU1826 | Surgical Instrument |
| Mayo dissecting scissors, straight | V. Mueller | SU1821 | Surgical Instrument |
| Medipore Dress-It Pre-Cut Dressing Covers by 3M | Medline | MMM2955Z | Surgical Disposable |
| Meloxicam | Patterson Veterinary | 14043-909-10 | Medication, Post-Operative |
| Mixter thoracic forceps, 9” | V. Mueller | CH1730-003 | Surgical Instrument |
| Mosquito hemostats | V. Mueller | 88-0301 | Surgical Instrument |
| Multi-Channel Research Consoles | Transonic | T402/T403 | Equipment, Perivascular Flow Meter |
| Multi-Lumen Central Venous Catheterization Kits | Medline | ARW45703XP1AH | Surgical Disposable |
| Multi-Parameter Vital Signs Monitor | Smiths Medical | SurgiVet Advisor 3 | Equipment |
| Needles: Hypodermic Needle with Regular Bevel, Sterile, 18 G x 1.5" | Medline | B-D305185Z | Surgical Disposable |
| No. 3 knife handle | V. Mueller | SU1403-001 | Surgical Instrument |
| No. 7 knife handle | V. Mueller | SU1407 | Surgical Instrument |
| Non-Vented Male Luer Cap | Qosina | 13614 | Disposable, Chronic PH |
| Octal Bridge Amplifier | ADInstruments | FE228 | Equipment |
| Ophthalmic Ointment | Akorn Animal Health | 59399-162-35 | Medication, Pre-Operative |
| Penrose Tubing, 6 mm x 46 cm, 11 mm Flat | Medline | SWD514604H | Surgical Disposable |
| Perma-Hand Black Braided Silk: 2-0 SH Taperpoint Needle, Control Release, 30" | Medline | ETHD8552 | Surgical Disposable |
| Perma-Hand Suture, Black Braided, Size 0, 6 x 30” | Medline | ETHA306H | Surgical Disposable |
| Perma-Hand Suture, Black Braided, Size 4-0, 12 x 30" | Medline | ETHA303H | Surgical Disposable |
| Phenylephrine | West-Ward | 0641-6142-25 | Medication, Intra-Operative |
| Polyhesive Cordless Patient Return Electrodes, Adult | Medline | SWDE7509 | Surgical Disposable |
| Port-A-Cath Huber Needle, Straight, 22 G x 1-1/2" | Medline | AAKM21200724 | Disposable, Chronic PH |
| PROLENE Monofilament Suture, Blue, Size 4-0, 36", Double Arm, RB-1 Needle | Medline | ETHD7143 | Surgical Disposable |
| PROLENE Polypropylene Monofilament Suture, Blue, Double-Armed, RB-1 Needle, Size 5-0, 24" | Medline | ETH8555H | Surgical Disposable |
| Regional Block Needles, 22-gauge | Medline | B-D408348Z | Surgical Disposable |
| Schnidt tonsil artery forceps | V. Mueller | M01700 | Surgical Instrument |
| Skin staple extractor | Medline | CND3031 | Disposable, Chronic PH |
| Skin stapler 35 wide, with counter | Medline | STAPLER35W | Surgical Disposable |
| Sphygmomanometer | Medline | Equipment | |
| Sponge bowl | V. Mueller | GE-75 | Surgical Instrument |
| Sponge, Lap: X-Ray Detectable Sterile Lap Sponge, 18" x 18", 5/Pack | Medline | MDS241518HH | Surgical Disposable |
| Sponge, Peanut: X-Ray Detectable Sterile Peanut Sponge, Small, 3/8" | Medline | MDS72038 | Surgical Disposable |
| Sterile Disposable Deluxe OR Towel, Blue, 17'' x 27'', 2/Pack | Medline | MDT2168202 | Surgical Disposable |
| Sterile Luer-Lock Syringe, 3 mL | Medline | SYR103010Z | Disposable, Chronic PH |
| Sterile Luer-Lock Syringe, 5 mL | Medline | SYR105010Z | Disposable, Chronic PH |
| Sterile Surgical Equipment Probe Covers | Medline | DYNJE5930 | Surgical Disposable |
| Stopcock: 3-Way Stopcock with Handle in OFF Position, Rotating Adaptor Male Collar Fitting, 45 PSI | Medline | DYNJSC301 | Surgical Disposable |
| Stopcock: 3-Way Stopcock with Handle in OFF Position, Rotating Adaptor Male Collar Fitting, 45 PSI | Medline | DYNJSC301 | Disposable, Chronic PH |
| Subcutaneous Port with 5-French Connector and Blue Boot | Access Technologies | CP2AC-5NC | Surgical Disposable |
| Super cut metzenbaum dissecting scissors | V. Mueller | CH2032-S | Surgical Instrument |
| Super cut nelson-metzenbaum dissecting scissors | V. Mueller | CH2025-S | Surgical Instrument |
| Syringes: Sterile Luer-Lock Syringe, 10 mL | Medline | SYR110010Z | Surgical Disposable |
| Thoracic Catheter, Straight, 28 Fr x 20" | Medline | SWD570549H | Surgical Disposable |
| Three-quarter surgical drape | Medline | DYNJP2414H | Surgical Disposable |
| Tiletamine + Zolazepam | Zoetis Inc | 54771-9050-1 | Medication, Pre-Operative |
| TourniKwik Tourniquet Set with Four 7.5" Bronze-Colored Tubes and 1 Snare, 12 French | Medline | CVR79013 | Surgical Disposable |
| Transducer clip | Edwards LifeScience | TCLIP05 | Equipment |
| Trigger Aneroid Gauge (Sphygmomanometer) | Patterson Veterinary | 07-815-0464 | Equipment |
| TruWave Disposable Pressure Transducer Kits by Edwards Lifesciences | Medline | VSYPX260 | Surgical Disposable and Chronic PH |
| TS420 Perivascular Flow Module | Transonic | TS420 | Equipment, Perivascular Flow Meter |
| Tubing, Suction: Sterile Universal Suction Tubing with Straight Ribbed Connectors, 1/4" x 12' | Medline | OR612 | Surgical Disposable |
| Tubing: Pressure Monitoring Tubing with Fixed Male Luer Lock and Female Fitting, Low Pressure, 72" L | Medline | DYNJPMTBG72MF | Surgical Disposable |
| Tubing: Pressure Monitoring Tubing with Fixed Male Luer Lock and Female Fitting, Low Pressure, 72" L | Medline | DYNJPMTBG72MF | Disposable, Chronic PH |
| Tubular Elastic Dressing Retainer | Medline | DERGL711 | Disposable, Chronic PH |
| Tuffier rib retractor | V. Mueller | CD1101 | Surgical Instrument |
| Tygon E-3603 Flexible Tubings | Fisher Scientific | 14-171-227 | Surgical Disposable |
| U.S.A retractor | V. Mueller | SU3660 | Surgical Instrument |
| Umbilical Tape, Cotton, 3-Strand, 1/8 x 36" | Medline | ETHU12TH | Surgical Disposable |
| Valleylab Button Switch Pencil | Medline | VALE2516H | Surgical Disposable |
| Vanderbilt deep vessel forceps | V. Mueller | CH1687 | Surgical Instrument |
| Veterinary Anesthesia Machine | Midmark | Matrx VMC | Equipment |
| Veterinary Anesthesia Ventilator | Hallowell EMC | Model 2000 | Equipment |
| Vicryl: Undyed Coated Vicryl 0 CT-1 36" Suture | Medline | ETHVCP946H | Surgical Disposable |
| Vicryl: Undyed Coated Vicryl 2 TP-1 Taper 54" Suture | Medline | ETHVCP880T | Surgical Disposable |
| Vicryl: Undyed Coated Vicryl 2-0 CT-1 18" Suture | Medline | ETHVCP739D | Surgical Disposable |
| Vital crile-wood needle holder, 10-3/8” | V. Mueller | CH2427 | Surgical Instrument |
| Vital mayo-hegar needle holder, 7-1/4” | V. Mueller | CH2417 | Surgical Instrument |
| Vital metzenbaum dissecting scissors, 14’’ | V. Mueller | CH2009 | Surgical Instrument |
| Vital metzenbaum dissecting scissors, 9” | V. Mueller | CH2006 | Surgical Instrument |
| Vital ryder needle holder, 9” | V. Mueller | CH2510 | Surgical Instrument |
| Yankauer, Bulb Tip: Sterile Rigid Yankauer with Bulb Tip, No Vent | Medline | DYND50130 | Surgical Disposable |
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Citar este artigo
Ukita, R., Stokes, J. W., Wu, W. K., Talackine, J., Cardwell, N., Patel, Y., Benson, C., Demarest, C. T., Rosenzweig, E. B., Cook, K., Tsai, E. J., Bacchetta, M. A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep. J. Vis. Exp. (173), e62694, doi:10.3791/62694 (2021).